Preliminary Classification of Viruses Based on Quantitative Comparisons of Viral Nucleic Acids

JOURNAL OF VIROLOGY, Apr. 1967, p. 245-259Copyright © 1967 American Society for Microbiology

Vol. 1, No. 2Printed in U.S.A.

Preliminary Classification of Viruses Based on

Quantitative Comparisons of ViralNucleic Acids

A. J. D. BELLETT

Departmenit of Microbiology, Johli C67rtiln School of Medical Research, Australian Nationial University, Canberra,Australia

Received for publication 21 November 1966

It is proposed that classifications used in science are of two main types; thosewhich are designed to solve practical problems and which are based on conventions,and those which are designed to solve theoretical problems, based on theories, andin which the classes are tested by experiment. An attempt has been made to con-

struct a preliminary classification of viruses which is of the second type. It is basedon the theories of molecular biology, with the use of computer-based comparisonsof the molecular weights and base ratios of viral nucleic acids to assign the viruses

to clusters which show a high degree of correlation with groupings based on nucleicacid hybridization, serological cross-reactions, and phenotypic properties.

Some classifications consist of a number ofscientific theories and hypotheses, where "scien-tific" is used to describe statements which can bedisproved by experiment or observation (61).These classifications will be described as "scien-tific" in this limited sense. Other classificationsare scientific in the wider sense since they arenecessary for the development and use of scien-tific knowledge and involve the use of experi-mental methods to determine the properties ofthe entities, but they are based on conventionsrather than on scientific theories. A "conven-tional" classification may itself suggest a theoryon which a "scientific" classification can bebased. In the section on Principles and Methodsin this paper, this philosophy of classification isapplied to the problem of classifying viruses.In the section on Data and Results, an attemptto construct a preliminary "scientific" classifi-cation of viruses is described.

PRINCIPLES AND METHODSConvention and theory. A convention is an

agreed usage designed to solve a practical prob-lem. It can be neither proved nor disproved be-cause it makes no assertion about reality; it willbe accepted if it is useful, ignored if it is not.A scientific theory is a universal statement

which explains a number of observed phenomenaand which can be disproved by an experiment;it contradicts a class of basic statements, the

class of potential falsifiers of the theory (61). Atheory is corroborated if, in spite of frequentexperimental challenge, we are unable to dis-prove it. A theory cannot be proved by "induc-tive" logic, nor can its degree of corroboration beequated with a probability (61).

Conventional and "scientific" classifications. Aconventional classification is designed to solve apractical problem; it is based on a convention asto which properties of a set of entities shall beused to divide them into classes.A "scientific" classification is designed to solve

a theoretical problem. It is based on a scientifictheory, the initial theory, which attempts to ac-count for the properties of the entities and thedistribution of those properties within the popula-tion of entities. The entities are put in classes,and an independent hypothesis is proposed foreach class. The hypotheses state that members ofa given class are related with respect to the initialtheory, but that no such relationship exists amongmembers of different classes. The class hypothesesand the initial theory are continually tested byexperiment. The classification is improved by theproposal of more and more informative andcorroborated theories and hypotheses, and by therejection or modification of theories and hy-potheses which are disproved; an example of thisprocess is the history of the periodic table of theelements.

Nomenclature and classification. All nomen-

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BELLETT

clature is conventional; it may be, but need notbe, based on a classification. A "scientific"classification changes constantly in a new andexpanding field, and is not a suitable basis fornomenclature under these conditions.

Class of all VirusesThe class of all viruses is a conventional class;

any precise definition of "virus" which is adoptedmust be arbitrary, and it cannot be concludedthat all entities which satisfy the definition arerelated, or that no virus is related to any entitywhich is excluded from the class (87). To con-struct a "scientific" classification of viruses, onecan begin by classifying those entities whichsatisfy the suggested definition of viruses butrelationships outside the conventional classshould also be sought. I shall use the term"viruses" to refer to a class of entities defined bythe sum of the properties listed under "viruses"in Table 3 of Lwoff (44), that is, to infectiousagents which contain one type of nucleic acidonly, which multiply from that nucleic acid, andwhich do not grow and divide (as defined byLwoff) or contain a Lipmann system.

Classification of Viruses

Proposals of the Provisional Committee forNomenclature of Viruses (PCNV) (4). The mem-bers of the PCNV were concerned primarily withnomenclature rather than classification, but theyproposed a classification which is Linnean in ap-proach and terminology; their system of nomen-clature does indeed require that viruses beclassified at least into genera before they can benamed. Objections to this system have beenpublished by others (9, 26). My main concern isto point out that the classification is conven-tional; once this is accepted, it follows that itssurvival should depend only on its utility. It doesnot claim to be "scientific," and can therefore notbe criticized on the grounds that it is arbitrary.

