INFECTION AND IMMUNITY, June 1972, p. 961-967 Vol. 5, No. 6Copyright (© 1972 American Society for Microbiology Printied in U.S.A.

Characteristics of a Swine PapovavirusJOSEPH T. NEWMAN' AND KENDALL 0. SMITH

Departmelit of Microbiology, University of Texas Medical School at Sani A,,tontio,Sani Anitoniio, 7exas 78229

Received for publication 11 February 1972

A new member of the papovavirus group has been isolated and appears to infectswine. The new agent, tentatively named swine papovavirus, appears to be very defec-tive and replicates only within a very narrow host cell range. The original source ofthe isolate is under investigation. Preliminary evidence suggests that the origin ofswine papovavirus is either a stable pig kidney cell line or pancreas-derived trypsin

Papovaviruses have been isolated from manyanimals including man (2, 6, 9, 12). The papovagroup of deoxyribonucleic acid (DNA) viruses isknown for its potential to transform cells in vitroand cause tumors in vivo (5, 11, 21). Althoughpicodna viruses have been reported from pigletand swine breeding herds (4, 10), there are noreports in the literature of a swine papovavirus(SPV) having been isolated in vitro or seen intissues by electron microscopy. Parrish (13, 14)reported a transmissable genital papilloma inswine, but the agent was not propagated in vitro.Recently, the agent causing this disease has beenclassified as a member of the poxviruses byAllison (1) and Andrewes and Pereira (2). Duringa preliminary study in which we were searchingfor adventitious agents in swine pancreas-derivedtrypsin, a virus was isolated whose biological andphysical characteristics are the subject of thiscommunication. We are still uncertain as to theexact source of this isolate, but experimentalevidence is presented which suggests that theisolate is a swine agent and a new member of thepapovavirus group.

MATERIALS AND METHODSCell cultures. The virus was propagated in two pig

kidney cell lines derived from different clones of aparent line, PK-2a, established by Stice (15). One clonehas been referred to as the PK-15 cell line (15) and thesecond clone as the PS cell line (7). The PK-15 cellline was purchased from Flow Laboratories, Inc.,Rockville, Md. This cell line was originally obtainedby Flow Laboratories, Inc. from the American TypeCulture Collection at the 154th passage level. The PScell line was provided by D. W. Trent of the Universityof Texas Medical School at San Antonio who re-ceived the cells from E. G. Westaway, Monash Medi-

I A portion of this work shall be presented in a dissertation to besuLbmitted hy J. T. Newman in partial fulfillment of the require-ments for the Ph.D. degree from the University of Texas MedicalSchool at San Antonio 78229.

cal School, Victoria, Australia. Other cell lines used inthis study were MDCK (dog), HA (human), MA-104(monkey), and L (mouse). Primary embryonic pigkidney cells and primary embryonic mouse cells wereprepared in our laboratory.

Cell monolayers were grown in 32-oz (ca. 0.95liters) glass bottles and Falcon disposable petridishes (10 by 35 mm). Growth media consisted of anautoclavable modification of Eagle basal medium(BME; Auto-POW-BME, Flow Laboratories, Inc.)supplemented with virus-screened 10c% fetal calfserum (FCS; Industrial Biological Laboratories,Rockville, Md.) and neomycin (20ug/mrnl). Con-fluent monolayers were maintained in BME with 1 %cFCS.

Viral assay: immunofluorescence. The technique weused in the immunofluorescence focus assay has beendescribed previously (20).

Antibody against SPV was prepared artificially byinoculating 0.2 ml of cesium chloride-purified virusand Freund's complete adjuvant (1:1) into youngadult guinea pigs. The inoculation sites (0.05 ml/site)were the rear foot pads and the upper flanks. After 1month, the guinea pigs received booster shots at thesame sites; 10 days later a series of weekly bleedingswas initiated. Anti-guinea pig globulin conjugated withfluorescein isothiocyanate was either purchased froma commercial source (Colorado Serum Co.) or pre-pared in our laboratories. Stained cells were examinedin a dark-field lluorescence microscope (Bausch &Lomb, Inc.) with an Osram HBO200 lamp. A Bausch& Lomb no. 5-58 exciter filter was used.

Source of swine sera. Individual lots of swine serawere provided by E. H. Bohl of the Ohio AgriculturalResearch and Development Center, Wooster, Ohio.Swine serum samples were selected from healthy pigsof different ages and herds.

