Characteristics of a Swine Papovavirus

Transcription

1 IFECTIO AD IMMUITY, June 1972, p Vol. 5, o. 6 Copyright ( 1972 Amerian Soiety for Mirobiology Printied in U.S.A. Charateristis of a Swine Papovavirus JOSEPH T. EWMA' AD KEDALL. SMITH Departmelit of Mirobiology, University of Texas Medial Shool at Sani A,,tontio, Sani Anitoniio, 7exas Reeived for publiation 11 February 1972 A new member of the papovavirus group has been isolated and appears to infet swine. The new agent, tentatively named swine papovavirus, appears to be very defetive and repliates only within a very narrow host ell range. The original soure of the isolate is under investigation. Preliminary evidene suggests that the origin of swine papovavirus is either a stable pig kidney ell line or panreas-derived trypsin Papovaviruses have been isolated from many animals inluding man (2, 6, 9, 12). The papova group of deoxyribonulei aid () viruses is known for its potential to transform ells in vitro and ause tumors in vivo (5, 11, 21). Although piodna viruses have been reported from piglet and swine breeding herds (4, 1), there are no reports in the literature of a swine papovavirus (SPV) having been isolated in vitro or seen in tissues by eletron mirosopy. Parrish (13, 14) reported a transmissable genital papilloma in swine, but the agent was not propagated in vitro. Reently, the agent ausing this disease has been lassified as a member of the poxviruses by Allison (1) and Andrewes and Pereira (2). During a preliminary study in whih we were searhing for adventitious agents in swine panreas-derived trypsin, a virus was isolated whose biologial and physial harateristis are the subjet of this ommuniation. We are still unertain as to the exat soure of this isolate, but experimental evidene is presented whih suggests that the isolate is a swine agent and a new member of the papovavirus group. MATERIALS AD METHODS Cell ultures. The virus was propagated in two pig kidney ell lines derived from different lones of a parent line, PK-2a, established by Stie (15). One lone has been referred to as the PK-15 ell line (15) and the seond lone as the PS ell line (7). The PK-15 ell line was purhased from Flow Laboratories, In., Rokville, Md. This ell line was originally obtained by Flow Laboratories, In. from the Amerian Type Culture Colletion at the 154th passage level. The PS ell line was provided by D. W. Trent of the University of Texas Medial Shool at San Antonio who reeived the ells from E. G. Westaway, Monash Medi- I A portion of this work shall be presented in a dissertation to be sulbmitted hy J. T. ewman in partial fulfillment of the requirements for the Ph.D. degree from the University of Texas Medial Shool at San Antonio al Shool, Vitoria, Australia. Other ell lines used in this study were MDCK (dog), HA (human), MA-14 (monkey), and L (mouse). Primary embryoni pig kidney ells and primary embryoni mouse ells were prepared in our laboratory. Cell monolayers were grown in 32-oz (a..95 liters) glass bottles and Falon disposable petri dishes (1 by 35 mm). Growth media onsisted of an autolavable modifiation of Eagle basal medium (BME; Auto-POW-BME, Flow Laboratories, In.) supplemented with virus-sreened 1% fetal alf serum (FCS; Industrial Biologial Laboratories, Rokville, Md.) and neomyin (2ug/mrnl). Confluent monolayers were maintained in BME with 1 % FCS. Viral assay: immunofluoresene. The tehnique we used in the immunofluoresene fous assay has been desribed previously (2). Antibody against SPV was prepared artifiially by inoulating.2 ml of esium hloride-purified virus and Freund's omplete adjuvant (1:1) into young adult guinea pigs. The inoulation sites (.5 ml/site) were the rear foot pads and the upper flanks. After 1 month, the guinea pigs reeived booster shots at the same sites; 1 days later a series of weekly bleedings was initiated. Anti-guinea pig globulin onjugated with fluoresein isothioyanate was either purhased from a ommerial soure (Colorado Serum Co.) or prepared in our laboratories. Stained ells were examined in a dark-field lluoresene mirosope (Baush & Lomb, In.) with an Osram HBO2 lamp. A Baush & Lomb no exiter filter was used. Soure of swine sera. Individual lots of swine sera were provided by E. H. Bohl of the Ohio Agriultural Researh and Development Center, Wooster, Ohio. Swine serum samples were seleted from healthy pigs of different ages and herds. Soure of SV4 and polyoma virus and antiserum. SV4 was a small-plaque strain, provided by P. Gerber, whih was propagated in MA-14 ells. Rhesus monkey serum served as a soure of naturally ourring antibodies to SV4. Anti-human globulin onjugated with fluoresein isothioyanate was purhased from Progressive Laboratories, In. Polyoma virus was grown in primary embryoni 961