Adansonian principle and the proposals of Gibbset al. (26). Gibbs et al. (26) are also more con-cerned with nomenclature than with classifica-tion, but treat the two problems separately. Theypropose that viruses should eventually be classi-fied on the Adansonian principle that all charac-ters have equal weight, by use of the computer-based numerical technique developed by Sneath(75).The Adansonian principle is a convention; it

proposes no theory as to what determines theproperties which are used in classification, orwhy they must all be considered equally im-portant. Williams and Lance (88) suggested that,if the population of elements is treated as finite

and defined, the classification produced by suchanalysis cannot be held to be true or false, or evenprobable or improbable; it can only be profitableor unprofitable. Goodall (29) defined the utilityof such a classification in terms of its ability topredict an attribute, a function suggested byGibbs et al. (26) for their proposed pheneticclassification of viruses. Computer-based Adan-sonian classifications have been described asobjective (26); they may be unbiased given idealdata but are not necessarily "scientific" as de-fined here.Modern classification programs can deal with a

small number of characters, either quantitativeor qualitative (40, 41, 88). They can be used toproduce conventional classifications for a de-fined purpose or an undefined and potentiallyinfinite number of purposes (29), or, given aninitial theory and appropriate empirical tests,they can be used to construct profitable classhypotheses.

Proposals for a Preliminary "Scientific"Classification of Viruses

The systems discussed in the previous sectionwere designed to solve the practical problems ofnaming and identifying viruses, and were notconcerned with their natural relationships. Someviruses have been shown to be genetically relatedby nucleic acid hybridization experiments (Table2), but as yet no general "scientific" classifica-tion of viruses has been attempted. Such aclassification will basically be a system for theproposal and testing of solutions to the theo-retical problems of what determines the proper-ties of viruses and the distribution of these prop-erties among different viruses, just as the periodictable has been concerned with theories whichaccount for the distribution of properties amongdifferent elements.The "central dogma" of molecular biology

is a highly informative and corroborated scien-tific theory and is suitable as an initial theory forthe classification of viruses. Similar approaches tothe classification of bacteria (48), mycoplasmas(62), and higher organisms (34) have been pro-posed. For the purpose of a "scientific" classifi-cation of viruses, the theory will be briefly re-stated as follows.

Initial theory. The properties of a virus are de-termined by the sequence of purine and pyrim-idine bases in its nucleic acid, which may besingle- or double-stranded, and of the deoxy-pentose or pentose type. During replication in asusceptible cell, the viral nucleic acid is exposed,copied by a process involving pairing of comple-mentary bases, and translated into polypeptideswith or without transcription into one or more

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VIRUS CLASSIFICATION BASED ON NUCLEIC ACIDS

messenger nucleic acids. The genetic code isuniversal. Progeny virions consist of progenynucleic acids protected by proteins and, in somecases, lipids specified by either the virus or itshost.The possible relationships among different

viruses can now be defined with respect to theinitial theory. It is convenient first to define termswhich describe the relationship between the in-formation contents of nucleic acids. Two codonsare isosemantic if they specify the same aminoacid in a polypeptide (91). Two polynucleotidesare equisemantic if they specify the same sequenceof amino acids; their base sequences will beidentical, or differ only in bases which result inthe substitution of one isosemantic codon byanother.Two viruses are isologous if their nucleic acids

are equisemantic; their nucleic acids will beidentical in molecular weight, and, since theirpossible use of different isosemantic codons willbe restricted by the population of transferribonucleic acid (RNA) molecules available tothem, they are also likely to have similar baseratios. Two viruses are heterologous if theirnucleic acids contain no equisemantic sequences;their proteins and phenotypic properties willshow few similarities. Two viruses are homol-ogous if their nucleic acids are equisemantic oversome, but not all, of their lengths. The degree ofhomology between two viruses is inversely pro-portional to the number of bases (or base-pairs)by which their nucleic acids differ. Homologousviruses will have some similar proteins andphenotypic properties; if the relationship is close,the viral nucleic acids will have similar baseratios and molecular weights and will formmolecular hybrids under appropriate experi-mental conditions.The object of this preliminary classification is

to identify groups of viruses for each of which itis reasonable to propose the hypothesis that allmembers are homologous with one another.Also included will be some groups consisting ofone virus, which is proposed as not being homol-ogous with any other virus in the classification.It is hoped that these hypotheses will then be testedby nucleic acid hybridization experiments andcomparison of nearest-neighbor base frequencies.The initial theory suggests that groups of

viruses have similar properties because theirnucleic acids are partially equisemantic, but doesnot account for the occurrence of equisemanticnucleic acids. In higher organisms, classificationsbased on homology agree with those based onphylogeny. The probability of chance homologyis very low, and it could be proposed that homol-ogous viruses have a common phylogeny. This

theory would give a more informative accountof the distribution of properties among differentgroups of viruses, but at present it is not directlytestable.

