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rus DNAs at different degrees of stringency. Intervirol 28:114121. 17. O’Banion MK, Sundberg JP, Shima AL, Reichmann ME: 1987, Venereal papilloma and papillomavirus in a Colobus monkey (Colobus guereza). Intervirology 28:232-237. 18. Pfister H, Hettich I, Runne U, Chilf GN: 1983, Characterization of human papillomavirus type 13 from focal epithelial hyperplasia Heck lesions. J Virol 47:363-366. 19. Sundberg JP: 1987, Animal papillomaviruses. In: Papillomaviruses and human disease, ed. Syrjanen K, Gissmann L, Koss L, pp. 40-103. Springer-Verlag, Heidelberg. 20. Sundberg JP, Junge RE, Lancaster WD: 1984, Immunoperoxidase localization of papillomaviruses in hyperplastic and neoplastic epithelial lesions of animals. Am J Vet Res 45: 14411446.

21. Sundberg JP, Reichmann ME: 1991, Papillomavirus infections in nonhuman primates. In: Diseases of nonhuman primates. Monographs in laboratory animal pathology, ed. Jones TC, Mohr U, Hunt RD, in press. Springer-Verlag, Heidelberg. 22. Syrjanen K, Gissmann L, Koss L: 1987, Papillomaviruses and human disease. Springer-Verlag, Heidelberg. 23. Syrjanen SM, Syrjanen KJ, Happonen RP: 1988, Human papillomaviruses (HPV) DNA sequences in oral precancerous lesions and squamous cell carcinoma demonstrated by in situ hybridization. J Oral Pathol 17:273-278. 24. Syrjanen SM, Syrjanen KJ, Happonen RP, Lamberg MA: 1987, In situ DNA hybridization analysis of human papillomavirus (HPV) sequences in benign oral mucosal lesions. Arch Dermatol Res 279:543-549.

J Vet Diagn Invest 4:74-77 (1992)

Interspecies polymorphism of double-stranded RNA extracted from reoviruses of turkeys and chickens Luis-Fernando Lozano, Salah Hammami, Anthony E. Castro, Bennie I. Osbum Reoviruses are ubiquitous in environments where turkeys and chickens are kept. These viruses are associated with enteric disorders, growth abnormalities, leg deformities, and respiratory disease conditions and are found in disease conditions of turkeys and chickens etiologically associated with other viruses and bacteria.’ Reoviruses are also associated with diseases in other avian species.3,11 Avian reoviruses are classified within the family Reoviridae, which is characterized by segmented double-stranded RNA (dsRNA). The genomic electropherotype of avian reoviruses consists of 10 segments classified as Large (L)l, L2, L3; Medium (M)l, M2, M3; Small (S)1, S2, S3, S4. The approximate molecular weight range of each genomic segment group is L1, L2, L3: 2.5-2.7 x 10-6 daltons; Ml, M2, M3: 1.8 x 10-6 daltons; and S1, S2, S3, S,: 0.71-1.1 x 10-6 daltons. To diagnose infections by reoviruses, viral nucleic acid extraction and segment separation by polyacrylamide gel electrophoresis has been used.2 This technique is also employed to obtain epidemiologic data. Further, genomic differences may exist between chicken and turkey reoviruses.1,7 The genomic profiles of 70 avian reovirus isolates were analyzed to test this hypothesis. This study compares the genomic profiles of isolates and/ or strains from 60 turkeys, 8 chickens, 1 canary, and 1 cockatiel to determine the differences in their mobility in gels of the dsRNA segments. Fifty-nine reovirus isolates were from specimens obtained From the California Veterinary Diagnostic Laboratory System (Lozano, Castro) and the Department of Veterinary Pathology (Hammami, Osbum), School of Veterinary Medicine, University of California, Davis, CA 95616. Received for publication December 6, 1990.

