APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1978, p. 1206-1210 0099-2240/78/0035-1206$02.00/0 Copyright © 1978 American Society for Microbiology

Vol. 35, No. 6

Printed in U.S.A.

NOTES Restriction Endonuclease Analysis to Distinguish Two Closely Related Nuclear Polyhedrosis Viruses: Autographa californica MNPV and Trichoplusia ni MNPVt LOIS K. MILLER* AND KATHERINE P. DAWES

Department of Bacteriology and Biochemistry, The University of Idaho, Moscow, Idaho 83843

Received for publication 31 January 1978

Restriction endonuclease fragment patterns of the deoxyribonucleic acid geof Autographa californica nuclear polyhedrosis virus (multiply embedded type) and Trichoplusia ni nuclear polyhedrosis virus (multiply embedded type) demonstrate that the two viruses are distinct but closely related variants. nomes

Nuclear polyhedrosis viruses (NPVs) are identification of NPVs (7). The NPVs have doumembers of the family Baculoviridae and have ble-stranded circular deoxyribonucleic acid considerable potential as biological insecticides. (DNA) genomes of reportedly 50 x 106 to 100 NPVs infecting the class Insecta generally have x 106 daltons (5, 7, 9, 11, 12). REN digestion of a narrow host range which is frequently limited the NPV DNA genomes followed by gel electroto species within a single genus, although some phoretic separation of the digestion products NPVs have a broader range which extends to provide easily distinguishable fragment patterns different genera within a single family. As a for the identification of the Heliothis zea SNPV, result, the baculoviruses are frequently named the Orgyia pseudotsugata MNPV, and the A. after the host from which they were isolated. californica MNPV (7). These viruses have Hence, Autographa californica MNPV and Tri- markedly different host ranges and markedly choplusia ni MNPV were originally isolated different REN fragment patterns. To determine if the A. californica MNPV and from A. californica and T. ni insects, respectively. Both insects are members of the order T. ni MNPV can be distinguished by REN analLepidoptera, subfamily Plusiinae in the family ysis, both MNPVs were propagated in T. ni larvae. Previous studies demonstrated that limNoctuidae. The A. californica MNPV and T. ni MNPV ited passage of A. californica MNPV through have been used extensively as experimental the T. ni host did not affect REN fragment NPV model systems, and precise knowledge con- patterns of the viral DNA (7). The viruses were cerning their relationship to each other is very purified from diseased T. ni larvae by a series of apropos at this time. The A. californica MNPV steps including centrifugation through a linear and T. ni MNPV are known to be closely related sucrose gradient (7). The polyhedral occlusion viruses, as judged by the similarity of their host bodies were then disrupted with 0.05 M Na2CO3, range (3, 6) and serological properties (personal and the viral DNAs were purified by phenol communication, G. Tompkins and J. Vaughn, extraction as previously described (7). The purU.S. Department of Agriculture, Agricultural ified viral DNAs were digested with appropriate Research Service, Beltsville, Md.). Differences RENs, and the resulting DNA fragments were between the two viruses, however, were detected fractionated by gel electrophoresis on 0.7% again peptide maps of the polyhedral proteins (2) rose gels (7) in B buffer. [B buffer contains 10.8 and in macrophage migration inhibition assays g of tris(hydroxymethyl)aminomethane (Tris) (1). An S or M preceding the "NPV" indicates base, 0.93 g ethylenediaminetetraacetic acid-sowhether the virions within the polyhedral occlu- dium salt (EDTA), and 5.5 g of boric acid in 1 sion body have one or more than one nucleocap- liter of water, pH 8.3.] sid per envelope, respectively. Digestion of the two viral DNAs with BamHI We have demonstrated the usefulness of re- REN resulted in identical fragment patterns as striction endonuclease (REN) analysis for the shown in Fig. 1. This result suggests that the T. t Research paper no. 77516, Idaho Agricultural Research ni MNPV and A. californica MNPV are very Station. closely related. Since the largest of these BamHI 1206

VOL. 35, 1978

NOTES

B

am

A FIG.

Fragments

Ac

fragments is approximately 59 x 106 daltons (Miller and Dawes, unpublished data from 0.2% agarose gels), RENs which produce a greater number of smaller fragments were used for finer structural analysis. Digestion of the two viral DNAs with SailI REN resulted in similar but nonidentical fragment patterns for T. ni MNPV and A. californica MNPV as shown in Fig. 2. Although the majority of fragments of T. ni MNPV matched with fragments of the A. californica MNPV, the largest fragment of A. californica MNPV (approximately 8.5 x 106 daltons) was missing in the T. ni MNPV pattem, whereas two smaller fragments appeared exclusively in the T. ni MNPV pattem. One of these fragments comigrated with the second largest fragment of the T. ni MNPV pattem and was visible only as greater staining intensity at this position. This fragment was approximately 4.5 x 106 daltons as determined by comparing mobility of the fragment in 0.7% agarose gels with the mobility of ADNA fragments of known size. The other smaller fragment, present only in the T. ni pattem, was clearly visible under the fifth largest fragment. The molecular weight of this fragment was approximately 4.0 x 106. These results suggest that the T. ni MNPV has an additional SalI cleavage site in the region of DNA corresponding to the largest (8.5 x 106 daltons) fragment of A. californica MNPV. Hence two smaller fragments, with additive molecular weights equal to 8.5 x 106, were observed in the T. ni MNPV pattem. Another minor difference between the T. ni MNPV and A. californica MNPV SailI patterns was apparent upon close inspection. A fragment of approximately 1.2 x 106 daltons was present in the A. californica MNPV pattern but was absent in the T. ni MNPV pattern. Instead, the T. ni MNPV had a 1.1 x 106-dalton fragment not present in the A. californica MNPV pattern. A fragment of 0.1 x 106 daltons would be difficult to detect in the T. ni NPV fragment pattern. Fragment patterns of the T. ni MNPV and A. californica MNPV DNA genomes produced by Hind III and EcoRI REN digestion are shown in Fig. 3. The differences between the A. californica MNPV and T. ni MNPV patterns are char-

