use of ultrafiltration- for animal virus grouping,
TRANSCRIPT
BACTERIOLOGICAL REVIEWS, Dec., 1965Copyright ( 1965 American Society for Microbiology
Vol. 29, No. 4Printed in U.S.A.
Use of Ultrafiltration- for Animal Virus Grouping,G. D. HSIUNG'
Department of Epidemiology and Public Health, Yale University School of Medicine,New Haven, Connecticut
INTRODUCTION.................................................................. 477
GENERAL CONSIDERATIONS ON BASIC CRITERIA FOR DEFINING MAJOR VIRUSGROUPS...................................................................... 477
Nucleic Acid Type............................................................ 477
Virus Size and Structure ..................................................... 478
Electron microscopy......................................................... 478
Ultrafiltration.............................................................. 478
Other Chemical and Biological Characters ...................................... 479
Ether sensitivity............................................................ 479
Acid pH lability............................................................ 479
Influence of receptor-destroying enzyme (RDE) treatment of erythrocytes onviral agglutination....................................................... 479
PROBLEMS FOR PLACING A "NEW" VIRUS IN A TAXONOMIC ORDER ........ 479
Recognition of the Vacuolating Virus of Monkeys............................... 480Isolation of a Strain of Parainfluenza Virus Type 5 from Man.................. 480
ULTRAFILTRATION AND VIRUS SIZE ESTIMATION .................. 480
Comparison of Millipore Membrane and Gradocol Membrane for Filtration ofViruses of Established Groups............................................... 481
Filtration of Viruses of Undefined Group-the Simian Viruses.................. 481
USE OF ULTRAFILTRATION FOR GROUPING ANIMAL VIRUSES: A SIMPLIFIED SCHEME. 483SUMMARY .............................. 484
LITERATURE CITED ............. 485
INTRODUCTIONWith the application of improved culture tech-
niques, the number of new virus isolations hasincreased rapidly during the past decade. It is ap-parent that a simple, meaningful basis for sys-tematic grouping of these viral agents is nowurgently needed. In August, 1962, the VirusSubcommittee of the International Nomencla-ture Committee made some recommendations onvirus nomenclature and grouping (51), but, pre-sumably because of incomplete information,they were unable to provide any procedure forclassifying new virus groups. This paper attemptsto provide a simple guide to animal virus group-ing, based upon virus size as determined byfiltration. The major groups of viruses whichresult are in no way different from the presenttaxonomic order but rather exist within it.
GENERAL CONSIDERATIONS ON BASIC CRITERIAFOR DEFINING MAJOR VIRUS GROUPS
Classification of viruses of vertebrates was re-cently reviewed and described in detail by
1 Presented at the Annual Meeting of theAmerican Society for Microbiology, Washington,D.C., 6 May 1964.
2 Present address: Department of Medicine,New York University School of Medicine, NewYork, N.Y.
Andrewes (2, 3). Certain criteria such as nucleicacid type, virus size and structure, and sensitivityto inactivation by physical and chemical meansare fundamental characteristics for defining themajor groups.
Nucleic Acid TypeAnimal viruses consist of a nucleic acid core
enclosed in a protein coat; the cores containeither ribonucleic acid (RNA) or deoxyribo-nucleic acid (DNA) (35). Because of this basicchemical difference, animal viruses have beendivided into two groups (15). Several techniquesmay be used to determine the type of constituentviral nucleic acid. For example, multiplication ofa DNA virus is inhibited by the presence ofhalogen derivatives of deoxyuridine (5-fluorode-oxyuridine or bromodeoxyuridine); multiplica-tion of a RNA virus is relatively unaffected bythese compounds (22). The appearance ofFeulgen-positive intranuclear inclusions in in-fected cultures is another indication that the virusis probably DNA in nature. In addition, exami-nation with ultraviolet light of virus-infectedcells stained with fluorochrome acridine orange,or electron microscopic examination of prepara-tions stained with uranyl acetate, may suggestthe nature of the virus nucleic acid type. In theacridine orange preparations, yellowish greenfluorescence is usually an indication of DNA, and
477
BACTERIOL. REV.
RELATIVE SIZE
DNAVIRUSES
RNAVIRUSES
OF ANIMAL VIRUSES
2 3
5 6 7 8
FIG. 1. Relative sizes of animal viruses (schematic diagram). Modified from Horne (26). DNA viruses:(1) poxvirus group, (2) herpesvirus group, (3) adenovirus group, (4) papovavirus group. RNA viruses:(5) myxovirus group (mumps virus), (6) myxovirus group (influenza virus), (7) reovirus group, (8) entero-virus group.
flame-red staining represents the RNA nature(39). Only DNA cores usually stain densely withuranyl acetate in the latter method (45). Thesetechniques, either individually or in combina-tion, have been used for the determination of thetype of nucleic acid of a variety of viruses.