2 962 EWMA AD SMITH IFECT. IMMUITY mouse ells. Antiserum to polyoma virus was prepared in rabbits. Both antiserum and virus were provided by H. D. Mayor. Anti-rabbit globulin onjugated with fluoresein isothioyanate was purhased from Progressive Laboratories, In. Virus purifiation, onentration, and buoyant density determinations. Infeted PK-15 ells were disrupted by a single freeze-thawing at -5 C, followed by soni osillation for 3 min at 38 ma with a Branson sonifier ell disruptor, model no. W14D. After removing large partiulate matter by low-speed entrifugation, the virus suspension was plaed on a preformed gradient and entrifuged for 3 hr at 1, X g in a Bekman model L-265 ultraentrifuge. A modified proedure for purifiation was also employed. onidet P-4, a nonioni detergent, was mixed with frozen-thawed rude virus to a final onentration of.1 %. This mixture was treated by soni osillation for 6 min and was larified by filtration through Celite no. 512 (Johns-Manville). Then the material was entrifuged on a preformed gradient at 1, X g for 14 to 17 hr. Frations were olleted through a punture in the tube bottoms, and eah fration was examined for virus partiles by eletron mirosopy. Buoyant density measurement were made on virus-ontaining frations with a densitometer. The densitometer was alibrated by plotting refrativeindex readings against densities of esium hloride solutions determined by weight-volume measurements. Eletron mirosopy. The tehnique for partile ounting was a modifiation of the Sharp method (17) for negative staining as desribed by Smith and Melnik (19). eutralization tests. eutralization tests were arried out by employing an immunofluoresene fous assay method desribed previously (18). A redution of 8% or greater in infetious virus titer was onsidered the serum neutralization end point. Ether sensitivity determination. We used a standard proedure desribed by Kapikian (8) for determining ether sensitivity of viruses. This onsisted of treating virus suspensions with equal volumes of ether at 4 C for 18 to 24 hr. Herpes simplex virus was used in the test system as a positive ontrol, and assay of this virus was performed as desribed by Roizman and Roane (16). Treated and untreated SPV was titrated in PS ells, and infetivity was measured by the immunofluoresene fous assay. ulei aid determination. PS ells were adsorbed with.2 ml of undiluted virus suspension for 1 hr at room temperature and then maintained on BME maintenane media ontaining.1 mg of 5-iodo-2'- deoxyuridine (IUDR) per ml. After 4 days the ell monolayers were sraped and frozen at -5 C. Thawed samples of IUDR-treated and untreated ontrol ells were then titrated in PS ells. Adenovirus type 2 propagated in HA ells was used as a known virus ontrol and reovirus type 3 propagated in PS ells served as a known ribonulei aid (RA) virus ontrol. All virus titers were determined by measuring infetivity with the immunofluoresene fous assay. RFSULTS Soure of virus. SPV was initially isolated by us during a searh for adventitious viruses in swinederived panreati trypsin. In this study we exposed PK-15 ells to onentrated trypsin whose ativity was inhibited by mixing with FCS. After removing the trypsin, the ells were maintained on BME with 1 % FCS. Some treated PK-1 5 ell ultures slowly developed ytopathi effets (CPE), whih we believed to be virus-indued beause eletron mirosopi examination of frozen and thawed material from these ultures revealed numerous ubially symmetrial partiles. Observation of virus-infeted ell ultures. Infeted PK-15 ells maintained in BME medium with 1%, FCS (normal ell growth medium) ould be propagated and held for prolonged periods of time without obvious CPE. If infeted ell monolayers were held in BME maintenane media (1% FCS) with weekly medium hanges, ell monolayers slowly degenerated within a period of 3 weeks. Virus titers of supernatant fluids from these ultures averaged about 14 infetious doses per ml when titrated in PS ells. Partile ounting