Tn

californica MNPV and T.

of A. niMNPVDNAs produced by 1.

HI

BamHIdigestion.

1207

Sam-

pies of viral DNA were digested in 0.01 M Tris (pH 7.6), 0.05 M NaCI, 0.01 M MgCl2, and 1 mM dithiothreitol at 37°C for 3 h with BamHI REN. Approximately 2 pug of digested viral DNA in 30-,ul volumes were loaded into slots on 0. 7% slab agarose gels and electrophoresed as previously described (7). The slab gels were then stained with ethidium bromide and photographed under ultraviolet light. The BamHI fragment patterns for lambda bacteriophage, A. californica MNPV, and T. ni MNPVDNAs are denoted as A, Ac, and Tn, respectively.

ENVIRON. MICROBIOL. NOTES NOTES ~~~~~~~~~~~~APPL. 1208 1208

S alI

13.8-~b

.b

00-

40

.lk

op-

12ab

01-

12 '1.

'A Ac Tn FIG. 2. Fragments of A. californica MNPV and T. ni MNPV DNAs produced by Sail digestion. Viral DNAs were digested with Sail restriction endonuclease and electrophoresed as described in Fig. 1. The Sail fragment patterns for lambda bacteriophage, A. californica MNPV, and T. ni MNPV DNAs are denoted as A, Ac, and Tn, respectively. Arrows on the right point to alterations in the fragment patterns of the two MNPVs. The numbers to the right are the approximate molecular weights (xJO -6) of the fragments specifically referred to in the text. The arrows on the left indicate the migration positions of EcoRI fragments of ADNA used as molecular weight standards. The AEcoRI fragment molecular weights (XlOb') are given by the numbers next to the arrows. acterized

by

ments in

one

the

pearance of other tern

or

vice

appearance

pattern

with

fragments

versa.

of distinct

congomitant

fragdisap-

in the second pat-

In both the Hind III and

EcoRI patterns, the variations observed

simply explained by alterations

in

a

can

be

few REN

recognition sites. The fragment patterns are sufficiently similar, however, to indicate a close evolutionary relationship between the two viruses.

The use of REN analysis to define evolutionary relationships must be applied with caution

VOL. 35, 1978

NOTES

Hind d

1209

E c o RT

14.1> 6.1

>

4.1->

1.5->_

Ac Tn

NAc TnA

FIG. 3. Fragptents of A. californica MNPV and T. ni MNPV DNAs produced by EcoRI and HindIII digestions. Viral DNAs were digested with EcoRI REN in 0.01 M Tris (pH 7.6), 0.01 M MgCl2, 0.10 M NaCl, and 1 mM dithiothreitol for 3 h at 37°C. Another set of viral DNA samples was digested with HindIII REN in 0.01 M Tris (pH 7.6), 0.05 M NaCl, 0.01 M MgCl2, and 1 mM dithiothreitol for 3 h at 37°C. Electrophoresis was through 0. 7% agarose slab gels as in Fig. 1. The enzyme used is denoted at the top of the lanes (EcoRI or HindIII), and the viral DNA is denoted at the bottom of each lane (lambda, A; A. californica MNPV, Ac; T. ni MNPV, Tn). The arrows and numbers on the left refer to the positions and molecular weights of the AHindIII fragment standards.

and can be utilized only in cases of very close relationships (variants). With regard to T. ni MNPV and A. californica MNPV, the close relationship is clear from the REN patterns,

since digestion with a number of RENs produced similar (although in most cases not identical) fragment patterns for the two viruses, and the variations in the fragment patterns are simply