Base sequences in nucleic acids cannot be de-termined yet. However, the base ratios andmolecular weights of viral nucleic acids are crudebut unbiased functions of the information con-tents of these molecules, can be determined ex-perimentally, and are more relevant to the initialtheory than are phenotypic properties. A pre-liminary classification of some viruses wastherefore attempted, the molecular weights andbase ratios of the viral nucleic acids being usedas the characters.

This approach has several disadvantages, themost serious of which is coincidence of molecularweights and base ratios of heterologous nucleicacids resulting in overlapping of values amongdifferent groups. Another problem is the dif-ficulty of comparing viruses which have double-stranded nucleic acids with those which havesingle-stranded nucleic acids. To avoid a con-vential division of viruses along these lines, it isproposed that no virus with single-strandednucleic acid is homologous with any virus withdouble-stranded nucleic acid, and these viruseswill be treated separately unless the hypothesisis disproved experimentally.

This analysis will not detect homology be-tween two viruses with nucleic acids that differgreatly in molecular weight, the smaller beingpartially equisemantic with a short section of thelarger. The degree of homology between suchviruses would be low; it could be detected bymolecular hybridization, but the result would beasymmetric in reciprocal tests.When comparing viruses with single-stranded

nucleic acids, it is assumed that the input (+)strand, and not its complementary (-) strand,specifies the sequence of amino acids in the viralproteins. This hypothesis may be untrue forsome groups of viruses, or some cistrons may beread from the + and some from the - strand.Because the simplest hypothesis has not beendisproved, the unmodified data on the + strandswere used in the analysis.

Computer programs. Computer-based classifi-cation techniques were used to calculate the dif-ferences between the characters for all possiblepairs of viruses, and to sort the viruses intoclusters in which these differences are minimal.The distance function used was the nonmetric co-efficient (NMC) of the MULTIST program, anddendrograms were produced by use of flexiblesorting, with a, = 0.625, a2 = 0.625, 3 = -0.25and y = 0 (40, 41, 88). In this case, each virusfuses with a neighboring virus or cluster of

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248~~~~~~BELLETTJ.VRL

TABLE la. Data for viruses which contain single-stranded nucleic acidsa

INucleic ac

Virus Strain Abbrev-iation Group Mol wvt Mol

(X 10-6) AI

zidb

les % ,References

C iU(T)

Turnip yellow mosaic. TYMV 0 1.9 22.5 17.4 38.0 22.3 46, 72, 53Wild cucumber mosaic. WCuM 0 2.4 18.3 15.9 40.0 25.8 90M13*.. ~~~~~~~~~~~M131 1.3 23.3 21.1 19.8 35.8 68

fd*..............fd 1 1.3 24.4 19.9 21.7 34.1 33,49OX 174*........... OX 174 2 1.7 24.6 24.1 18.7 32.8 74QO ........... Qf 3 1.1 22.3 23.7 24.7 i29.4 32Tobacco ringspot... TRS 4 1.5 23.9 24.7 23.2 28.2 72Newcastle disease NDV 5 7.5 23.8 23.8 23.0 29.4 21Influenza...........AC fluA 6a 2.9 23.0 19.0 23.0 31.5 69Influenza....A,7 PR8 PR8 6a 2.9 23.1 20.1 24.0 32.8 1,72Influenza........ A,WSE WSE 6a 2.9 22.5 20.1 24.1 33.3 72Influenza......A, swine SW 6a 2.9 122.8 20.4 24.5 32.4 72Influenza.....A, CAM CAM 6a 2.9 22.8 19.3 24.5 33.5 72Influenza........... A, MEL MEL 6a 2.9 23.0 19.8 25.3 32.0 72Influenza....Bc flu B 6b 2.9 22.0 17.5 23.0 37.0 69Influenza...........B, LEE LEE 6b 2.9 23.0 18.3 23.1 35.6 72Influenza.......... B, ROB ROB 6b 2.9 22.5 18.5 23.5 35.5 72Influenza..........B, MIL MIL 6b 2.9 22.8 17.5 23.8 36.0 72Avian myeloblastosis.....AVM 7a 12.0 25.3 28.5 23.6 22.8 64Rous sarcoma..... Bryand RSV 7a 12.0 25.1 28.3 '24.2 22.4 64Rauscher murine leukemia. Rau 7b 13.0 25.5 25.1 26.7 22.7 54Mammary tumor....... MT 7c 12.0 19.0 30.0 22.0 29.0 20, 43Squash mosaic.......,SqM 8 2.4 31.6 22.5 16.2 29.5 51Tobacco mosaic.... "Normal" TMV 9a 2.1 29.8 25.3 18.5 26.3 72, 46Aucuba mosaic.... AuM 9a 2.2 29.7 25.4 18.5 26.4 72Ribgrass mosaic... Rib 9a 2.2 29.3 25.8 18.0 27.0 72Cucumber mosaic ......3 and 4 ,Cu 3, 4 9b 2.2 '27.6 25.6 24.6 22.3) 46PotatoX PX 10 2.0 34.3 21.8 .22.8 21.3 72,46Sindbis...........Sin 11 '-2.0 29.6 25.8 24.9 119.7 58Dengue.... Type 2 Den 2 11 3.3 30.7 26.4 21.3 ~21.6 78Foot-and-mouth disease.. .. A 119 FMD '12 '-2.0 25.7 24.0 28.0 22.3 5f2..........f2... 13 1.8 22.1 26.8 25.9 ~25.1 42R17 (+M12)e R17 13 1.1 23.1 26.3 124.9 25 .753,23M52............M52 13 1.1 22.8 27.1 124.9 25.2 79fr ~~ ~ ~~~~~~~~~fr13 1.2 '24.3 27.1 124.9 123.7 33,49

