Characteristics of Neonatal Calf Diarrhea Virus Ribonucleic Acid B. B. Barnett, L. N. Egbert and R. S. Spendlove* ABSTRACT The nucleic acids of neonatal calf diarrhea virus were characterized by isopycnic centrifugation in Cs2SO4 electron microscopy, ultraviolet absorbance temperature profiles and polyacrylamide gel electrophoresis. These studies indicated that the neonatal calf diarrhea virus genome consists of 11 segments of double stranded RNA with a total molecular weight of 10.75 million daltons.

R1JSUME Cette experience visait 'a caracteriser les acides nucleiques du virus de la diarrhee neonatale du veau. A cette fin, les auteurs utiliserent la centrifugation isopycnique au Cs2SO4, la microscopie electronique, les profils de denaturation thermique et l'electrophoriese sur gel de polyacrylamide. Les resultats reve'lerent que le genome de ce virus consiste en 11 segments d'ARN a double brin dont le poids moleculaire total atteint 10.75 millions de daltons.

et al (18) have presented similar data for NCDV. Rodger et al (21) suggested that both NCDV and human infant diarrhea virus (reoviruslike agent) should be classified in a genus separate from the reoviruses and the orbiviruses but within the family Reoviridae. Newman et al (18), who use the term calf rotavirus in place of NCDV, determined the molecular weights for the 11 or 12 segments that they observed and reported a possible coding relationship between the RNA segments and the viral polypeptides. Here we present the results of electron microscopy (EM) studies of the NCDV genome segments including electron micrographs of multiple segments being extruded from individual NCDV particles. Since there is considerable interest in the size distribution of the NCDV genome segments (12, 22) we present data correlating EM contour length data with polyacrylamide gel electrophoresis (PAGE) molecular weight data. We also report the results of studies on the buoyant density and rigidity of the NCDV nucleic acids.

MATERIALS AND METHODS

INTRODUCTION VIRUSES Early physicochemical characterization of neonatal calf diarrhea virus (NCDV) indicated an RNA genome which was possibly double stranded (27, 28). We reported that the NCDV genome consists of 11 segments of double-stranded RNA (3,4). Subsequently, Rodger et al (21) and Newman *Department of Biology, UMC 55, Utah State Logan, Utah 84322.

Submitted June 26, 1976.

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University,

The Lang strain of reovirus type 1 was propagated in Madin-Darby bovine kidney cells (American type culture collection, Rockville, Maryland). Cell culture fluids containing NCDV were a gift of Norden Laboratories, Lincoln, Nebraska. Both reovirus and NCDV were purified by differential centrifugation followed by two cycles of isopycnic banding in CsCl density gradients. NCDV was assayed by an immunofluorescent cell assay (5).

Can. J. comp. Med.

ISOLATION OF VIRAL NUCLEIC ACIDS

ELECTRON MICROSCOPY

A suspension of purified virus in 0.15 M NaCl, 0.015 M sodium citrate, pH 6.8 (SSC), was made 1% in sodium dodecyl sulfate (SDS), incubated 15 min at 25°C then extracted three times with equal volumes of redistilled phenol. Residual phenol was removed from the aqueous phase by three ether extractions. Nitrogen was gently bubbled through the aqueous phase to remove the remaining ether. The last traces of phenol and ether were removed by extensive dialysis against 0.1 x SSC.

Electron microscopy of the viral nucleic acids was performed by the protein monolayer technique as described by Davis et al (8). The nucleic acids were released from the purified virus suspension by spreading on a 4M urea subphase (25). The electron microscope magnification was calibrated with a cross-ruled grating replica (54,800 lines/inch, Ted Pella Co.). The RNA strands were measured by projecting the electron micrographs, tracing the filaments onto paper and then measuring the tracings with a map measure. The tracings were at a final magnification of 200,000x.