12 10

NOTES

accounted for by loss or gain of a few REN recognition sites in each case. It is interesting to note that the variations, observed in the SalI, Hind III, and EcoRI fragment patterns, occurred at REN sites separated by at least 4 x 106 daltons (see the SailI patterns) and up to 12 x 106 daltons (see the Hind III patterns). The variations, however, did not apparently extend through the entire length of these DNA regions, since REN sites between the altered REN sites were left unchanged. As an example of this, we note that in Fig. 2, the DNA region within the 8.5 x 106-dalton Sal I fragment of the A. californica MNPV was altered in the T. ni MNPV such that an additional Sal I recognition site was generated. This alteration was not so extensive that it affected the SailI recognition sites at the ends of the 8.5 x 106-dalton fragment. Nevertheless, there is an additional difference detected between the A. californica MNPV and T. ni MNPV fragment patterns (the 1.2 x 106- and 1.1 X 106-dalton fragments) which must be at least 4.1 x 106 daltons from the other altered Sall cleavage site. The REN fragment patterns of the A. californica MNPV and T. ni MNPV DNA genomes indicate that these two viruses are closely related but differ enough to be considered variants. Since there is no indication that they differ in host range or differ markedly in phenotype, it is questionable whether these viruses should be considered different viruses and whether they deserve distinct names. We note that variants of other animal viruses such as polyoma viruses A3 and P16 are simply designated by alphabetical or numerical references (8). Such variants can actually differ both genotypically (additional REN sites and/or altered fragment sites) and pheno-

typically (plaque morphology, hemagglutination properties, etc.) without considering them different viruses(8). Since over 300 baculoviruses have been reported in the literature, there is need to begin a systematic classification of the viruses themselves. We also note that from an applied perspective relating to the use of these viruses as biological pesticides, the knowledge that two viruses are variants suggests that efforts toward registration of only one of the viruses should be pursued. The DNA fragment patterns of A. californica MNPV and T. ni MNPV genomes reflect their

APPL. ENVIRON. MICROBIOL.

close evolutionary relationship while providing a method for easily distinguishing the two viruses. As an identification technique, REN analysis may excel serological techniques in cases of close evolutionary relationships as exemplified in the research reported herein. ACKNOWLEDGMENTS We thank James Vaughn (U.S. Department of AgricultureAgricultural Research Service [USDA-ARS], Beltsville, Md.) for supplying the original T. ni MNPV in the form of hemolymph from diseased Spodoptera frugiperda larvae, Marion Bell (USDA-ARS, Phoenix, Ariz.) for supplying the original A. californica MNPV in the form of diseased larvae, and Brad Harmon for his technical assistance in rearing T. ni as hosts for both the T. ni MNPV and the A. californica MNPV. This research was supported by Public Health Service grant AI-01567 from the National Institutes of HealthEnvironmental Health Sciences.

LITERATURE CITED 1. Benton, C. V., C. F. Reicheldefer, and F. M. Hetrick. 1973. Differentiation of T. ni MEV and A. californica MEV by macrophage migration inhibition tests. J. Invertebr. Pathol. 22:42-49. 2. Cibulsky, R. J., J. D. Harper, and R. T. Gadauskas. 1977. Biochemical comparison of polyhedral protein from five NPVs infecting plusiine larvae (Lepidoptera noctuidae). J. Invertebr. Pathol. 29:182-191. 3. Harper, J. D. 1976. Cross-infectivity of six NPV isolates to plusiine hosts. J. Invertebr. Pathol. 27:275-277. 4. Harrap, K. A., C. C. Payne, and J. S. Robertson. 1977. The properties of three baculoviruses from closely related hosts. Virology 79:14-31. 5. Kelly, D. C. 1977. The DNA contained by NPVs isolated from four Spodoptera Sp. (Lepidoptera noctuidae): genome size and homology assessed by DNA reassociation kinetics. Virology 76:468-471. 6. Knudson, D. L., and S. M. Buckley. 1977. Invertebrate cell culture methods for the study of invertebrate-associated animal viruses. Methods Virol. 6:323-391. 7. Miller, L. K., and K. P. Dawes. 1978. Restriction endonuclease analysis for the identification of baculovirus pesticides. Appl. Environ. Microbiol. 35:411-421. 8. Miller, L. K., and M. Fried. 1975. Construction of infectious polyoma hybrid genomes in vitro. Nature (London) 259:598-601. 9. Rohrmann, G. F., J. W. Carnegie, M. E. Martignoni, and G. S. Beaudreau. 1977. Characterization of the genome of the nuclear polyhedrosis bundle virus pathogenic for 0. pseudotsugata. Virology 79:334-338. 19. Scharnhorst, D. W., K. L. Saving, S. B. Vuturo, P. H. Cooke, and R. F. Weaver. 1977. Structural studies on the polyhedral occlusion bodies, virions, and DNA of the nuclear polyhedrosis virus of the cotton bollworm Heliothis zea. J. Virol. 21:292-300. 11. Summers, M. D. 1977. Deoxyribonucleic acids of baculoviruses, p. 233-246. In J. A. Romberger (ed.), Virology in agriculture. Universe Books, New York. 12. Summers, M. D., and D. L. Anderson. 1973. Characterization of NPV DNAs. J. Virol. 12:1336-1346.

Restriction endonuclease analysis to distinguish two closely related nuclear polyhedrosis viruses: Autographa californica MNPV and Trichoplusia ni MNPV.

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1978, p. 1206-1210 0099-2240/78/0035-1206$02.00/0 Copyright © 1978 American Society for Microbiology Vol...
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