of the same material resulted in ounts of 11 virus partiles/ml. Therefore, the alulated partile-infetivity ratio approximated 16 partiles per infetious dose. There was no observable CPE in primary pig embryoni kidney ells. Immunofluoresene staining of pig embryoni kidney ells 4 days postinfetion showed sattered foi of fluoresing ells. Fluoresent foi were onfined almost entirely to pathes of flat epithelial-like ells; spindle-shaped, fibroblast-like ells were rarely involved. The SPV did not pass suessfully in other ell lines, i.e., MDCK, HA, MA-14, and L ells. Growth of virus during passage was determined by immunofluoresene staining of SPV-infeted ells of different ell lines. Satisfatory passage was obtained in PK-1 5 ells, whih served as a positive ontrol. Immunofluoresene. The main detetion system for SPV was the indiret Coons' method of staining virus-infeted ells. Antiserum against SPV, whih was produed in guinea pigs, ould usually be used suessfully for fluoresene work at a dilution of 1:3. Stained, infeted ell nulei displayed maximum fluoresene 96 to 12 hr after virus inoulation. Antisera against SV4 and polyoma virus did not stain SPV-infeted PK-15 ells. either embryoni mouse ells infeted with polyoma virus nor MA-14 ells infeted with SV4 were stained with antiserum against SPV. In all instanes infeted ells stained positively with homologous antisera. eutralization tests. Anti-SPV made in guinea pigs neutralized SPV at a maximum dilution of 1:6. All 1 lots of different swine sera from ani-

3 VOL. 5, CHARACTERISTICS OF A SWIE PAPOVAVIRUS 963 mals 6 months to 4 years of age neutralized SPV at a dilution of >1:2. Seven of these swine serum samples neutralized SPV at a dilution of > 1 :1 and one serum lot neutralized SPV at a > 1:3 dilution. We onlude, therefore, that SPV ommonly infets swine. Eletron mirosopy. The SPV partile, when negatively stained, exhibited ubi symmetry and did not appear to possess an envelope (Fig. ib). In many instanes, however, membrane-like material was found assoiated with single partiles, as well as with lusters of partiles (Fig. IC). The apsomere number and arrangement losely resembled that observed with other A FIG. 1. Morphology of swivne papovavirus. (A) a filamnenztous fjrm showing apsomeres; (B) ubi symmetry partiles showintg apsomeres; (C) virus partiles assoiated withnmembraniouts material. Bars equal 1 inim.

4 964 EWMA AD SMITH IFECT. IMMUITY papovaviruses. The average virus size, based on the measurement of 13 negatively stained partiles, was 39.6 nm. Approximately 8% of the partiles examined had diameters between 36 and 44 nm. (Fig. 2). Filamentous forms were observed whih measured 35.8 nm in diameter (Fig. 1A). Buoyant density. Sonially treated rude virus, larified by low-speed entrifugation, banded with a peak infetivity at a density of 1.22 g/ml, as an be seen in Fig. 3. Eletron mirosopi examination of these frations showed membrane-like material assoiated with virus partiles similar to that seen in Fig. 1C. Ether treatment of banded virus, when rebanded, did not show a signifiant hange in density. When the rude virus was prepared for purifiation and onentration by a modified proedure employing Celite filtration and addition of onidet P-4, the infetivity peak was at a density of 1.35 g/ml. Eletronmirosopi examination of these frations showed virus partiles free of membrane-like material. 2 o \ Ether sensitivity. Treatment of SPV with ether resulted in no signifiant derease in infetivity, whih suggests that the virus does not ontain essential lipids. Optimum growth temperature. PS ells infeted with SPV and held at 33, 36, and 39 C for different time intervals yielded similar titers of infetious virus (a. 5 X 14/ml) when harvested at the same time intervals. ulei aid type. IUDR at a onentration of.1 mg/ml was found to be inhibitory for SPV and adenovirus repliation, whereas reovirus type 3 repliated freely under the same onditions (Table 1). RA viruses whih depend on synthesis for the prodution of new virus progeny might also be inhibited by IUDR. However, no nonenveloped, ubially symmetrial RA virus is known to be inhibited by IUDR. Therefore, the results of the IUDR experiment would imply that the SPV genome is omposed of FIG. 2. Diameter of Partiles (nm) Distribiitionz ofpartile sizes of swinie papovaviruis based oni the measuiremenit of 13 virius partiles.