from turkey flocks located in California. One reference strain, turkey avian reovirus type 3,a was analyzed with this group. The 8 chicken specimens included 4 field isolates from flocks in California and 4 reference strains. Additionally, 1 isolate from a canary and 1 from a cockatiel were used in the genomic comparisons. Five prototype avian reoviruses were included within their respective species for this analysis. The prototype avian reoviruses used were the Fahey-Crawley strain,a,b avian enteric reovirus type I, and turkey enteric reovirus type 3 and the S-1133 strain.c Except for the reovirus type 3 originally isolated from turkeys, all other prototype strains were originally isolated from chickens. The prototype strains provided the reference genomic electropherotype for all comparisons. Cell cultures used for propagating these viruses provided the uninfected negative controls. Primary chicken embryo kidney (CEK) cell monolayer was used for virus isolation from field specimens.6 Tissues submitted were diluted 1/10 (v/v) in viral transport media (VTM) consisting of minimum essential medium with 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10 µg/ml kanamycin sulfate. After dilution in VTM, the tissues were homogenized for 10-15 sec. A sample aliquot of 10 ml was centrifuged at low speed (250 x g ), filtered through a 0.2-µm filter, and stored at 4 C for further cell inoculation. The remaining homogenized tissue was stored at -20 C. Primary cell cultures of CEK grown in 25-ml flasks were washed once with VTM, and each culture was inoculated with 0.5 ml of the filtered supernatant. As negative controls, flasks containing a monolayer of CEK cells received 0.5 ml of VTM. After incubation for 1 hour at 37 C for adsorption of virus, CEK cells were fed with Dulbecco’s maintenance medium supplemented with 4 mM L-glutamine, 5% bovine fetal serum,

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Figure 1. Comparison of electropherotypes of 5 prototype avian reoviruses isolated from chickens and turkeys. Lane 1: chicken S-1133 (SPAFAS); lane 2: chicken enteric reovirus (ATCC); lane 3: chicken Fahey-Crawley strain (ATCC); lane 4: chicken Fahey-Crawley strain (NVSL); lane 5: turkey enteric reovirus (ATCC); lane 6: reovirus field isolate from a canary; lane 7: reovirus field isolate from a cockatiel.

100 µg/ml streptomycin, and 10 µg/ml kanamycin sulfate. Inoculated cell cultures were observed daily for 6 days for virus cytopathic effect (CPE). When extensive CPE (>90%) was observed in the monolayer of CEK cells, replication of virus was confirmed by electron microscopy (EM) and RNA extraction methods. The virus isolates were further cloned by terminal dilution by 3 serial passages in CEK cells, and

the viral RNA was extracted from the third cell culture passage. Cross-contamination of isolates was avoided by keeping flasks in separate incubators. Flasks were handled individually and stored at -80 C for subsequent analysis. Each virus isolated was titered by the end point method, and the amount of virus used to inoculate each cell monolayer was standardized to 5 x 103 TCID50 per inoculation volume of 0.5 ml.

Figure 2. Comparison of electropherotypes of field isolates of avian reovirus from chicken and turkey flocks in California. Lanes 1-4: electropherotype of reovirus field isolates from chicken flocks; lanes 5-8: electropherotypes of reovirus field isolates from turkey flocks.

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Brief communications

Figure 3. Electropherotypes of field isolates of reovirus from turkey flocks in California. Lane C: primary chicken embryo kidney cell culture control; lanes 1-9: electropherotype of field isolates of turkey reoviruses.