Virus Size and StructureVirus size has always been a parameter em-
ployed in classification, but the value given forthe size of individual viruses has varied greatlyfrom laboratory to laboratory, depending uponthe method used. Several techniques have beenused, including various electron microscopicmethods, ultrafiltration, sedimentation, and ir-radiation. Only the first two will be dealt withhere.
Electron microscopy. Size and shape of a viruscan often be determined by electron microscopicexamination when the necessary facilities areavailable. Ultrathin sections of virus-infectedtissues permit a direct-approach to the determi-nation of virus size,- but distortion of the virus
particle may occur during the process of fixationand dehydration. These techniques have been re-
viewed and discussed by Williams (54) and, morerecently, by Valentine (50). The development byHorne and his colleagues of negative stainingtechniques with the use of electron-dense salts,such as potassium phosphotungstate, has per-
mitted visualization of virus fine structures (11,25). Previously unseen details in both surfaceand interior structures of many types of virushave been revealed (Fig. 1 and 2). The contribu-tions of this technique to the understanding ofvirus structures are manifold, and aid considera-bly in devising systems of virus classification (36).
Ultrafiltration. One of the simplest and oldestmethods for estimating virus particle size is ultra-filtration. This is usually done by passing a virussuspension through a series of collodion mem-
branes of graded porosity. The size of the virusis related to the size of the pores in the finest filterthrough which infectious virus particles pass.Several limitations and complications have beenencountered (17, 18), in addition to the absence
4
478 HSIUNG
ULTRAFILTRATION FOR ANIMAL VIRUS GROUPING
3
5 6 8
FIG. 2. Relative sizes of animal viruses (negative-stained virus particles). Modified from Horne (26),Howatson (27), and Waterson (62). DNA viruses: (1) poxvirus group (vaccinia virus), (2) herpesvirus group(herpes simplex), (3) adenovirus group, (4) papovavirus group (polyoma virus). RNA viruses: (6) myxovirusgroup (sendai virus), (6) myxovirus group (influenza A virus), (7) reovirus group, (8) enterovirus group(poliovirus).
of any commercial supply of graded membranes.The difficulty of producing these membranes ona small scale in the laboratory has limited theusefulness of the method. Recently, the availa-bility of Millipore membranes and suitable filterholders has brought the method within reach ofeven the smallest laboratory (for details see latersection).
Other Chemical and Biological CharactersEther sensitivity. Determinations of the essen-
tial lipid of a virus can be carried out readily bythe ether-sensitivity test (4). This is performedby adding diethyl ether, 20% by volume, to anundiluted virus suspension, shaking, refrigeratingat 4 C for 18 to 24 hr, and testing for reductionof virus infectivity. Instead of ether, a solutionof sodium deoxycholate (1:1,000) can be used ina similar manner to test for essential viral lipids(47).Acid lability. Sensitivity to low pH may be
used to distinguish certain virus subgroups. Asreported by Hamparian et al. (22), some virusesretain full infectivity when exposed to pH 3.0 for
30 min, whereas other viruses are completelyinactivated by such treatment.
Influence of receptor-destroying enzyme (RDE)treatment of erythrocytes on viral agglutination.Certain groups of viruses will not agglutinateerythrocytes that have been treated with RDE,although the untreated cells were readily agglu-tinable. There are, however, other types of viralagglutination which are not affected by this treat-ment. Thus, hemagglutinin sensitivity to theRDE-treated erythrocytes is characteristic ofcertain groups of viruses (12).
PROBLEMS OF PLACING A "NEW" VIRUS IN ATAXONOMIC ORDER
The basic criteria of chemical composition andphysical nature cannot be used practically inroutine work of differentiating new isolates. Anexperienced worker usually can recognize anisolate by the type of cultures in which the agentgrows and the type of cytopathic changes pro-duced. Isolates are usually grouped by their bio-logical characteristics, even though these criteriaare less stable. Finally, identification is accom-
VOL. 29, 1965 479
* ,ivg p bSP.4F: b :ss. 9 ...- .:. :.:,.. #. . ..t .Kit i.o*x- Of .....
BACTERIOL. REV.
TABLE 1. Filtration of vacuolating virusstrain PA-67*
Gradocol membrane APD Virus titer in filtrate(log TcMso/ml)
Unfiltered 6.5303 6.388 3.852 1.244 038 0
* From Hsiung and Gaylord (30).
plished by one or more serological tests. However,when a "new" virus or a new variant of a knownagent emerges, the identification and classifica-tion may present difficulties. In the followingsections, I shall discuss our experiences on twooccasions in which difficulties were encountered.