Southern bean mosaic. .SBM 14 1.4 25.8 26.0 i23.0 :25.3 46Tomato bushy stunt.. Un-named BS 15 1.4 25.0 28.0 22.0 25.0 46Tomato bushy stunt.. Type 3 BS 3 ,15 1.7 25.7 27.9 20.8 25.7 72Tomato bushy stunt... Type 9 BS 9 15 1.7 25.7 28.2 20.5 25.5 72Tomato bushy stunt... Type 10 BS 10 15 1.7 25.9 28.1 20.4 25.6 72Coxsackievirus ........ A9 Cox A9 16a '-2.0 27.0 28.0 20.0 25.0 50Coxsackievirus ........ AIO Cox AIO 16a -..2 .0 128.0 28.0 21.0 123.0 50Coxsackievirus.... BI Cox Bi 16b 1'--2.0 ~29.0 24.0 23.0 i24.0 69Murine encephalomyo-

carditis ..........K2 EMC 17a --2.0 27.4 23.6 23.5 25.6 .17,24Poliomyelitis type 1 .....Mahoney Po I 17b '-2.0 28.5 24.8 21.8 '25.0 70) 72Poliomyelitis type 2. MEFL Po 2 17b 2.0 128.8 23.3 22.5 ~25.5 70,~72Poliomyelitis type 3 .Saukett Po 3 I17b -'2.0 '28.0 24.3 21.8 26.3 70, 72Tobacco necrosis.. TNec 18 1.6 27.9 24.4 22.0 .25.7 46, 72

aAll the viruses listed in Table Ila contain RNA except for members of groups 1 and 2 (*,whichcontain DNA.

b A, adenine; G, guanine; C, cytosine; U(T), uridine (thymidine).Mode of the range of values quoted (69).

d Contains avian leukosis virus.M12 differs from RI17 by only one amino acid residue in the protein coat (23).

-i

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TABLE lb. Data for viruses which contain double-stranded nzucleic acidsa

Nucleic acid

Virus Strain (or host) Abbreviation Group w ReferencesMol wt Moles%e(X 10-6) G + C

Shope papilloma..Polyoma................Simian virus 40.........Human papilloma......Nuclear polyhedrosis...Reovirus*.Wound tumor*.........029...Adenovirus (human)....Adenovirus (human)....Adenovirus (human)....

Adenovirus (human)....Adenovirus (human)....Adenovirus (human)....Adenovirus (human)....

777

(Bombyx mori)Type 3

(Bacillus subtilis)Types 1, 23Type 5Types 13, 17, 22

Type 25Type 2Type 4Types 6, 9, 10, 15, 19,

24, 26

Adenovirus (human).... Types 20, 27, 28

Adenovirus (human)....Adenovirus (human)....Adenovirus (human)....Adenovirus (human)....Adenovirus (human)....Adenovirus (human)....Adenovirus (human)....Adenovirus (human)....T3............T7......................+......................

X dg....................080.....................+80 pt1.................P22..Ti..Infectious bovine rhino-

tracheitis.............Pseudorabies.Herpes simplex.........Human cytomegalovirus.LK (equine herpesvirus

2) ...................Equine abortion (equinerhino pneumonitis)

T5...SPO-1 ..................SP8, SP82, 2C..........SP50 ...................