Source of SV40 and polyoma virus and antiserum.SV40 was a small-plaque strain, provided by P. Gerber,which was propagated in MA-104 cells. Rhesus mon-key serum served as a source of naturally occurringantibodies to SV40. Anti-human globulin conjugatedwith fluorescein isothiocyanate was purchased fromProgressive Laboratories, Inc.

Polyoma virus was grown in primary embryonic

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NEWMAN AND SMITH

mouse cells. Antiserum to polyoma virus was pre-pared in rabbits. Both antiserum and virus were pro-vided by H. D. Mayor. Anti-rabbit globulin conju-gated with fluorescein isothiocyanate was purchasedfrom Progressive Laboratories, Inc.

Virus purification, concentration, and buoyant den-sity determinations. Infected PK-15 cells were dis-rupted by a single freeze-thawing at -50 C, followedby sonic oscillation for 3 min at 38 ma with a Bransonsonifier cell disruptor, model no. W140D. After re-moving large particulate matter by low-speed centrif-ugation, the virus suspension was placed on a pre-formed gradient and centrifuged for 3 hr at 100,000 Xg in a Beckman model L-265 ultracentrifuge. Amodified procedure for purification was also em-ployed. Nonidet P-40, a nonionic detergent, wasmixed with frozen-thawed crude virus to a final con-centration of 0.1 %. This mixture was treated by sonicoscillation for 6 min and was clarified by filtrationthrough Celite no. 512 (Johns-Manville). Then thematerial was centrifuged on a preformed gradient at100,000 X g for 14 to 17 hr. Fractions were collectedthrough a puncture in the tube bottoms, and eachfraction was examined for virus particles by electronmicroscopy. Buoyant density measurement were madeon virus-containing fractions with a densitometer.The densitometer was calibrated by plotting refractive-index readings against densities of cesium chloridesolutions determined by weight-volume measurements.

Electron microscopy. The technique for particlecounting was a modification of the Sharp method (17)for negative staining as described by Smith andMelnick (19).

Neutralization tests. Neutralization tests werecarried out by employing an immunofluorescencefocus assay method described previously (18). Areduction of 80% or greater in infectious virus titerwas considered the serum neutralization end point.

Ether sensitivity determination. We used a standardprocedure described by Kapikian (8) for determiningether sensitivity of viruses. This consisted of treatingvirus suspensions with equal volumes of ether at 4 Cfor 18 to 24 hr. Herpes simplex virus was used in thetest system as a positive control, and assay of thisvirus was performed as described by Roizman andRoane (16). Treated and untreated SPV was titratedin PS cells, and infectivity was measured by the im-munofluorescence focus assay.

Nucleic acid determination. PS cells were adsorbedwith 0.2 ml of undiluted virus suspension for 1 hr atroom temperature and then maintained on BMEmaintenance media containing 0.1 mg of 5-iodo-2'-deoxyuridine (IUDR) per ml. After 4 days the cellmonolayers were scraped and frozen at -50 C.Thawed samples of IUDR-treated and untreated con-trol cells were then titrated in PS cells. Adenovirustype 2 propagated in HA cells was used as a knownDNA virus control and reovirus type 3 propagated inPS cells served as a known ribonucleic acid (RNA)virus control. All virus titers were determined bymeasuring infectivity with the immunofluorescencefocus assay.

RFSULTSSource of virus. SPV was initially isolated by us

during a search for adventitious viruses in swine-

derived pancreatic trypsin. In this study we ex-posed PK-15 cells to concentrated trypsin whoseactivity was inhibited by mixing with FCS. Afterremoving the trypsin, the cells were maintainedon BME with 1 % FCS. Some treated PK-1 5 cellcultures slowly developed cytopathic effects(CPE), which we believed to be virus-inducedbecause electron microscopic examination offrozen and thawed material from these culturesrevealed numerous cubically symmetrical par-ticles.

Observation of virus-infected cell cultures. In-fected PK-15 cells maintained in BME mediumwith 10%, FCS (normal cell growth medium)could be propagated and held for prolongedperiods of time without obvious CPE. If infectedcell monolayers were held in BME maintenancemedia (1% FCS) with weekly medium changes,cell monolayers slowly degenerated within aperiod of 3 weeks. Virus titers of supernatantfluids from these cultures averaged about 104infectious doses per ml when titrated in PS cells.Particle counting of the same material resulted incounts of 1010 virus particles/ml. Therefore, thecalculated particle-infectivity ratio approximated106 particles per infectious dose.There was no observable CPE in primary pig

embryonic kidney cells. Immunofluorescencestaining of pig embryonic kidney cells 4 days post-infection showed scattered foci of fluorescing cells.Fluorescent foci were confined almost entirely topatches of flat epithelial-like cells; spindle-shaped,fibroblast-like cells were rarely involved.The SPV did not pass successfully in other cell

lines, i.e., MDCK, HA, MA-104, and L cells.Growth of virus during passage was determinedby immunofluorescence staining of SPV-infectedcells of different cell lines. Satisfactory passagewas obtained in PK-1 5 cells, which served as apositive control.