Recognition of the Vacuolating Virus of MonkeysIn 1957, a vacuolating virus, PA-57, a strain
of SV4o belonging to the papovavirus group, was-isolated from uninoculated patas monkey kidneycultures (30). At the time of the original recogni-tion of a cytopathic agent in the patas culture, itwas noted that the cellular changes were differentfrom the changes caused by any virus known atthat time. Extensive vacuolation was present inpatas cultures 15 days after infection. Intra-nuclear inclusions were seen throughout the cul-ture when stained preparations were examined.These inclusions were Feulgen-positive, suggest-ing that the virus was possibly DNA in nature(20). Physicochemical properties were studied.The virus was stable after several cycles of freez-ing and thawing. No change in infectivity titersoccurred when the agent was exposed to ether.The size of the virus was determined by Gradocolmembrane filtration (Table 1). Infectious viruspassed through a membrane with an averagepore diameter (APD) of 52 mju, although con-siderable virus was retained by this membrane.No infectious virus was recovered in the filtratesfrom 44- or 38-mju membranes. When infectedtissue culture cells were sectioned and examinedunder the electron microscope, low electrondensity particles 300 A in diameter were seen inthe nucleus and cytoplasm throughout the prep-aration. The virus was tentatively placed in agroup with tumor-producing viruses because itsmorphological structure resembled that of theother members (20). Later experiments by others(16, 21) showed that this virus too was indeedcapable of inducing tumors in experimental ani-mals, and the name of papovavirus group was
TABLE 2. Filtration of parainfluenza virustype 5, DA strain
Gradocol membrane APD Virus titer (log PFU/ml)
myA
Unfiltered 5.5680 4.2310 4.0150 096 054 0
proposed (40). Although it was evident that asmall DNA, ether-resistant virus was involved,there was no simple method for placing this virusin a taxonomic class.
Isolation of a Strain of Parainfluenza Virus Type 5from Man
When the DA virus was first isolated fromhuman blood by the plaque technique (28), itsidentification posed a difficult problem. Theagent produced no cytopathic effect (CPE) influid kidney cell cultures, but did producedistinct plaques in parallel cultures overlaid withan agar medium. Since CPE was then the stand-ard method for recognizing the presence of a viralagent in tissue culture, this pattern hampered itsidentification. Subsequent studies indicated thatthe virus was heat-labile and sensitive to ethertreatment. RDE-treated erythrocytes were notagglutinable by the virus (31), and stained prep-aration of infected cells showed no intranuclearinclusions. Upon filtration through Gradocolmembranes, the virus was found to be large,passing through a 310-mA membrane but notthrough a 150 mA filter (Table 2). Again, thevirus could not be placed into a systematic order,although it was noted that a large, ether-sensitiveviral agent, probably a myxovirus, was involved.The final classification of this agent as parainflu-enza 5 was not accomplished until recently (32).A similar situation might be encountered in any
virus laboratory, especially one dealing withclinical specimens from varied sources. Hence,there is great need for simple tools for grouping"new"l agents to place them into a taxonomicposition.
ULTRAFILTRATION AND VIRUS SIZE ESTIMATIONUltrafiltration with Gradocol membranes has
been employed for the past decade to determinethe size of viruses (9, 17, 18, 46). Unfortunately,the complexity of the filter apparatus and thelimited supply of the Gradocol membranes re-stricted the use of this technique. Now, however,commercially produced membranes (Millipore
480 HSIUNG
ULTRAFILTRATION FOR ANIMAL VIRUS GROUPING
FIG. 3. Filter set consists of a 2-ml syringe, twoSwinny adapters, a hypodermic needle, and a
vacutainer tube.
filters) are available that greatly facilitate filtra-tion procedures. A crude estimate of the size ofthe viruses can be made readily. The equipmentfor filtration is illustrated in Figure 3. (Gradocolmembranes were supplied by the Wright-Fleming Institute, St. Mary's Hospital, London,and were kindly made available by M. Harris,National Institutes of Health, Bethesda, Md.Millipore filters were purchased from the Milli-pore Filter Corp., Bedford, Mass. Swinny filteradapters and vacutainer tubes were purchasedfrom Becton Dickinson & Co., Rutherford, N. J.)A 2-ml syringe was attached to two Swinnyadapters connected in series with a hypodermic
needle. This was autoclaved; then either a Sietzmembrane or a collodion membrane of large porediameter was placed in the upper adapter, andthe test membrane was placed in the lower. Avacutainer tube was used to collect the finalfiltrate. Millipore membranes used were: VC type,100-m~t APD; VM type, 50-mii; and VF type,10-mtt. These membranes were sterilized by ex-posure to ultraviolet light for 10 min on each side.Virus infectivity in the suspension was deter-mined before and after filtration, and virus sizerange was estimated according to the virus infec-tivity retained by the filters of the smallest porediameters.
