Journal of Yirological Methods, 9 (1984) 227-235

Elsevier

JVM 00340

227

DIFFERENTIATION OF NEWCASTLE DISEASE VIRUS STRAINS BY ONE-

DIMENSIONAL PEPTIDE MAPPING

&A NAGY and B. LOMNICZI

Veterinary Medical Research fnstitute. Hungarian Academy of Sciences, P. 0. Box 18, H-1581 Budapest,

Hungary

(Accepted 2 July 1984)

One-dimensional peptide mapping was used for the differentiation of Newcastle disease virus (NDV)

strains. Virions were purified in one step, and digested with Szaphylococcus aureus V8 protease or

chymotryps~n without prior separation of their proteins. Peptides were separated by polyacrylamide gel

electrophoresis and stained with Coomassie blue. This method proved to be a simple, economic and

reproducible means of differentiating NDV strains.

Newcastle disease virus strain differentiation one-dimensional peptide mapping

INTRODUCTJON

Based upon pathogenicity for the chicken and chick embryo, Newcastle disease

virus (NDV) strains were assigned to three virulence groups, i.e. lentogenic (aviru-

lent), mesogenic (moderately virulent) and velogenic (highly virulent, Hanson and

Brandly, 1955; Waterson et al., 1967). Certain lentogenic strains can also be distin-

guished on the basis of their heat-stability (Hanson et al., 1949; McFerran and Nelson,

1971; Lomniczi, 1973, their adsorption to brain cells and elution from red blood cells

(Hanson et al., 1967; Spalatin et al., 1970). Irrespective of virulence, antigenic differ-

ences between strains were also demonstrated (Schloer, 1974; Avery and Niven, 1978;

Pennigton, 1978). Recently, using monoclonal antibodies, it has become possible to

establish at least 8 groups comprising strains with common epidemiolo~i~a1 character-

istics (Russell and Alexander, 1983).

NDV strains have also been compared on biochemical grounds by partial digestion

of isolated radioiabelled proteins with Staphylococcus aweus V8 protease followed by

one-dimensional peptide mapping (Nagai et al., 1980). Most recently RNA fingerprint-

ing has also been used for the differentiation of virulent NDV strains (McMillan and

Hanson, 1982).

In the present paper we report how NDV strains can be anaiysed by one-dimension-

al electrophoresis of peptides obtained by partial proteolysis (Cleveland et al., 1977).

Olhh-t)934/X4/$0?.00 8 1984 Elsevirr Science Publishers B.V

228

In this study non-radiolabelled whole virions were digested directly without previous

separation of the individual virion proteins. Virus was purified by one ultracentrifuga-

tion step and one run in polyacrylamide gel. The method is simple, rapid and

reproducible for strain differentiation, and in some cases even for positive identifica-

tion of certain strains.

MATERIALS AND METHODS

Virus strains The origin of the majority of the NDV strains was described earlier (Lomniczi,

1973). The ‘Dessau’ line of the LaSota strain was supplied by Dr. Heinicke (Institut

fur Impfstoffe, Dessau, G.D.R.), line ‘Biol’byDr.E.Kaleta(Klinikfi.irGefliigelkrank-

heiten der Justus-Liebig-Universitat, Giessen, F.R.G.). Clone-30 was obtained from

Intervet International B.V. (Boxmeer, The Netherlands), while NDV-38, NDV-39 and

NDV-40 were obtained from Dr. Zsuzsanna Mate (Veterinary Institute, Miskolc,

Hungary). The representative avian paramyxovirus (PMV) strains (PMV-2: Yucaipa

56; PMV-4: Duck 75; PMV-6: Duck 77) were supplied by Dr. D.J. Alexander(Central

Veterinary Laboratory, Weybridge, U.K.).

Virus propagation and purification The virus was propagated in the allantoic cavity of 9- to 1 l-day-old embryonated

fowls’ eggs and clarified by centrifueation at 5,000 Xg for 20 min. Thesupernatant was

centrifuged through 6 ml 30% w/v sucrose at 20,000 rpm for I .5 h at 5°C in an SW 28

Beckman rotor and the virus pellet resuspended in NTE (100 mM NaCI, 10 mM

Tris-HCl, pH 7, 1 mM EDTA) buffer. The purity of preparations was adequate for

further analysis (Fig. 1).

Partial digestion Approximately 50 1.18 viral protein was digested with 2 ~8 of Staphylococcus aureus

V8 enzyme (Miles) or with 0.1 pg of chymotrypsin (BDH) in 50 ul at 37°C in the

presence of 1% SDS (BDH) as described (Cleveland et al., 1977). The reaction was

stopped by boiling.