ISOPYCNIC BANDING OF NCDV NUCLEIC ACID IN Cs2SO4

The method of Szybalski (24) was used to determine the buoyant density of the viral nucleic acid in CS2SO4. Two samples of the NCDV nucleic acid in 0.1 x SSC were adjusted to densities of 1.566 g/ml and 1.622 g/ml respectively with neutralized saturated CS2SO4. The densities of the solutions were determined by refractometry (24) using a Bausch and Lomb Abbe - 3L - refractometer. The samples were centrifuged at 44,770 rpm in an An-D rotor in a Spinco Model E analytical ultracentrifuge. After 72 hrs photographs of the nucleic acid bands were taken with the ultraviolet absorption optical system of the Model E. Densitometric tracings of these films were made using a modified Gilford Model 2410 linear transport (9) attached to a Gilford Model 2000 spectrophotometer.

ULTRAVIOLET ABSORBANCE TEMPERATURE PROFILE

The thermal denaturation studies of NCDV RNA were conducted as described by Mandel and Marmur (16). The samples of NCDV RNA were extensively dialyzed against 0.1 x SSC. The RNA solution was placed in a tightly sealed quartz cuvette and the thermal denaturation profile was determined using a Gilford automatic melting curve apparatus. The temperature increase was programmed at 0.5°C per min. The absorbance measurements at a wavelength of 260 nm were corrected for solvent expansion.

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POLYACRYLAMIDE GEL ELECTROPHORESIS

(PAGE) The PAGE and subsequent staining of viral RNA was performed as described by Adesnik (1) using recrystallized acrylamide and N, N' - methyl-enebisacrylamide (15). The viral RNA was prepared by dissociating purified virus at 50°C for 90 min in a 2% SDS, 0.1% 2-mercaptoethanol, 10% sucrose solution. The gels were cast in 0.6 cm x 10.0 cm quartz tubes. The distribution of the RNA on the polyacrylamide gels was determined by densitometry. Gels were scanned at a wavelength of 260 nm without staining or at 540 nm following staining with toluidine blue 0. The molecular weights of the NCDV genome segments were determined by comparing their electrophoretic mobilities to those of the genome segments of reovirus type 1 Lang (29). The densitometric peaks corresponding to RNA segments were traced on homogeneous paper, cut out and weighed. These weights were then divided by the molecular weights of the respective RNA segments to obtain the molar ratios. The number of segments represented in each peak was determined by dividing the molar ratios by the molar ratio for the most distinct single peak.

RESULTS VIRUS PURIFICATION An electron micrograph of the purified NCDV is shown in Fig. 1. It was noted that

47

the infectivity of the NCDV preparations dropped over 100-fold after banding in CsCl. All fractions were titered by the immunofluorescent cell assay of Barnett et al (5).

CS2SO4 A sample of purified NCDV nucleic acid was analyzed by isopycnic centrifugation in CS2SO4 density gradients. The bouyant density of the NCDV nucleic acid in Cs2S04 was calculated to be 1.61 g/ml.

THERMAL DENATURATION PROFILES

The midpoint (Tm) of the thermal transition in duplicate thermal denaturation experiments was 75°C. The hyperchromicity was 35 % in one experiment and 34 % in the other.

Fig. 1. Electron

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micrograph

ELECTRON MICROSCOPY OF VIRAL NUCLEIC ACIDS When either NCDV or reovirus was spread on 4M urea the viral RNA was gradually released. When NCDV or reovirus were spread on a nondissociating subphase (0.25M ammonium acetate) no nucleic acid filaments were visible, indicating a viral origin for the nucleic acids. Multiple RNA segments were often released from a single NCDV or reovirus particle (Fig. 2). The contour length distribution of ureareleased viral RNA is illustrated for reovirus and for NCDV in Figs. 3 and 4 respectively. The reovirus distribution exhibits three major peaks corresponding to the small, medium and large size classes of reovirus dsRNA. The peaks in the NCDV RNA contour length distribution profile do not form well defined groups as do those for reovirus RNA. The rigidity of nucleic acid molecules has been cited as indicative of their strandedness (13). Double-stranded nucleic

of purified NCDV, stained with 0.5% uranyl acetate, marker represents 100 nm.