Comparison of Millipore Membrane and GradocolMembrane for Filtration of Viruses of
Established GroupsViruses of representative groups commonly en-
countered in a virus laboratory or with humandisease were selected for the comparison. Tables 3and 4 show the data obtained with Milliporemembranes as compared with results reported inthe literature for Gradocol membranes. Table 3is concerned with the filtration of DNA viruses.Poxvirus and herpesvirus are considered to belarge viruses, since infectious viruses were not re-covered in their filtrates after passing fluidsthrough 100-, 50-, and 10-mi APD filters. Adeno-viruses belong to the medium size range becausethey passed 100-mi/ APD even though at re-duced titer. The papovaviruses are in the smallsize range. Similar results were obtained withRNA viruses (Table 4). Parainfluenza, respira-tory syncytial (RS), and measles viruses wereconsidered to be large viruses since they did notpass through the 100-, 50-, or 10-min filters. Reo-virus types 1, 2, and 3 belong to the medium sizerange; enteroviruses are in the small size group.Two arbovirus serotypes, Bunyamwera andEastern equine encephalitis (EEE), were alsoincluded with the filtration experiments and wereplaced into large (Bunyamwera) and small(EEE) virus categories (Table 4). This observa-tion confirmed a previous report that the sizes ofthese two arboviruses were different (46). Atpresent, arboviruses are classified primarily onthe basis of their serological properties (13), buteventually this large group may be subdividedby use of other criteria, as was the case with thesimian viruses.
Filtration of Viruses of an Undefined Group-the Simian Viruses
Simian viruses comprise a large group of agentsisolated from monkey kidney tissues taken fromapparently healthy asymptomatic animals (24,
481VOL. 29; 1965
TABLE 3. Ultrafiltration of DNA viruses
Millipore membranes (mu) Infectivity titration Virus size reported for Gradocol
Virus group Virus strain
Unfil- 300 100 5O 10 Cell Assay APD hold- APD pass- Refer-tered systema method ing virus ing virus ence
Pox virus Vaccinia 50." 1.0 0 0 0 RhMK Plaques 125-300 260-650 17
Herpes Herpesvirus simplex
(5756)c 4.5 3.5d 0 0 0 HK CPE 150 and 250 and 18(6212) c 6.5 5.0 0 0 0 Hep-2 CPE 210 300
Adeno- Type 1 7.0 7.Oe 4.0 0 0 Hep-2 CPEvirus Type 2 5.0 4. Od 2.5 0 0 Hep-2 CPE
Type 5 6.5 6.5d 2.5 0 0 Hep-2 CPE 140 160 23fType 7 6.8 5.5e 2.8 0 0 Hep-2 CPEType 16 6.7 5.0 1.5 0 0 Hep-2 CPE
Papova- SV40virus PA-57 6.3 6.0d" 5.5 4.7 0 GrMK CPE 52 44 30
a RhMK = rhesus monkey kidney; GrMK = green monkey kidney; HK = human kidney culture;Hep-2 = Hep-2 cell line.
b Virus titers (log TCD50 or plaque-forming units per milliliter).c New isolates.d Seitz filter pad was used for clarification.e Millipore membrane (220 mu) was used for clarification.f Adenovirus type 4 was used.
TABLE 4. Ultrafiltration of RhNA viruses.. .. . . .. ~~~~~~~Virus size reported for GradocolMillipore membranes (mju) Infectivity titration membrane filtrationVirus group Virus strain
Unfil- 300 100 s0 10 Celk. Assay APD hold- APD pass- Refer-tered systemP method ing virus ing virus ence
Myxo- Para-5 6.8b 4.0 0 0 0 RhMK Hads 96 and 310 31virus (DA) and 150
plaquesMeasles 5.2 3.5c 0 0 0 Hep-2 CPE 210 320 9RS 4.8 2.8 0 0 0 Hep-2 CPE
Reovirus Type 1 5.5 4.5 3.1 0 0 Rh CPE 110 and 130 44Type 2 4.5 4.3 1.3 0 0 Rh CPE 120Type 3 5.6 4.5 0 0 0 Rh CPE
Entero- Polio-1 7.0 5.5d 4.8 3.8 0 Rh Plaques 38, 27 50, 40 9virus Cox A9 7.0 7. Od 7.0 6.5 0 Rh CPE 38 50 9
Cox B5 6.5 6.5d 6.0 5.5 0 Rh CPEEcho-1 7.0 6.5 6.0 5.0 0 Rh CPE 38 50 9Echo-7 7.9 8.1 7.1 6.5 0 Rh Plaques 29 50 9Echo-8 7.2 7.1 6.7 5.2 0 Rh Plaques
Arbovirus EEE 9.3 9.5 8.7 8.7 0 CE Plaques 19-24 53 46Bunyam- 10.0 8.0 0 0 0 CE CPE 121-142 151 46wera
See Table 5; CE = chick embryo fibroblast.b Virus titers (TCD50 or plaque-forming units per milliliter).c Seitz filter pads were used for clarification.d Millipore membrane (220 mu) was used for clarification.