5 VOL. 5, 1972 CHARACTERISTICS OF A SWIE PAPOVAVIRUS 965 LL- 't -:2 =3.21,Z; (L) (M (L) (L) a- - < Density of Frations (gm/ml) FIG. 3. Distributioni of swinie papova virionis after enitrifiugationz over a preformed esium hloride dentsity gradientt. () Crude virus treated by sonii osillationi aiid larified by low-speed enitrifugationi; () rude virus nmixed with onzidet P-4, treated by sonii osillationt, anid larified by Celite filtrationi. TABLE 1. Effets of 5-iodo-2'-deoxyuridine oni the repliationl of reoviruts type 3, adenioviruis type 2, anid swinie papovavirus in tissue ultuirea Virus Cell substrate IUDR treated Control Reovirus 3 PS ells 1.4 X 1Yb 1. X 18 Ad 2 HA ells <1.8 X X 17 SPV PS ells <6.3 X X 13 aabbreviations: IUDR, 5-iodo-2'-deoxyuridine; Ad 2, adenovirus 2; SPV, swine papovavirus. bfluoresing foi per ml. DISCUSSIO A new member of the papovavirus group has been isolated and tentatively named swine papovavirus. The virus has been propagated in two ell lines, PS and PK-15, whih were both derived as separate lones from a parent ell line PK-2a. Virus repliation has also been demonstrated in primary embryoni pig kidney ell ultures, although virus multipliation seems to be largely limited to epithelial-like ells in these ultures. SPV did not repliate to detetable levels 1.4 in ell lines from animal speies other than swine. In addition to a narrow host range, SPV appears to be a very defetive virus beause only one partile in approximately 16 is infetious in the assay system we use. The reason for its apparent defetiveness is presently unknown. Two possibilities are apparent: (i) the PK-15 ells are naturally ineffiient produers of infetious partiles, or (ii) optimal multipliity of infetion has not yet been ahieved for maximum infetious virus yields. The reasons for plaing the new isolate in the papovavirus group were based on the harateristis summarized in Table 2. Morphology of the ubi symmetry partile, size, and the presene of filamentous forms are distintive harateristis of the papovavirus group. The bouyant density of the swine papova virion appears to be within the range of values reorded for other members of the papovavirus group. However, density determinations for others have been done under a variety of onditions that makes strit omparisons diffiult. For instane, density measurements have been orrelated with physial partiles in some ases

6 966 EWMA AD SMITH IFECT. IMMUITY' TABLE 2. Papovavirus group" Papovavirus Size (nm) Cubi symmetry Filamen tous forms ulei aid type Boyn Assem- Growth deuynstyi density bly in site vitro atural host Latent and Tumorohroni infetions Lipid (envelope) gei geni SPV... Polyoma... SV4... Rabbit kidney vauolating virus Papilloma (rabbit)... Papilloma (Human wart)... K virus... Bovine papilloma Swine Mouse Monkey Rabbit Rabbit Man Mouse Bovine amodified from Melnik (referene 11). Abbreviations: SPV = swine papovavirus; = nuleus. I Probable. and infetivity measurements in others. Ether sensitivity tests and growth inhibitory studies employing a analogue suggest that SPV is nonenveloped and ontains a ore; these are properties of the papovavirus group. Serologial studies suggest that the present isolate is not a previously reognized papovavirus beause SPV-infeted PK-15 ells are not stained in the indiret Coons' test by antisera to SV4 and polyoma virus. Evidene has been obtained that neutralizing antibodies to SPV are widely distributed in apparently healthy swine. It is likely, therefore, that swine are naturally infeted with this virus, possibly without obvious disease (lateny is harateristi of papovaviruses in vivo). In most instanes the neutralization titers were greater than we were able to obtain artifially in guinea pigs. We onlude that this agent is SPV on the basis of (i) its physial harateristis, (ii) its nulei aid type, (iii) the absene of essential lipids, (iv) its repliation being restrited to swine ells, and (v) the presene of naturally ourring antibody in swine. At this time, studies