SP3 ....................T2 .....................T4 .T6......................Tipula iridescent........

Type 8Type 14Types 16, 21Type 3Type 7Type 11Type 12Type 18(Escherichia coli)(E. coli)(E. coli)(E. coli)(E. coli)(E. coli)(Salmonella)(E. coli)

DekkingHFEM

(E. coli)(B. subtilis)(B. subtilis)(B. subtilis)(B. subtilis)(B. subtilis)(E. coli)(E. coli)(E. coli)(Tipula paludosa)

ShPPySV40HuPNPhReo 3WT029Ad 1, 23Ad 5Ad 13, 17,22

Ad 25Ad 2Ad 4Ad 6, 9, 10,

15,19,24,26

Ad 20, 27,28

Ad 8Ad 14Ad 16, 21Ad 3Ad 7Ad 11Ad 12Ad 18T3T7xXdg080+80 phP22Tl

IBRPsRHpHCM

LK

EAVT5SPO-1SP8, 82, 2c(SPSO)03SP3T2T4T6TIV

19a19a19b19b2021a21b2223a23a23a

23a23a23a23a

23a

23b23b23b23b23b23b23c23c242425a25a25b25b2627

28a28a28a28b

28c

28c2930a30ad30bd313132323233

4.23.53.25.32.010.010.011.821.Oc21.022.0

22.023.024.021.0

21.0

22.022.021.0123.021.021.021.021.027.027.034.032.629.331.037.042.0

54.070.068.064.0

84.0

92.083.0110.0110.0110.0120.0120.0130.0137.0139.0140.0

49.048.041.041.042.044.Ob37.635.058.057.058.0

59.057.057.559.0

60.0

52.053.052.051.051.050.049.047.049.648.050.050.053.053.050.048.0

11, 8515, 131412, 1556282863, 2595959

59595959

59

595959595959595977, 4780, 78, 4777, 80, 880898977, 8377

71.0 6774.0 6768.0 6758.0 16

56.0 67

55.0 67, 7639.0 77, 8345.0 55e43.0 31, 57,43.0 25e36.0 84e35.0 65e34.0 77, 6634.5 77, 2234.0 7732.0 82f

37, 18a

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TABLE lb-(Continued)

Nucleic acid

Virus Strain (or host) Abbreviation Group ReferencesMol wt MIoles%/(X 10-6) G + C

Sericesthis iridescent... (Sericesthis pruinosa) SIV 33 140.0 31.0 -IChilo iridescent (Chilo suppressalis) CIV 33 140.0 28.0 -Vaccinia. CL Vac 34a ~'160 37.5 35gCowpox ..Brighton) CP, RP 34a K,160 37.0 35Rabbitpox ... Utrecht f 35, 36'Ectromelia. .Dohi A Ect 34a -160 36.0 35Fowlpox FP 34b 200 35.0 81

a All the viruses listed in Table lb contain DNA except members of group 21, which contain RNA (*).Hydroxymethyluracil replaces thymine in group 30a, and hydroxymethyl cytosine replaces cytosinein group 32.

b Quoted as 39.8% G + C (27).c Molecular weights of adenovirus DNA calculated from per cent DNA except for types 2 and 18.d SP8, 82, and 2c are related serologically and by DNA homology. SP50 has identical values for DNA

molecular weight and base ratios, but is put in a separate subgroup because it is not serologically re-lated to 2c.

e See acknowledgments.f A. J. D. Bellett and R. B. Inman, J. Mol. Biol. i)t press.g Also, J. F. McCrea, personial communicationt.h Also, J. K. Tomkins, A. J. D. Bellett, and M. C. Taylor, unpuiblished data.

viruses so that the distance function on fusion isminimal. It was also useful to map the differ-ences between the characters for viruses whichcontain single-stranded nucleic acid, by use ofthe ordination procedure of Gower (30). Ordinarycomponent analysis cannot be used with theNMC because the distances are noneuclidean,but the program identifies a Euclidean space inwhich the distances between entities coincide withthe nonmetric coefficients or simple multiples ofthem.The computer was used to process the data in

a way which has been profitable in the construc-tion of class hypotheses. The analysis does notyield a unique solution, and it is necessary toconsider whether each cluster or group ofclusters is meaningful in the light of existingnucleic acid hybridization experiments, serolog-ical cross-reactions, and phenotypic propertiesof the viruses concerned. Two types of anomalyare likely to occur in the dendrograms; clustersmay be formed from entities which are alike onlyin their dissimilarity from members of all otherclusters, and atypical members of a group may beassigned to a neighboring cluster which is widelyseparated at higher levels of the classification.Such anomalies occur in all computer-basedclassification, and, since the computer was usedas an aid in the construction of class hypothesesrather than to produce a final and binding solu-tion, there is no compulsion to accept meaninglessclusters and propose class hypotheses for them.