Immunofluorescence. The main detection systemfor SPV was the indirect Coons' method ofstaining virus-infected cells. Antiserum againstSPV, which was produced in guinea pigs, couldusually be used successfully for fluorescence workat a dilution of 1:3. Stained, infected cell nucleidisplayed maximum fluorescence 96 to 120 hrafter virus inoculation.

Antisera against SV40 and polyoma virus didnot stain SPV-infected PK-15 cells. Neitherembryonic mouse cells infected with polyomavirus nor MA-104 cells infected with SV40 werestained with antiserum against SPV. In all in-stances infected cells stained positively withhomologous antisera.

Neutralization tests. Anti-SPV made in guineapigs neutralized SPV at a maximum dilution of1:6. All 10 lots of different swine sera from ani-

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CHARACTERISTICS OF A SWINE PAPOVAVIRUS

mals 6 months to 4 years of age neutralized SPVat a dilution of >1:2. Seven of these swine se-rum samples neutralized SPV at a dilution of> 1 :10 and one serum lot neutralized SPV at a> 1:30 dilution. We conclude, therefore, that SPVcommonly infects swine.

Electron microscopy. The SPV particle, when

A

negatively stained, exhibited cubic symmetry anddid not appear to possess an envelope (Fig. iB).In many instances, however, membrane-likematerial was found associated with single par-ticles, as well as with clusters of particles (Fig.IC). The capsomere number and arrangementclosely resembled that observed with other

FIG. 1. Morphology of swivne papovavirus. (A) a filamnenztous fJrm showing capsomeres; (B) cubic symmetryparticles showintg capsomeres; (C) virus particles associated withnmembrcaniouts material. Bars equal 100 inim.

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NEWMAN AND SMITH

papovaviruses. The average virus size, based onthe measurement of 103 negatively stained par-ticles, was 39.6 nm. Approximately 80% of theparticles examined had diameters between 36 and44 nm. (Fig. 2). Filamentous forms were observedwhich measured 35.8 nm in diameter (Fig. 1A).Buoyant density. Sonically treated crude virus,

clarified by low-speed centrifugation, banded witha peak infectivity at a density of 1.22 g/ml, as canbe seen in Fig. 3. Electron microscopic examina-tion of these fractions showed membrane-likematerial associated with virus particles similar tothat seen in Fig. 1C. Ether treatment of bandedvirus, when rebanded, did not show a significantchange in density. When the crude virus was pre-pared for purification and concentration by amodified procedure employing Celite filtrationand addition of Nonidet P-40, the infectivitypeak was at a density of 1.35 g/ml. Electron-microscopic examination of these fractions showedvirus particles free of membrane-like material.

32-

28-

24-

20

o \16-

Ether sensitivity. Treatment of SPV with etherresulted in no significant decrease in infectivity,which suggests that the virus does not containessential lipids.Optimum growth temperature. PS cells infected

with SPV and held at 33, 36, and 39 C for differenttime intervals yielded similar titers of infectiousvirus (ca. 5 X 104/ml) when harvested at thesame time intervals.

Nucleic acid type. IUDR at a concentration of0.1 mg/ml was found to be inhibitory for SPVand adenovirus replication, whereas reovirus type3 replicated freely under the same conditions(Table 1). RNA viruses which depend on DNAsynthesis for the production of new virus progenymight also be inhibited by IUDR. However, nononenveloped, cubically symmetrical RNA virusis known to be inhibited by IUDR. Therefore, theresults of the IUDR experiment would imply thatthe SPV genome is composed of DNA.

32 36 40 44 48 52 56

Diameter of Particles (nM)

FIG. 2. Distribiitionz ofparticle sizes of swinie papovaviruis based oni the measuiremenit of 103 virius particles.