482 HSIUNG BACTERIOL. REV.
ULTRAFILTRATION FOR ANIMAL VIRUS GROUPING
33, 34, 37, 38). Classification of these viruses hasbeen a problem. During our studies on characteri-zation of simian viruses, Millipore membraneshave been used for estimating the size of theseviruses (8). Results obtained from several lots ofmembranes were reproducible, and representa-tive experiments on the filtration of simianviruses are shown in Table 5. No infectious viruscould be detected in the filtrates of SV5 after thevirus fluid was passed through Millipore filters of100-, 50-, and 10-m, pore sizes. A significant re-sidual titer remained in fluid containing SV1,SV51, SV12, SV17, SV23, SV25, SV27, SV30, SV32, orSV33 after passing through a 100-mIA APD filter,and no infectious virus was recovered in thefiltrates passed through 50-mn APD filters. Onthe other hand, infectious virus was found infiltrates of SV16, SV18, SV26, SV29, SV35, and SV4opassed through both the 100- and 50-mIA APDfilters. For confirmation, Gradocol membranesof appropriate pore diameters were selected ac-cording to each virus size range as determined bythe preliminary Millipore filtration. For example,Gradocol membranes with APD greater than100 mrn were used for filtering SV5. Membranesof APD smaller than 50 my but larger than 10 mIAwere used for SV16 or SV40. For SV11 and SZV12
TABLE 5. Use of Millipore membranes forultrafiltration of simian viruses*
Strain
SV5t
SV12SV17SV23SV25SV27SV30SV31SV32SV33
SV16SV18SV26SV29SV35SV40
Unfil-tered
6.8
6.45.35.56.76.75.16.75.56.56.74.1
7.16.86.15.35.76.3
Virus titer (log TwDrs per ml)
Seitz orMilliporemembrane(220 mju)
5.ot
5.75.15.16.16.14.55.74.55.76.53.5
6.56.55.75.Ot5.5t6.Ot
Millipore membranes,avg pore diam (mja)
100
0
4.72.72.71.73.52.72.72.72.92.12.4
5.15.15.34.85.15.5
50
0
00000000000
2.13.53.12.83.13.7
10
0
00000000000
000000
Estimatedvirus size
Large
MediumMediumMediumMediumMediumMediumMediumMediumMediumMediumMedium
SmallSmallSmallSmallSmallSmall
TABLE 6. Combination of Millipore and Gradocolmembrane filtration*
Avg pore diam SV5 SVs| SVs6
mjs
Unfiltered + + +310 + + +240 + + +
M-100 0 + +87 0 + +62 0 0 +
M-50 0 0 +40 0 0 +30 0 0 0
M-10 0 0 0
* M = Millipore membranes; others wereGradocol membranes; + = virus infectivitypassed through filter; 0 = virus infectivity re-tained by filter.
TABLE 7. Ultrafiltration and virus size estimation*
Millipore membrane, avg porediam (mis)
Virus size Seitz filter
100 50 10
Large. + 0 0 0Medium.. + + 0 0Small ..... + + + 0
* Virus infectivity passed through filter, +;virus infectivity retained by filter, 0.
Gradocol membranes of APD between 50 and100 m~i were used. Comparative experiments aresummarized in Table 6. It appears that SV5 wasin the size range of 240 to 100 m.t, SV11 in therange of 87 to 62 mju, and SV16 about 40 to 30 m/A.If a value of 0.64, as suggested by Black (10), isused, these results, in fact, cover the size antici-pated in each case, since SV5 is a myxovirus (31),SVli is an adenovirus (33), and SV16 is an entero-virus (24).
USE OF ULTRAFILTRATION FOR GROUPINGANIMAL VIRUSES: A SIMPLIFIED SCHEME
Results obtained with Millipore membranefiltrations (Tables 3, 4, 5, and 6) indicate thatanimal viruses can readily be grouped into threesize categories as summarized in Table 7. Largeviruses are those that did not pass through afilter of 100-miA porosity; medium size virusespassed through a 100-mtt filter with significantresidual infectivity titers; and small virusespassed through both 100- and 50-mA filters.Based on the results in Tables 3 and 4, and takinginto account other properties such as ether sensi-tivity, stability in acid pH, and the ability toagglutinate RDE-treated erythrocytes, in addi-
* Modified from Atoynatan, Hsiung (8).t Results were obtained by hemadsorption.I Seitz filter discs were used for clarification.