TABLE 2. Nucleic acid htybridizationz antd serologicalcross-reactions amonig viruses,

Nucleic acid hybridizationh' Serological cross-reactionsb

Virus groups References V;irus groups References

3, 9, 13 86 0 4519 12 1, 13, 32, 3 68, 7323 38,39 0,4,8,9, 1-)24, 27, 29, 32 71 14, 15, 18 _ 630 C 6, 12, 16d, 17d_,25 10 23, 28, 34 -J

28 6024, 27, 29, 32 7730 -C

33 19

a Table 2 lists references for the relationshipsindicated in Fig. 2, 4, and 6.

e Groups listed together are not related, butdata on relationships within the groups are re-ported in the same reference. The proposed virusgroups are indicated in Fig. 2, 3, and 4, and theirmembers are listed in Tables la and b.

c See acknowledgments.d Occasional heterotypic reactions with some

sera.

Taxonomic units. According to the initial theory,the ultimate taxonomic units in the classificationshould be isologous clones. At this stage, theyare viruses which can be distinguished by someexperimental test, usually serological. If some of

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the taxonomic units are mixtures of highly ho-mologous variants rather than isologous clones,this will not affect the result, since the variantswill by definition fall within the same homologousgroup.

DATA AND RESULTSData. Data for viruses with single-stranded

nucleic acids are shown in Table la, and data forviruses with double-stranded nucleic acids, inTable lb. For convenience, viruses are arrangedin groups proposed as a result of analysis of thedata. Determination of all the characters is sub-ject to experimental error, and, in addition, there

are uncertainties in relating sedimentation co-efficients of nucleic acids to molecular weight,particularly for the single-stranded forms. Insome cases, molecular weights have been recal-culated from experimental results or refer tochemical determination of the mass of nucleicacid per virus particle. Base ratios have been re-ported for the nucleic acid of each virus strainlisted, but in a few cases the molecular weight ofthe nucleic acid of one strain has been assumed toapply to other strains of the same virus [e.g., themolecular weight of influenza virus RNA (1) isthat reported for strain PR8]. Abbreviations ofvirus names are also listed in Table la and b.

4.1,

I1l

t

FIG. 1. Photograph of a model in which the relative differences among viruses which contain single-strandednucleic acid are represented in a three-dimensional map. Data, Table la; program, Ordination, NMC. Y axis,ordinates in the first dimension of the output; each division is a distance of 0.02. Z and X axes, ordinates in thesecond andfourth dimensions of the output; each division is a distance of0.01.

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BELLETIT

*08 *04 0

Cu.M

I 1 M13fd

J. VIROL.

S P Gp.1 1 ]0] ] I1

-2

-5

I6b

6a

iERau

1 C~~~AVMRSV

_______Sq M

TMV

LAu M

Rib

_______Cu.3,4

*Cox.A9

.Cox.AlO*Cox.Bl

*EMC

*Po.2

.Po. 1

Po .3

T. Nec.

I

IiiI] 7C

7b

i 7a

-8

9a

i 9b-10-11-12

113-14

115I 16aJ 16b17a

.17b-18

FIG. 2. Dendrogram of viruses which contain single-stranded nucleic acids. Data, Table la; program, NMC andflexible sorting, with a, = 0.625, a2 = 0.625, = -0.25, y = 0. S, serological cross-reactions; P, similarhenotypic properties; Gp, proposed homologous group number.

*12

*38 .134

!35

!29

18

*13

252

4--

.%

i

MIL

ROB

LEE

-flu B

MEL

CAM

SWI WSE

PR8I flu A


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In deciding whether to propose a group hy-pothesis for viruses which are clustered in thecomputer output, published nucleic acid hy-bridization experiments were considered, alongwith serological cross-reactions, which were takento imply homology in at least one cistron. Refer-ences to these data are listed in Table 2. When noresults of this type were available, group hypothe-ses were not proposed for clusters of viruseswhich differ greatly in their phenotypic properties,because such differences suggest that the virusesare not homologous in spite of gross similarityin their nucleic acids. The phenotypic propertiesconsidered were those which have been used inconventional classifications (4, 26).

Results and Discussion

Viruses which contain single-stranded nucleicacid. Analysis of the data according to twoprograms is shown in Fig. 1 and 2. Figure 1 is aphotograph of a three-dimensional map in whicheach virus is plotted according to its co-ordinatesin the first, second, and fourth of the five dimen-sions available in the computer output (Program:

100 L

5005

E_.