Can. J. comp. Med.

Fig. tein The ker

2. Electron micrograph of NCDV spread on 4M urea. NCDV in 1M ammonium acetate was spread by the promonolayer technique on 4M urea which denatured the capsid proteins, allowing the NCDV RNA to be released. monolayer was transferred to grids, stained with uranyl acetate and rotary shadowed with platinum. The marrepresents 500 nm.

acids are more rigid than single-stranded nucleic acids. The rigidity of reovirus dsRNA and of NCDV RNA was analyzed by comparing the end-to-end distance to the contour length for individual RNA molecules. The data in Fig. 5 indicate that the rigidity of the NCDV RNA was essentially identical to that of reovirus dsRNA.

ELECTROPHORESIS OF VIRAL RNA The RNA genome segments of both NCDV and reovirus were fractionated by polyacrylamide gel electrophoresis using several different acrylamide concentrations. Densitometric tracings of 7.5% gels on which NCDV and reovirus RNA were run in parallel are illustrated in Fig. 6. The NCDV RNA was fractionated into nine bands. The molecular weights of the NCDV RNA segments were determined by comparing their relative electrophoretic mobilities to those of the reovirus genome segments. The molar ratios of the bands obtained by PAGE were determined by tracing, cutting out and weighing the peaks from the densitometric tracings. Data was collected by scanning the gels at 260 nm and also by scanning at 540 nm following staining. In the case of NCDV RNA, each peak accounts for one genome segment with the exception that peak number seven rep-

Volume 42 - January, 1978

resents three segments. Thus the intact NCDV virion contains eleven genome segments. The molecular weights for the NCDV genome segments were determined by averaging the data from experiments using 2.6%, 5.0% and 7.5% gels. The molecular weights of the eleven segments give a total of 10.75 x 106 daltons for the molecular weight of the NCDV genome. These data are listed along with the corresponding data for other diplornaviruses in Table I.

DISCUSSION During the purification of NCDV there was over a one-hundredfold loss of infectivity when the virus was exposed to CsCl. Similar observations have been reported for bluetongue virus (17). It has been reported that both the morphology and polypeptide composition of bluetongue virus is altered following exposure to CsCl (26). Welch (27) reported that the morphology of NCDV was not altered by centrifugation in CsCl gradients. Welch did not mention the effect of CsCl on NCDV infectivity. Bridger and Woode (7) have characterized

49

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CONTOUR LENGTH (Mm Fig. 3. Reovirus type 1 Lang RNA cont length tribution. Reovirus was spread by the prcotein monolayer technique on 4M urea. The virus-protein monolayer was transferred to grids, stained with uran iyl acetate and rotary shadowed with platinum. ElectrcDn micrographs (x 8,000) of the resulting RNA strands were traced and measured (final magnificati( in of 200m000). Only RNA molecules with both ends visihibl were measured. 351 molecules were measured.

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two NCDV particle types which differ from each other in density, infectivity and the presence or absence of an outer capsid layer. Bridger and Woode (7) reported no loss of infectivity when NCDV was exposed to CsCl. They did report that the incomplete particle was approximately a thousandfold less infectious than the complete particle. Two possibilities to account for the drop in infectivity in our studies are that some of the intact particles may have been converted to the less infective incomplete particles or that the purification step on CsCl may have selected for the incomplete particles. However, examination of Fig. 1 reveals the purified NCDV used in our study closely resembles the 66 nm complete particle described by Bridger and Woode (7). Szybalski (24) has tabulated the buoyant densities in CS2SO4 for a variety of nucleic acids. The buoyant densities of DNA's range from 1.42 to 1.46 g/ml, singlestranded RNA's from 1.60-1.69 g/ml and dsRNA's from 1.57-1.65 g/ml. Thus the buoyant density of a nucleic acid in CS2SO4 is a unique method for differentiating be-

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50

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CONTOUR LENGTH (pm) Fig. 5. Comparison of rigidity of NCDV RNA segments and reovirus type 1 Lang double-stranded RNA segments. The solid line represents the relation shown by a rigid rod in a two-dimensional system (straight line with a slope of two) the dashed line is the relation shown by the double-stranded RNA from reovirus. Each data point represents the average of measurements from 20 individual RNA molecules. The RNA was spread by the protein monolayer technique on a 4M urea subphase. The contour lengths and end-to-end distances were measured from tracings of electron micrographs at a magnification of 200,000. Logarithmic plot.