483VOL. 29, 1965
BACTERIOL. REV.
ANIMAL VIRUSES
DNA
LARG E MEDIUM SMALL>100* 50-100 <50
ETHER ETHER ETHER
I I I I IRES. SENS. RES. SENS. RES. SENS.
POX- HERPES- ADENO- rAPVVA-
RNA
l I ILARGE MEDIUM SMALL>100 50-100 <50
ETHER ETHER ETHER
SE NS. RES. SENS. RES. SENS. RES.
II I I H1' H30
RDE RDE pH 3.0 pH3.0SENS. RES. SENS. RES.
RHINO-MYXO- MEASLES- REO- ARBO- ENTERO-
LIMITING PORE DIAMETER (fm7,J OF MILLIPORE MEMBRANE
FIG. 4. Simplified scheme for the classification of animal viruses (Hsiung, 29). DNA and RNA virusesare subdivided on the basis of filtration through Millipore filters: large, viruses which do not pass througha 100-mr Millipore filter; medium, those which pass a 100- but not a 50-mM filter; small, those which go througha 50-mM millipore filter. Sens. = sensitive; Res. = resistant; RDE = receptor-destroying enzyme.
tion to nucleic acid types, a simplified scheme foranimal virus classification has been derived. Thisis summarized in Fig. 4. An outline of the pro-posed taxonomic scheme is given below. For de-tailed description of each virus group and sub-divisions within each group, the reader is re-ferred to the recent publications by Andrewes(2, 3) and by Hsiung (29).RNA VIRUS
Small RNA virus, <50 mjuEther-sensitive (not recognized to date;
certain arboviruses, for example,EEE, may belong to this group)
Ether-resistant, picornavirus (41)pH 3.0-sensitive, rhinovirus (48, 49)pH 3.0-resistant, enterovirus (14)
Medium RNA virus, 50 to 100 m~iEther-sensitive (not recognized to date)Ether-resistant, reovirus (44)
Large RNA virus, >100 m,4Ether-sensitive, myxovirus and myxo-
like virus (5, 6, 53)RDE-treated cells not agglutinable,
myxovirus (12)RDE-treated cells agglutinable,
measles, etc. (42)Ether-resistant (not recognized to date)
DNA VIRUSSmall DNA virus, <50 m~i
Ether-sensitive (not recognized to date)Ether-resistant, papovavirus (40)
Medium DNA virus, 50 to 100 m1AEther-sensitive (not recognized to date)Ether resistant, adenovirus (43)
Large DNA virus, >100 mjiEther-sensitive, herpesvirus (1)Ether-resistant, poxvirus (19)
SUMMARY
The use of ultrafiltration has facilitated thegrouping of animal viruses. Millipore filters of100-, 50-, and 10-miA pore sizes were found to besatisfactory for estimating the size of animalviruses and grouping into large, medium, andsmall size categories. A simplified scheme forgrouping animal virus is derived and unknownagents can be placed in a logical taxonomicposition.
ACKNOWLEDGMENTSThe original work was supported by Public
Health Service research grants AI-05313 andAI-05577 from the National Institute of Allergyand Infectious Diseases. Filtration experimentson arboviruses and measles virus were done in
484 HSIUNG
r-
ULTRAFILTRATION FOR ANIMAL VIRUS GROUPING
collaboration with J. R. Henderson and F. L.Black, Department of Epidemiology and PublicHealth, Yale University School of Medicine.
LITERATURE CITED1. ANDREWES, C. H. 1954. Nomenclature of
viruses. Nature 173:620-621.2. ANDREWES, C. H. 1962. Classification of
viruses of vertebrates. Advan. Virus Res.9:271-296.
3. ANDREWES, C. H. 1964. Viruses of vertebrates.The Williams & Wilkins Co., Baltimore.
4. ANDREWES, C. H., AND D. M. HORSTMANN.1949. The susceptibility of viruses to ethylether. J. Gen. Microbiol. 3:290-297.
5. ANDREWES, C. H., F. B. BANG, AND F. M.BURNET. 1955. A short description of themyxovirus group (influenza and relatedviruses). Virology 1:176-184.
6. ANDREWES, C. H., F. B. BANG, R. M. CHA-NOCK, AND V. M. ZHDANOV. 1959. Parain-fluenza viruses 1, 2, and 3: suggested namesfor recently described myxoviruses. Virol-ogy 8:129-130.