For EM of each specimen, primary CEK cells were grown in a 75-ml cell culture flask and inoculated with the third virus passage. Cell culture monolayers showing extensive CPE (>90%) were stored by freezing the flask at -70 C until further processed by a procedure previously described.10 After 3 cycles of freezing and thawing, each cell culture supernatant was sonicated, clarified by low speed centrifugation (250 x g), filtered through a 0.2-µm filter, and ultracentrifuged at 340,000 x g for 45 minutes at 4 C using a 55.2 Ti rotor. Each pellet was resuspended in 0.4 ml distilled water and placed in a glass nebulizer containing 8 drops distilled water and 1 drop 4% phosphotungstic acid at pH 7. The mixture was nebulized onto two 200-mesh formvar-coated carbon-stabilized copper grids. The grids were examined by transmission EM in a liquid nitrogen-cooled column (-192 C) of a Zeiss 10C electron microscope. The dsRNA was extracted from each virus propagated in cell culture. Briefly, the cell monolayer at the third serial virus passage was scraped and pelleted at low speed centrifugation (500 x g ), and the dsRNA was sequentially extracted using one treatment with sodium acetate-saturated phenol and one with Tris-saturated phenol. This emulsion was centrifuged in a microcentrifuge for 10 minutes at 14,000 x g, and the upper aqueous layer was removed. The dsRNA was precipitated from the aqueous layer by adding 1/2 volume of ammonium acetate (7.5 M) and 3 volumes of ethanol. The resulting solution was mixed by inverting the tube several times then incubated for 30 minutes at -70 C. After incubation, the tubes were spun at 14,000 x g for 30 minutes in a microcentrifuge. The resultant pellet was air dried at room temperature and resuspended in 35 µ1 distilled water and 9 µ1 sample application buffer (SAB). As reference, dsRNA was extracted from the prototype avian reoviruses by the same procedure. Specimens were analyzed by species. Thus, specimens from one species at a time were loaded onto discontinuous polyacrylamide gels (5% stacking gel/10% resolving gel) containing sodium dodecyl sulfate (SDS) in Tris-glycine-SDS buffer and electrophoresed for 30 minutes at 16 mA/gel followed by 20 hours at 24 mA/gel. The gels were stained by the silver

nitrate method described elsewhere.9 Briefly, the gels were soaked in a solution of 10% ethanol and 1% acetic acid on a platform shaker for 15 minutes. The washing solution was discarded and replaced by a 1.9 g/liter silver nitrate solution. The gels were then soaked in the staining solution for 30 minutes. The gels were rinsed rapidly 3 times with doubledistilled water to remove the excess silver and then placed in a reducing solution containing 0.1 M NaBH4, 10 ml/liter HCHO (37%), and 10 mM NaOH until dsRNA bands were visualized. To stop the staining reaction, the gels were placed into a solution of 5% glacial acetic acid. The length of migration and variability of each virus genome segment by species was determined by calculating the mean and standard deviation of the distance between the upper line of each separating gel and the respective genomic segment. For comparative analysis, mean values and their respective standard deviations were arranged as a graph. Reoviruses isolated and propagated in primary cell culture were identified morphologically by EM (virions ranged in size from 65 to 75 nm-data not shown) and by their electropherotype ( 10 bands arranged by size: L1, L2, L3; M1, M2, M3; S1, S2, S3, S4). The genomic profiles of the species prototype reoviral strains electrophoresed on a gel are presented in Fig. 1. Greater heterogeneity was seen with the turkey reovirus prototype strain within the highest and lowest molecular weight segments when compared with the chicken reovirus prototype strains. There were no major differences in the migration patterns of field isolates from a canary and a cockatiel. Greater heterogeneity of the migration pattern was seen in the 60 turkey reoviruses examined as compared with the pattern of the 8 chicken reovirus electropherotypes (Figs. 2, 3). The distance of band migration was measured after simultaneous electrophoresis of standard reoviruses with field isolates. Mean values and standard deviations were calculated. The genomic segments S1, S2, S3, and S4 of turkeys and chickens showed higher heterogeneity in their migration pattern (Fig. 4). A characteristic migration pattern by species could not be determined because of the high polymorphism existing in the mobility patterns of the field reoviruses from

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Brief communications


quency in these species due to differences in management and population densities when compared with those of chicken and turkey flocks. Although coelectrophoresis of the dsRNA extracted from the canary and cockatiel reoviruses was not done, it would provide more data on the similarities of the migration profiles of these 2 reovirus isolates.