are being onduted to determine the original soure of the SPV we isolated. We are onsidering the possibilities (i) that the agent is indigenous in the PK-15 ell line we obtained for these studies and (ii) that the agent is present in some lots of panreas-derived trypsin to whih our PK-15 ells were exposed. If the latter is orret, high-passage levels of stable swine ells (suh as the PK-15 line) would be exposed repeatedly to swine agents during routine trypsinization for passage. The presene of virus in swine panreas-derived trypsin would not ± ± -F be surprising beause swine myoplasma are ommon ontaminants of ell ultures whih are exposed to swine-derived trypsin (3). ow that we have developed a method for deteting SPV, we are examining trypsin and swine ells from many soures for the presene of this agent. ACKOWLEDG METS This investigation was partially supported by grant I-9A from the Amerian Caner Soiety. We thank H. D. Mayor, P. Gerber, D. W. Trent, and E. H. Bohl for providing speifi antisera, virus strains, aind ell ultures used in this study. LITERATURE CITED 1. Allison, A. C Viruses induinig skin tumiiors in animals, p In A. J. Rook and G. E. Wailton (ed.), Comparative physialogy and pathology of the skini. F. A. Davis Co., Philadelphia. 2. Andrewes, C., aind H. G. Pereira Viruses of vertebrates. Williams & Wilkins Co., Baltimore. 3. Barile, M. F Myoplasmai anid ell ultures. ait. Caner Inst. Moniogr. 29: Cartwr-ight, S. F., M. Luas, and R. A. Huk A small haemagglutinating porine virus. I. Isolaition and properties. J. Comp. Pathol. 79: Eddy, B. E Simian virus 4 (SV-4): an onogeni virus. Progr. Exp. Tumor Res. 4: Gardner, S. D., A. M. Field, D. V. Colemiian, and B. Hulme ew humilan papovavirus (B. K.) isolated from urine after renal tr-ansplantation. Lanet 1: Inoue, Y. K., and M. Yamada Clonal line of porine kidney stable ells for assay of Japanese enephalitis virus. J. Bateriol. 87: Kapikian, A. Z Rhinoviruses, p In E. H. Lennette and. J. Shinidt (ed.). Diagnosti proedures for viral and r-ikettsial diseases. Amiierian Publi Health Assoiation, ew York. 9. Koprowski, H., G. Barbainti-Brodano, and M. Katz Interation between papova-like virus and paramyxovirus in human brain ells: a hypothesis. ature (London) 225: Mayr, A., P. A. Bahmann. G. Siegl, H. Mahnel. and B. E.

7 VOL. 5, 1972 CHARACTERISTICS OF A SWIE PAPOVAVIRUS 967 Sheffy Charaterization of a small porine virus. Arh. Gesamte Virusforsh. 25: Melnik, J. L Papova virus group. Siene 135: Padgett, B. L., G. M. ZuRhein, D. L. Walker, R. J. Ekroade, and B. H. Dessel Cultivation of papova-like virus from human brain with progressive multifoal leuoenephalopathy. Lanet 1: Parrish, W. E A transmissible genital papilloma of the pig resembling ondyloma auminatum of man. J. Pathol. Bateriol. 81: Parrish, W. E An immunologial study of the transmissible genital papilloma of the pig. J. Pathol. Bateriol. 83: Registry of animal ell lines ertified by the Cell Culture Collet on Committee, Amerian Type Culture Colletion Cell Respository Rokville, Md. 16. Roizman, B., and P. R. Roane A physial differene between two strains of herpes simplex virus apparent on sedimentation in esium hloride. Virology 15: Sharp, D. G Sedimentation ounting of partiles via eletron mirosopy, p Eletron Miros., Pro. Int. Cong. E. M Berlin. 18. Smith, K. O., R. C. Dunlap, J. F. Thiel, J. T. ewman, and A. E. Palmer Isolation of viruses from primary dog ell ultures and the ourrene of viral antibody in donor animals. Pro. So. Exp. Biol. Med. 133: Smith, K. O., and J. L. Melnik A method for staining virus partiles and identifying their nulei aid type in the eletron mirosope. Virology 17: Smith, K. O., J. F. Thiel, J. T. ewman, and K. J. Dunn Searh for infetious agents in ell ultures by fluoresene mirosopy. eurology 18: Stewart, S. E., B. E. Eddy, A. M. Gohenour,. G. Borgese, and G. Grubbs The indution of neoplasms with a substane released from mouse tumors by tissue ulture. Virology 3:38-4. Downloaded from on Otober 31, 218 by guest