@3L 10

C*

E 5

Ordination, NMC). Figure 2 is a dendrogramproduced by flexible sorting according to NMC.Viruses which show serological cross-reactions (S)or similar phenotypic properties (P) are brack-eted. The 47 virus strains have been provisionallyassigned to 19 homologous groups shown inFig. 2 and Table la. Of these, groups 0 (TYMV),1 (M13, fd), 2 (4>X), 5 (NDV), 6 (flu), 9 (TMV),10 (potato X), 11 (Sindbis), and 13 (RNAphages) were recognized as families or sub-families in the proposals of the PCNV, althoughit is doubtful whether group 1 [highly anisometricbacteriophages which contain cyclic single-stranded deoxyribonucleic acid (DNA)] shouldhave been included in the class Deoxycubica.Group 7 consists of enveloped viruses with helicalnucleocapsids which contain 12 to 13 milliondaltons of RNA; all members cause neoplasia invertebrate animals. Cucumber mosaic viruses 3and 4 differ in their base ratios from other mem-bers of group 9 (TMV group), and were originallyclustered with Sindbis virus (group 11) in thedendrogram; they have been reallocated togroup 9 because of weak serological cross-reac

CCIV SIV T )

OZ APs .

tiDED

3

LN Phj.

I

30 40 50 60 70

% GCFIG. 3. Plot of base ratios and molecular weights of the nucleic acids of viruses which contain double-stranded

nucleic acid. Data, Table lb. Proposed groups and subgroups are outlined. GC, gutanine plus cytosine.

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254 BELLETIT

tions with members of the group. Data on Denguevirus 2 were published after the analysis wascompleted; its nearest neighbor is Sindbis, withwhich it fuses with a NMC of 0.04, and it hastherefore been placed with Sindbis in group 11.The remaining viruses belong to the conven-

tional picornavirus group, from which groups 0(TYMV and WCuM) and 13 (fr, MS2, R17,and f2) have already been separated. Most of theremaining groups form a loose cluster in the map

*5 *4 *3 *2

2-5

J. VIROL.

and dendrogram, from which SqM, Q6, FMD,and TRS can be separated. Relationships withinthe loose cluster [groups 14 (SBM), 15 (BS), 16(Cox), 17 (EMC, polio), and 18 (TNec)] aredifficult to determine. It is possible that the groupsare subgroups of a single homologous group,but separate groups are proposed until data onnucleic acid hybridization are available. Thereappears to be a close similarity in the base ratiosof poliovirus, EMC, and coxsackievirus Bi

-2 hom. SrSh.PLPy.SV 40

- Hu.P- N Ph.- REO 3- WT-/ 29

I Ad 1,23lAd SAd 13,17,22

Ad 25Ad 2Ad 4

EAd 6.9.10.1519 .24 .26

Ad 20,27,28Ad 8

I Ad 14Ad 16,21

- Ad 3

r Ad 7Ad 11Ad 12

-Ad 18

{ dAd

-P22-T 1-IBR- Ps .R- Hp-HPM I-HCM

- EAV

-T 5rSPOISP8 ,82,2C0 P50)

4SP3-T 2[T 4LT 6

CIV

Vac.CP,RPEct.

- FP

I 19a

19b-20]21-22

II.

23a

23b

J23c

]24

125-26*27

28a

28b

-29

O30aLEOb]J31

I32

33

34a

34b

FIG. 4. Dendrogram of viruses which contain double-stranded nucleic acids. Data, Table lb; program andcolumn headings as for Fig.2; hom, homology presumedfrom nueleic acid hybridization (Table 2).


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RNA, but this does not extend to coxsackie-viruses in the A group; these possible relation-ships are indicated in Fig. 2 and Table la.The only experimental investigation of homol-

ogy among viruses which contain single-strandednucleic acid showed (86) that the RNA of MS2(group 13) hybridizes with that of f2 (group 13)but not with RNA from Q$ (group 3) or TMV(group 9)

Viruses which contain double-stranded nucleicacid. Viruses which contain double-strandednucleic acids can immediately be placed in groupswhich form clusters on a simple plot of molecularweight against moles per cent guanine plus cyto-sine (G + C) in the nucleic acid (Fig. 3). Thereare some anomalies in a dendrogram produced byuse of flexible sorting according to NMC (Fig. 4),but most of the viruses which form clusters inFig. 3 and 4 are related by nucleic acid hybridiza-tion (hom) and serological cross-reaction (S) andhave similar phenotypic properties (P). The 69virus strains listed in Table lb have been assignedto 16 provisional homologous groups shown inthis table and in Fig. 4. Of these, groups 19

20 .