Sources and manufacturers a. American Type Culture Collection, Rockville, MD. b. National Veterinary Services Laboratory, Ames, IA. c. Specific Pathogen-Free Avian Supply, Storrs, CT. Figure 4. Electropherotype bands of avian reovirus genomes by species with their average segment mobility and standard deviation.


l r*

chickens and turkeys. Although the specimens from the canary and the cockatiel were processed separately and the virus isolation time was different, these two reovirus field isolates had similar electropherotypes which were different from those of chicken and turkey reoviruses (Fig. 1). The RNA extraction methods that have been useful for the differentiation of other dsRNA viruses have been equivocal in determining the species type of avian reoviruses, emphasizing the genetic diversity present among isolates of avian reoviruses. 5 However, these techniques were most valuable for the diagnostic detection of the presence of these viruses in tissues and for epidemiological studies.4,5,8 Differences in migration patterns of genomic segments were observed in individual reovirus isolates; however, the high polymorphism of the genomes of reoviruses of chickens and turkeys prevents the establishment of consistent migration patterns to determine interspecies differences. The low variability observed within the higher molecular weight segments of the field isolates is probably due to the structural stability of these segments and to their slow migration during the separation of the segments. The use of longer gels and an extended time for electrophoresis of the dsRNA may provide further information on the molecular stability associated with heavy genomic segments. Striking similarities between the genomic profiles of the canary and the cockatiel reoviral isolates may reflect a low probability of recombination fre-

1. Femandez-Larson RP: 1988, Development of reassortant strains of avian reovirus for use in live vaccines and examination of genomic RNA field isolates. Diss Abstr Int B Sci Eng 49(3):631. 2. Gouvea VS, Schnitzer TJ: 1982, Polymorphism of the genomic RNAs among the avian reoviruses. J Gen Virol 6187-91. 3. Graham DL: 1987, Characterization of a reo-like virus and its isolation from and pathogenicity for parrots. Avian Dis 31:411419. 4. Huang DD, Nugent MA, Rosenberger JK, et al.: 1987, Association of avian reovirus M and S genes with viral behavior in vivo. I. Viral characterization. Avian Dis 31:438-445. 5. Huang DD, Nugent MA, Rosenberger JK, et al.: 1987, Association of avian reovirus M and S genes with viral behavior in vivo. II. Viral pathogenicity. Avian Dis 31:446-454. 6. Nwajei BNC, Afaleq A, Jones RC: 1988, Comparison of chick embryo liver and Vero cell cultures for the isolation and growth of avian reoviruses. Avian Pathol 17:759-766. 7. Olson NO: 1984, Reovirus infections. In: Diseases of poultry, ed. Hofstad MS, Barnes JH, Calneck BW, et al., 8th ed., pp. 560-566. Iowa State University Press, Ames, IA. 8. Saif LJ, Theil KW: 1990, Viral diarrheas of man and animals. CDC Press, Boca Raton, FL, pp. 65-128. 9. Sammons DW, Lonnie DA, Nishzawa EE: 1981, Ultrasensitive silver-based color staining of polypeptides in polyacrylamide gels. Electrophoresis 2:135-141. 10. Spandidos DA, Graham AF: 1976, Physical and chemical characterization of an avian reovirus. J Virol 19:968-976. 11. Wilson RB, Holscher M, Hodges JR, Thomas S: 1985, Necrotizing hepatitis associated with a reo-like virus infection in a parrot. Avian Dis 29:568-571.

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Interspecies polymorphism of double-stranded RNA extracted from reoviruses of turkeys and chickens.

Brief communications 74 rus DNAs at different degrees of stringency. Intervirol 28:114121. 17. O’Banion MK, Sundberg JP, Shima AL, Reichmann ME: 198...
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