Can. J. comp. Med.

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Fig. 6. Ultraviolet optical density profiles of 7.5% polya crylamide gels with fractionated viral RNA segments. Approximately 20 qg of RNA was electrophoresed for 20 hr at 2.5 mA/gel (9 cm x 0.6 cm cylindrical gels). The gel was then scanned at a wavelength of 260 nm. Direction of migration was left to right. Upper tracing is NCDV RNA; lower profile is that of Reovirus Type 1 Lang RNA. Profiles are properly aligned for comparison of electro-

phoretic mobilities.

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51

tween DNA and RNA. Shatkin (26) determined the buoyant density of reovirus dsRNA in CS1SO4 to be 1.61 g/ml. We determined the buoyant density of the NCDV

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CONTOUR LENGTH (pm) Fig. 7. Comparison of the NCDV RNA contour length

distribution as determined by electron microscopy to the contour lengths calculated from PAGE experiments. The histogram is the contour length distribution determined by electron microscopy of NCDV RNA spread by a protein monolayer technique. The arrows indicate the contour lengths calculated for the NCDV RNA segments using the PAGE data (assuming 2.3 x 106 daltons4/m). The number above each arrow is the NCDV genome segment number.

nucleic acid to be 1.61 g/ml. This is in agreement with investigations which have shown by chemical means and through the use of metabolic inhibitors that the NCDV genome is RNA (21, 27). Our thermal denaturation data are indicative of double stranded nucleic acid. There is some variation in the reported thermal denaturation data (all in 0.1 x SSC): present study Tm = 75°C, hyperchromicity = 35%; Welch and Thompson (28) Tm = 81°C, hyperchromicity = 27% and Rodger et al (21) Tm = 78°C, hyperchromicity = 45%. The rigidity of the NCDV RNA as measured by the relationship between the end-to-end distance and contour length also indicated doublestrandedness. Similar rigidity experiments have been reported for reovirus dsRNA and wound tumor virus dsRNA (13). By converting the molecular weights for the NCDV RNA segments as determined by PAGE to contour lengths, the PAGE data and electron microscopy data can be compared. The reported molecular weight per unit length for dsRNA varies among investigators: 2.1 x 106 daltons/gm (6), 2.3 x 106 daltons/,um (14), 2.39 x 106 daltons/gm (11) and 2.6 x 10' daltons/gum (2). Using 2.3 x 106 daltons/,Lm for dsRNA, the moleular weights for the NCDV segments (Table I) were converted to contour lengths to yield: 0.10 jum, 0.13 p1m, 0.22 ,um (three segments of this length), 0.33 ,um, 0.41 ,gm, 0.64 um, 0.71 ,um, 0.75 ,um, and 0.95

TABLE I. Molecular Weights of Genome Segments of Selected Diplornaviruses (x 106 daltons)

NCDVS 2.18 1.73 1.64 1.48 0.94 0.77 0.50 0.50 0.50 0.29 0.22

± .06 i .07 .06 ± .06 ± .03 ± .02 .03 i .03 i .03 ± .03 .02

Reovirusb type 1

Silkwormc polyhedrosis virus

Riced dwarf virus

2.50 2.37 2.30 1.58 1.51 1.45 0.82 0.73 0.67 0.62

2.55 2.42 2.32 2.03 1.82 1.12 0.84 0.62 0.56 0.35

3.10 2.50 2.25 1.90 1.80 1.15 1.13 0.86 0.78 0.75 0.52 0.52 17.26

14.63 14.55 Sum 10.75 PAGE using determinations four -Average of bWood and Streissle (29) cFujii et at (10) dReddy et al (19) eMartin and Zweerink (17) fRedolfi and Boccardo (20)