7. ANDREWES, C. H., F. M. BURNET, J. F. EN-DERS, S. GARD, G. K. HIRST, M. M. KAPLAN,AND V. M. ZHDANOV. 1961. Taxonomy ofviruses infecting vertebrates: present knowl-edge and ignorance. Virology 15:52-55.
8. ATOYNATAN, T., AND G. D. HSIUNG. 1964.Ultrafiltration of simian viruses. Proc.Soc. Exptl. Biol. Med. 116:852-856.
9. BENYESH, M., E. C. POLLARD, E. M. OPTON,F. L. BLACK, W. D. BELLAMY, AND J. L.MELNICK. 1958. Size and structure of ECHO,poliomyelitis, and measles viruses deter-mined by ionizing radiation and ultra-filtration. Virology 5: 256-274.
10. BLACK, F. L. 1958. Relationship between virusparticle size and filterability throughgradocol membranes. Virology 5:391-392.
11. BRENNER, S., AND R. W. HORNE. 1959. Anegative staining method for high resolutionelectron microscopy of viruses. Biochim.Biophys. Acta 34:103-110.
12. BURNET, F. M., AND J. D. STONE. 1947. Thereceptor-destroying enzyme of V. cholerae.Australian J. Exptl. Biol. Med. Sci. 25:227-333.
13. CASALS, J. 1957. Viruses: the versatile para-sites; the arthropod-borne group ofanimal viruses. Trans. N.Y. Acad. Sci. 19:219-235.
14. COMMITTEE ON THE ENTEROVIRUSES. 1957.National Foundation for Infantile Paralysis.Am. J. Public Health 47:1556-1566.
15. COOPER, P. D. 1961. A chemical basis for theclassification of animal viruses. Nature190:302-305.
16. EDDY, B. E., G. S. BORMAN, G. E. GRUIBBS,AND R. D. YOUNG. 1962. Identification ofthe oncogenic substance in rhesus monkeykidney cell cultures as simian virus 40.Virology 17:65-75.
17. ELFORD, W. J., AND C. H. ANDREWES. 1932.Filtration of vaccinia virus through Gradocolmembranes. Brit. J. Exptl. Pathol. 13:36-42.
18. ELFORD, W. J., J. R. PERDRAU, AND W. SMITH.1933. The filtration of herpes virus throughgraded collodion membranes. J. Pathol.Bacteriol. 36:49-54.
19. FENNER, F., AND F. M. BURNET. 1957. A shortdescription of the poxvirus group (vac-cinia and related viruses). Virology 4:305-314.
20. GAYLORD, W. H., AND G. D. HSIUNG. 1961.The vacuolating virus of monkeys. II. Virusmorphology and intranuclear distributionwith some histochemical observations. J.Exptl. Med. 114.-987-996.
21. GIRARDI, A. J., B. H. SWEET, V. B. SLOTNICK,AND M. R. HILLEMAN. 1962. Developmentof tumors in hamsters inoculated in theneontal period with vacuolating virusSV4o. Proc. Soc. Exptl. Biol. Med. 109:649-660.
22. HAMPARIAN, V. V., M. R. HILLEMAN, AND A.KETLER. 1963. Contributions to characteri-zation and classification of animal viruses.Proc. Soc. Exptl. Biol. Med. 112:1040-1050.
23. HILLEMAN, M. R., A. G. TousIMIs, AND J. H.WERNER. 1955. Biophysical characteriza-tion of the RI (RI-67) viruses. Proc. Soc.Exptl. Biol. Med. 89:587-593.
24. HOFFERT, W. R., M. E. BATES, AND F. S.CHEEVER. 1958. Study of enteric viruses ofsimian origin. Am. J. Hyg. 68:15-30.
25. HORNE, R. W., AND P. WILDY. 1963. Virusstructure revealed by negative staining.Advan. Virus Res. 10:101-170.
26. HORNE, R. W. 1963. The structure of viruses.Sci. Am. 208:48-56.
27. HOWATSON, A. F. 1962. Architecture anddevelopment of tumor viruses and theirrelation to viruses in general. FederationProc. 21:947-953.
28. HSIUNG, G. D. 1959. The use of agar overlaycultures for detection of new virus isolates.Virology 9: 717-719.
29. HSIUNG, G. D. 1964. Diagnostic virology.Yale Univ. Press, New Haven, Conn.
30. HSIUNG, G. D., AND W. H. GAYLORD. 1961.The vacuolating virus of monkeys. I. Isola-tion, growth characteristics, and inclusionbody formation. J. Exptl. Med. 114:975-986.