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CHARACTERISTICS OF A SWINE PAPOVAVIRUS

- __

<1.10 1.10 1.20 1.30

Density of Fractions (gm/ml)

FIG. 3. Distributioni of swinie papova virionis after cenitrifiugationz over a preformed cesium chloride dentsitygradientt. (0) Crude virus treated by soniic oscillationi aiid clarified by low-speed cenitrifugationi; (0) crude virusnmixed with Nonzidet P-40, treated by soniic oscillationt, anid clarified by Celite filtrationi.

TABLE 1. Effects of 5-iodo-2'-deoxyuridine oni thereplicationl of reoviruts type 3, adenioviruis type 2,

anid swinie papovavirus in tissue cultuirea

Virus Cell substrate IUDR treated Control

Reovirus 3 PS cells 1.4 X 10Yb 1.0 X 108Ad 2 HA cells <1.8 X 102 2.6 X 107SPV PS cells <6.3 X 10 7.4 X 103

aAbbreviations: IUDR, 5-iodo-2'-deoxyuri-dine; Ad 2, adenovirus 2; SPV, swine papovavirus.

bFluorescing foci per ml.

DISCUSSIONA new member of the papovavirus group has

been isolated and tentatively named swinepapovavirus. The virus has been propagated intwo cell lines, PS and PK-15, which were bothderived as separate clones from a parent cell linePK-2a. Virus replication has also been demon-strated in primary embryonic pig kidney cellcultures, although virus multiplication seems tobe largely limited to epithelial-like cells in thesecultures. SPV did not replicate to detectable levels

in cell lines from animal species other than swine.In addition to a narrow host range, SPV appearsto be a very defective virus because only one

particle in approximately 106 is infectious in theassay system we use. The reason for its apparentdefectiveness is presently unknown. Two pos-sibilities are apparent: (i) the PK-15 cells arenaturally inefficient producers of infectiousparticles, or (ii) optimal multiplicity of infectionhas not yet been achieved for maximum infectiousvirus yields.The reasons for placing the new isolate in the

papovavirus group were based on the char-acteristics summarized in Table 2. Morphology ofthe cubic symmetry particle, size, and the presenceof filamentous forms are distinctive characteristicsof the papovavirus group. The bouyant density ofthe swine papova virion appears to be within therange of values recorded for other members of thepapovavirus group. However, density determina-tions for others have been done under a variety ofconditions that makes strict comparisons difficult.For instance, density measurements have beencorrelated with physical particles in some cases

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NEWMAN AND SMITH

TABLE 2. Papovavirus group"

Papovavirus

SPV.............Polyoma .........

SV40.............Rabbit kidneyvacuolating virus

Papilloma (rab-bit) ............

Papilloma (Hu-man wart) ...

K virus..........Bovine papil-loma...........

Size(nm)

4040-5040-50

47

40-50

40-5040-50

47

Cubicsym-metry

+

-1

Filamentousforms

+

Nucleicacidtype

DNADNADNA

DNA

DNA

DNADNA

DNA

Lipid(enve-lope)

Boyn Assem- GrowthdeuynstyI bly indensity site vitro

1.35 N +1.33 N +1.32 N +

1.29-1.34

1 .33

N

N

NN

N

+

Naturalhost

SwineMouseMonkey

Rabbit

Rabbit

ManMouse

Bovine

Latentand Tumoro-

chronic geiinfec- genic

tions

+ +

±

+

±+

-F

+

+

aModified from Melnick (reference 11). Abbreviations: SPV = swine papovavirus; N = nucleus.I Probable.

and infectivity measurements in others. Ethersensitivity tests and growth inhibitory studiesemploying a DNA analogue suggest that SPV isnonenveloped and contains a DNA core; theseare properties of the papovavirus group.

Serological studies suggest that the presentisolate is not a previously recognized papovavirusbecause SPV-infected PK-15 cells are not stainedin the indirect Coons' test by antisera to SV40 andpolyoma virus. Evidence has been obtained thatneutralizing antibodies to SPV are widely dis-tributed in apparently healthy swine. It is likely,therefore, that swine are naturally infected withthis virus, possibly without obvious disease(latency is characteristic of papovaviruses in vivo).In most instances the neutralization titers weregreater than we were able to obtain artifically inguinea pigs.We conclude that this agent is SPV on the

basis of (i) its physical characteristics, (ii) itsnucleic acid type, (iii) the absence of essentiallipids, (iv) its replication being restricted to swinecells, and (v) the presence of naturally occurringantibody in swine.At this time, studies are being conducted to

determine the original source of the SPV weisolated. We are considering the possibilities (i)that the agent is indigenous in the PK-15 cell linewe obtained for these studies and (ii) that theagent is present in some lots of pancreas-derivedtrypsin to which our PK-15 cells were exposed.If the latter is correct, high-passage levels ofstable swine cells (such as the PK-15 line) wouldbe exposed repeatedly to swine agents duringroutine trypsinization for passage. The presence ofvirus in swine pancreas-derived trypsin would not

be surprising because swine mycoplasma arecommon contaminants of cell cultures which areexposed to swine-derived trypsin (3). Now thatwe have developed a method for detecting SPV,we are examining trypsin and swine cells frommany sources for the presence of this agent.