52

Bluetonguee virus

Maizef rough dwarf virus

2.73 2.04 1.94 1.29 1.12 1.07 0.61 0.55 0.49 0.28

2.58 2.33 2.24 2.06 1.73 1.42 1.21 1.19 1.11

12.12

15.87

Woundd tumor virus 2.90 2.48 2.25 1.80 1.80 1.20 1.15 0.90 0.63 0.61 0.60 0.35 16.67

Can. J. comp. Med.

,am. In Fig. 7 the contour lengths calculated

from the PAGE data are compared to the electron microscopy data. Kalica et al (12) have reported a contour length distribution for NCDV RNA based on measurements of 128 molecules, their data being roughly similar to that which we report here which is based on measurements of 1,112 molecules.

ACKNOWLEDGMENTS This investigation was supported by funds from the Utah State University office for Research and by the Utah Agriculture Experiment Station. One of the authors (BBB) was supported by an NIH predoctoral traineeship in genetics. This article is number 2018 from the Utah Agriculture Experiment Station. The authors thank Dr. E. P. Bass of Norden Laboratories for supplying the virus culture fluids.

REFERENCES 1. ADESNIK, M. Polyacrylamide gel electrophoresis of viral RNA. In Methods in Virology. K. Maramo-

rosch and H. Kiproski, Eds. Vol. 5. pp. 125-177. New York: Academic Press. 1971. 2. ARNOTT. S., M. H. F. WILKINS, W. FULLER and R. LANGRIDGE. Molecular and crystal structures of double helical RNA. J. Molec. Biol. 27: 535-548. 1967. 3. BARNETT, B. B., L. N. EGBERT and R. S. SPENDLOVE. Neonatal calf diarrhea virus (NCDV): Characterization of the genome by electron microscopy. Abstracts Annual Meet. Am. Soc. Microbiol. p. 230. 1975. 4. BARNETT, B. B., L. N. EGBERT and R. S. SPENDLOVE. Partial characterization of the neonatal calf diarrhea virus (reolike agent) genome. Abstracts Internat. Congress Virol, (3rd). p. 150. 1975. B. B., R. S. SPENDLOVE, M. W. PEBARNETT, 5. TERSON, L. Y. HSU, V. A. LASALLE and L. N. EGBERT. Imnmunofluorescent cell assay of neonatal calf diarrhea virus. Can. J. comp. Med. 39: 462-465. 1975. 6. BELLAMY, A. R., L. SHAPIRO. J. T. AUGUST and W. K. JOKLIK. Studies on reovirus RNA. I. Characterization of reovirus genome RNA. J. Molec. Biol. 29: 1-17. 1961. 7. BRIDGER, J. C. and G. N. WOODE. Characterization of two particle types of calf rotavirus. J. gen. Virol. 31: 245-250. 1976.