31. HSIUNG, G. D., P. E. ISACSON, AND R. W.MCCOLLUM. 1962. Studies of a myxovirusisolated from human blood. I. Isolationand properties. J. Immunol. 88:284-290.
32. HSIUNG, G. D., P. W. CHANG, R. R.CUADRADO, AND P. ISACSON. 1965. Studiesof parainfluenza viruses. III. Antibodyresponses of different animal species follow-ing immunization. J. Immunol. 94: 62-73.
33. HULL, R. N., J. R. MINNER, AND J. W. SMITH.1956. New viral agents recovered from tissuecultures of monkey kidney cells. I. Originand properties of cytopathogenic agents.
VOL. 29, 1965 485
BACTERIOL. REV.
S.V.1, S.V.2, S.V.4, S.V.5, S.v.6, S.V.11,S.V.12, and S.V.15. Am. J. Hyg. 63:204-215.
34. HULL, R. N., J. R. MINNER, AND C. C.MASCOLI. 1958. New viral agents recoveredfrom tissue cultures of monkey kidneycells. III. Recovery of additional agentsboth from cultures of monkey tissues anddirectly from tissues and excreta. Am. J.Hyg. 68:31-44.
35. LWOFF, A. 1957. The concept of virus. J.Gen. Microbiol. 17:239-253.
36. LWOFF, A., R. HORNE, AND P. TOURNIER.1962. A system of viruses. Cold SpringHarbor Symp. Quant. Biol. 27:51-55.
37. MALHERBE, H., AND R. HARWIN. 1957. Sevenviruses isolated from the Vervet monkey.Brit. J. Exptl. Pathol. 38:539-541.
38. MALHERBE, H., AND R. HARWIN. 1963. Thecytopathic effects of Vervet monkey viruses.S. African Med. J. 37:407-411.
39. MAYOR, H. D., AND J. L. MELNICK. 1961-62.Intracellular and extracellular reactions ofviruses with vital dyes. Yale J. Biol. Med.34:340-358.
40. MELNICK, J. L. 1962. Papova virus group.Science 135:1128-1130.
41. MELNICK, J. L., W. C. COCKBURN, G. DAL-DORF, S. GARD, J. H. S. GEAR, W. McD.HAMMON, M. M. KAPLAN, F. P. NAGLER,N. OKERBLOM, A. J. RHODES, A. B. SABIN,J. D. VERLINDE, AND H. VON MAGNUS.1963. Picornavirus group. Virology 19:114-116.
42. NORRBY, E. 1962. Hemagglutination bymeasles virus. II. Properties of the hemag-glutinin and the receptors on the erythro-cytes. Arch. Ges. Virusforsch. 12:164-172.
43. PEREIRA, H. G., R. J. HUEBNER, H. S. GINS-BERG, AND J. VAN DER VEEN. 1963. A short
description of the adenovirus group. Vi-rology 20:613-620.
44. SABIN, A. B. 1959. Reoviruses. A new groupof respiratory and enteric viruses formerlyclassified as ECHO type 10 is described.Science 130:1387-1389.
45. SMITH, K. O., AND J. L. MELNICK. 1962. Amethod for staining virus particles andidentifying their nucleic acid type in theelectron microscope. Virology 17:480-490.
46. SMITHBURN, K. C., AND J. C. BUGHER. 1953.Ultrafiltration of recently isolated neuro-tropic viruses. J. Bacteriol. 66:173-177.
47. THEILER, M. 1957. Action of sodium desoxy-cholate on arthropod-borne viruses. Proc.Soc. Exptl. Biol. Med. 96:380-382.
48. TYRRELL, D. A. J., M. L. BYNOE, G. HITCH-COCK, H. G. PEREIRA, AND C. H. ANDREWES.1960. Some virus isolations from commoncolds. I. Experiments employing humanvolunteers. Lancet 1:235-237.
49. TYRRELL, D. A. J., AND R. M. CHANOCK. 1963.Rhinoviruses: a description. Science 141 :152-153.
50. VALENTINE, R. C. 1961. Contrast enhancementin the electron microscopy of viruses.Advan. Virus Res. 8:287-318.
51. VIRUS SUBCOMMITTEE OF INTERNATIONALNOMENCLATURE COMMITTEE. 1963. Recom-mendations on virus nomenclature. Virol-ogy 21:516-517.
52. WATERSON, A. P. 1961. Introduction to animalvirology. Cambridge Univ. Press, NewYork.
53. WATERSON, A. P., J. G. CRUICKSHANK, G. D.LAURENCE, AND A. D. KANAREK. 1961.The nature of measles virus. Virology 15 :379-382.
54. WILLIAMS, R. C. 1954. Electron microscopy ofviruses. Advan. Virus Res. 2:183-239.
486 HSIUNG