ACKNOWLEDGMENTS

This investigation was partially supported by grant IN-90Afrom the American Cancer Society. We thank H. D. Mayor, P.Gerber, D. W. Trent, and E. H. Bohl for providing specific anti-sera, virus strains, aind cell cultures used in this study.

LITERATURE CITED

1. Allison, A. C. 1965. Viruses inducinig skin tumiiors in animals,p. 665-684. In A. J. Rook and G. E. Wailton (ed.), Compara-tive physiAlogy and pathology of the skini. F. A. Davis Co.,Philadelphia.

2. Andrewes, C., aind H. G. Pereira. 1967. Viruses of vertebrates.Williams & Wilkins Co., Baltimore.

3. Barile, M. F. 1968. Mycoplasmai anid cell cultures. Nait. Can-cer Inst. Moniogr. 29:201-214.

4. Cartwr-ight, S. F., M. Lucas, and R. A. Huck. 1969. A smallhaemagglutinating porcine DNA virus. I. Isolaition andproperties. J. Comp. Pathol. 79:371-377.

5. Eddy, B. E. 1964. Simian virus 40 (SV-40): an oncogenic virus.Progr. Exp. Tumor Res. 4:1-26.

6. Gardner, S. D., A. M. Field, D. V. Colemiian, and B. Hulme.1971. New humilan papovavirus (B. K.) isolated from urineafter renal tr-ansplantation. Lancet 1:1253-1257.

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9. Koprowski, H., G. Barbainti-Brodano, and M. Katz. 1970.Interaction between papova-like virus and paramyxovirusin human brain cells: a hypothesis. Nature (London) 225:1045-1047.

10. Mayr, A., P. A. Bachmann. G. Siegl, H. Mahnel. and B. E.

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11. Melnick, J. L. 1962. Papova virus group. Science 135:1128-1130.

12. Padgett, B. L., G. M. ZuRhein, D. L. Walker, R. J. Eckroade,and B. H. Dessel. 1971. Cultivation of papova-like virusfrom human brain with progressive multifocal leucoen-cephalopathy. Lancet 1:1257-1260.

13. Parrish, W. E. 1961. A transmissible genital papilloma of thepig resembling condyloma acuminatum of man. J. Pathol.Bacteriol. 81:331-345.

14. Parrish, W. E. 1962. An immunological study of the trans-

missible genital papilloma of the pig. J. Pathol. Bacteriol.83:429-442.

15. Registry of animal cell lines certified by the Cell Culture Col-lect on Committee, American Type Culture Collection CellRespository. 1964. Rockville, Md.

16. Roizman, B., and P. R. Roane. 1961. A physical difference

between two strains of herpes simplex virus apparent on

sedimentation in cesium chloride. Virology 15:75-79.17. Sharp, D. G. 1960. Sedimentation counting of particles via

electron microscopy, p. 542-548. Electron Microsc., Proc.Int. Cong. E. M. 1958. Berlin.

18. Smith, K. O., R. C. Dunlap, J. F. Thiel, J. T. Newman, andA. E. Palmer. 1970. Isolation of viruses from primary dogcell cultures and the occurrence of viral antibody in donoranimals. Proc. Soc. Exp. Biol. Med. 133:560-567.

19. Smith, K. O., and J. L. Melnick. 1962. A method for stainingvirus particles and identifying their nucleic acid type in theelectron microscope. Virology 17:480-490.

20. Smith, K. O., J. F. Thiel, J. T. Newman, and K. J. Dunn. 1968.Search for infectious agents in cell cultures by fluorescencemicroscopy. Neurology 18:165-167.

21. Stewart, S. E., B. E. Eddy, A. M. Gochenour, N. G. Borgese,and G. Grubbs. 1957. The induction of neoplasms with a

substance released from mouse tumors by tissue culture.Virology 3:380-400.

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