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8. DAVIS, R. W., M. SIMON and N. DAVIDSON. Electron microscope hetero-duplex methods for mapping regions of base sequence homology in nucleic acids. In Methods in Enzymology. L. Grossman and K. Moldave, Eds. Vol. 21. pp. 413428. New York: Academic Press. 1971. 9. EGBERT, L. N. Densitometry of analytical ultracentrifuge films utilizing a modified Gilford Linear Transport. Analyt. Biochem. 56: 1-9. 1973. 10. FUJII-KAWATA, I., K. MIURA and M. FUKE. Segments of genome of viruses containing double stranded ribonucleic acid. J. Molec. Biol. 51: 247253. 1970. 11. GOMATOS, P. J. and W. STOECKENIUS. Electron microscope studies on reovirus RNA. Proc. Natn. Acad. Sci. 52: 1449-1455. 1964. 12. KALICA, A. R., C. F. GARON, R. G. WYATT, C. A. MEBUS, D. H. VAN KIRK, R. M. CHANNOCK and A. Z. KAPIKIAN. Differentiation of human and calf reoviruslike agents associated with diarhhea using polyacrylamide gel electrophoresis of RNA. Virology 74: 86-92. 1976. 13. KLEINSCHMIDT, A. K., T. H. DUNNEBACKE, R. S. SPENDLOVE, F. L. SCHAFFER and R. F. WHITCOMB. Electron microscopy of RNA from reovirus and wound tumor virus. J. Molec. Biol. 10: 282-288. 1964. 14. LANGRIDGE, R. and P. J. GOMATOS. The structure of RNA. Science 141: 694-698. 1963. 15. LOENING, U. E. The fractionation of high molecular weight ribonucleic acid by polyacrylamide gel electrophoresis. Biochem. J. 102: 251-257. 1967. 16. MANDEL, M. and J. MARMUR. Use of ultraviolet absorbance-temperature profile for determining the guanine plus cytosine content of DNA. In Methods in Enzymology. L. Grossman and K. Moldave, Eds. Vol. 12-B. pp. 195-206. New York: Academic Press. 1968. 17. MARTIN, S. A. and H. J. ZWEERINK. Isolation and characterization of two types of bluetongue virus particles. Virology 50: 495-506. 1972. 18. NEWMAN, J. F. E., F. BROWN, J. C. BRIDGER and G. N. WOODE. Characterization of a rotavirus. Nature, Lond. 258: 631-633. 1975. 19. REDDY, D. V. R., I. KIMURA and L. M. BLACK. Co-electrophoresis of dsRNA from wound tumor and rice dwarf viruses. Virology 60: 293-296. 1974. 20. REDOLFI, P. and G. BOCCARDO. Fractionation of the double-stranded RNA of maize rough dwarf virus subviral particles. Virology 59: 319-322. 1974. 21. RODGER, S. M.. R. D. SCHNAGL and I. H. HOLMES. Biochemical and biophysical characteristics of diarrhea viruses of human and calf origin. J. Virol. 16: 1229-1235. 1975. 22. SCHNAGL, R. D. and I. H. HOLMES. Characteristics of the genome of human infantile enteritis virus (rotavirus). J. Virol. 19: 267-270. 1976. 23. SHATKIN, A. J. Inactivity of purified reovirus RNA as a template for E. coli polymerases in vitro. Proc. Natn. Acad. Sci. 54: 1721-1728. 1965. 24. SZYBALSKI, W. Use of cesuim sulfate for equilibrium density gradient centrifugation. In Methods in Enzymology. L. Grossman and K. Moldave, Eds. Vol. 12-B. pp. 330-360. New York: Academic Press. 1968. 25. VASQUEZ, C. and A. K. KLEINSCHMIDT. Electron microscopy of RNA strands released from individual reovirus particles. J. Molec. Biol. 34: 137-147. 1968. 26. VERWOERD, D. W., H. J. ELS, E. DE VILLIERS and H. HUISMANS. Structure of the bluetongue virus capsid. J. Virol. 10: 783-794. 1972. 27. WELCH, A. B. Purification, morphology and partial characterization of a reoviruslike agent associated with neonatal calf diarrhea. Can. J. comp. Med. 35: 195-202. 1971. 28. WELCH, A. B. and T. L. THOMPSON. Physicochemical characterization of a neonatal calf diarrhea virus. Can. J. comp. Med. 37: 295-301. 1973. 29. WOOD, H. A. and G. STREISSLE. Wound tumor virus: Purification and fractionation of the doublestranded ribonucleic acid. Virology 40: 329-334. 1970.

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Characteristics of neonatal calf diarrhea virus ribonucleic acid.

Characteristics of Neonatal Calf Diarrhea Virus Ribonucleic Acid B. B. Barnett, L. N. Egbert and R. S. Spendlove* ABSTRACT The nucleic acids of neonat...
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