0A

E

E

(papovavirus), 21 (reovirus), 23 (adenovirus), 28(herpesvirus), 33 (iridescent), and 34 (poxvirus)were recognized as families in the proposals ofthe PCNV. There is some doubt as to the statusof group 19; these viruses have similar nearest-neighbor base frequencies (Subak-Sharpe, per-sonal communication), but it has not been possibleto detect nucleic acid hybridization within thegroup, perhaps because of the cyclic structure ofthe DNA (12). Group 20 contains only nuclearpolyhedrosis of the silkworm.The remaining nine groups are all tailed bac-

teriophages which were included in a singlefamily by the PCNV. Their nucleic acids differmore in molecular weight than in base ratios.The provisional groups in which they have beenplaced agree well with nucleic acid hybridizationand serological results (Fig. 4, Table 2), butpossibly the groups are related by a low degreeof homology not yet detected.Data on 480 and 480pt1 were published after

the dendrogram (Fig. 4) was completed. Theyform a pair with NMC of 0.01, and then fusewith the X plus X dg pair. The 480 virus recom-

0% GC

FIG. 5. Molecular weights and base ratios of the nucleic acids of some viruses which contain double-strandednucleic acid compared with values expected for the double-stranded forms of viral nucleic acids which are single-stranded in the virion. Where possible, proposed groups are outlined. GC, guanine plus cytosine.

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BELLETT

bines with X, but is not serologically related to it(89). It is proposed that the two pairs form sub-groups of group 25. Viruses of this group couldbe homologous but vary in the molecular weightand base composition of their DNA by replacingparts of their genome with host DNA.

Comparisons between viruses which containsingle-stranded and double-stranded nucleic acid.We have proposed the hypothesis that no viruswhich contains single-stranded nucleic acid ishomologous with any virus which containsdouble-stranded nucleic acid. The experimentalvalues for viruses which contain double-strandednucleic acids were compared with the molecularweights and base ratios expected for the double-stranded equivalents of single-stranded viralnucleic acids (Fig. 5). The only viruses for whichthe hypothesis is at all likely to be untrue arepolyoma and coxsackievirus A9, and Shopepapilloma and coxsackievirus AIO. Even in thesecases the phenotypic differences are marked, andthere seems to be no reason to reject the hypothe-sis unless experimental evidence of homology isobtained.

Genetic heterogeneity of viruses. The con-ventional class of all viruses is extremely hetero-geneous. Viral nucleic acids vary more than 100-fold in molecular weight and from 28 to 74% inG + C, which is as much as the variation in baseratios of bacterial DNA and very much morethan the variation in base ratios of DNA fromall other living things. Extreme variation in base

100

z0

c

E

= 503._

0

0E0

40 50%GC

FIG. 6. Degree ofgenetic heterogeneity of vertebrateanimals (a) compared with that of adenoviruses (b).Data from reference 34, 38, 39, and 59. Per centhomology is the amount of each DNA bound by theDNA with the lowest per cent G + C in each group,expressed as percentage of that bound in the self-control. GC = guanine plus cytosine.

ratios may be partly a consequence of the lowmolecular weight of viral nucleic acids, but evenwithin groups of homologous viruses there ismarked heterogeneity. The human adenoviruses(group 23) differ in their nucleic acids as much asmammals, or possibly all vertebrate animals(Fig. 6). It is therefore dangerous to generalizefrom one group of homologous viruses toanother, or to propose a general theory of theevolutionary origin of viruses. However, it may beprofitable to propose that a particular group ofviruses is homologous with a group excluded fromthe conventional class of viruses; there is alreadydirect evidence that viruses of groups 25 (X) and31 (43) are homologous with a number of bac-teria (10; J. Marmur, unpublished data), and in-direct evidence that viruses of group 19 (papova-virus) but not those of group 28 (herpesvirus)are related to mammalian cells (H. Subak-Sharpe,personal communication). The available evidenceclearly contradicts any monophylectic hypothesisfor the origin of viruses.

ACKNOWLEDGMENTSI am very grateful to W. T. Williams of the

C.S.I.R.O. Computing Research Section, Canberra,for advice and for carrying out the computer analyses.

In the data on Bacillus subtilis bacteriophages(groups 30 and 31), use has been made of unpublishedexperiments by B. E. Reilly, W. R. Romig, and T. A.Trautner quoted by J. Marmur in his preliminaryclassification of B. subtilis phages, which was circu-lated as scientific memo no. 317 of the InformationExchange Group No. 7.

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Documents

Preliminary Classification of Viruses Based on Quantitative Comparisons of Viral Nucleic Acids