Electrophoresis The samples were electrophoresed in 15% polyacrylamide gels (Laemmli, 1970)

with 100 V for 20 h. The gels were stained with Coomassie blue.

RESULTS

The optimal condition of digestion was determined by varying the concentrations

of the enzymes and the length of time. Since in most cases 20 min was enough to digest

the major polypeptides of the virus and by increasing the duration of digestion the

229

number of peptides decreased with certain strains (e .g. LaSota), 20 min was chosen for

further analysis.

As can be seen in Fig. ,l, relatively small differences exist between the undigested

protein patterns of six laboratory strains of NDV, whereas strains belonging to

PMV-2, PMV-4 and PMV-6, and Sendai virus are easily distinguishable. The same

NDV strains can be readily differentiated on the basis of their peptide patterns

obtained by digestion with the S. aureus V8 enzyme, while PMV-2, PMV-4, PMV-6

and Sendai virus exhibit further major differences (Fig. 2). The NDV strains used

represent the lentogenic (LaSota and F), the mesogenic (H and L) and the velogenic

(Texas GB and Herts 33) groups, but the differences in peptide patterns are character-

istic of the individual strain rather than of the virulence group they belong to. By

increasing the length of digestion from 20 to 60 min the strains are still easily

distinguishable (not shown). Thus, the observed differences reflect dissimilarities

existing in the protein structure of the strains rather than disparities in the conditions

of digestion. Patterns of the peptides obtained by chymotrypsin digestion from the

same NDV strains were also compared and again individual peptide distribution was

123456

L

HN

NP A

M

7 891011

-P -HN -El ‘NP

-F

-M

Fig. 1. Polypeptides of different strains of NDV, avian paramyxoviruses (PMV-2, PMV-4, PMV-6) and

Sendai virus in a 10% polyacrylamide gel. Lane I, LaSota; lane 2, F; lane 3, Hertfordshire, H; lane 4, Lederle

L; lane 5, Texas GB; lane 6, Herts 33; lane 7, Texas GB; lane 8, PMV-2; lane 9, PMV-4; lane 10, PMV-6; lane

11, Sendai virus. A: actin. Polypeptide labels refer to NDV and Sendai virus.

230

L-

HN-

NP-

M-

Ul 2345 6 7 8 91011 wtwmd&P--,

Fig. 2. Peptide mapping of NDV strains, avian paramyxoviruses and Sendai virus after digestion with S.

aureus V8 protease in a 15% polyacrylamide gel. Digestion was for 20 min. U: undigested strain LaSota.

Other lanes are as in Fig. 1.

obtained (Fig. 3). The peptide maps of the strains digested with chymotrypsin did not

change significantly when digested for a longer time. The high resistance both to V8

protease and chymotrypsin of the haema,, ~olutinin of Texas GB is remarkable.

The identity or dissimilarity of strains based on V8 digestion could be also confirm-

ed by chymotrypsin digestion (Fig. 4).

The reproducibility of the method and of the genetic stability of the strain is

illustrated by comparing LaSota ‘lines’, which have been maintained in different

laboratories for a number of years. The peptide patterns obtained both by V8 and

chymotrypsin digestion were very similar (Fi g. 5). On the other hand when strain

Texas was digested and run on different days its characteristic pattern was easily

recognizable (Fig. 6).

231

123456

- HN

- M

Fig. 3. Peptide mapping of NDV strains after digestion with chymotrypsin in a 15% polyacrylamide gel.

Digestion was for 20 min. Lanes 1 to 6 are as in Fig. 1, lanes 7 and 8, Texas GB after 20 and 60 min digestion,

respectively.

DlSCUSSlON

The analysis of peptides obtained by partial digestion of proteins with proteolytic

enzymes (Cleveland et al., 1977) has been successfully used for the identification of

different virus polypeptides (e .g. Lamb and Choppin, 1978). Likewise, the method has

proved to be suitable for the differentiation of strains of foot-and-mouth disease virus

(Robson et al., 1979), influenzavirus (Nakamura et al., 1981) and NDV (Nagai et al.,

1980) but as used by these workers, the method involves the digestion of radiolabelled

polypeptide bands separated on, and excised from polyacrylamide gels. However,

when the need arises to analyse a large number of strains, e.g. for epidemiological

investigation, the above method is too complicated and too slow because it requires

232

1 12 3

HN

Fig. 4. Peptide mapping of NDV strains isolated from the same outbreak after digestion with S. aweus V8

(A) and chymotrypsin (B). Lanes 1, NDV-38; lanes 2, NDV-39, lanes 3, NDV-40.

radiolabelling of the virus, at least two gel runs and other manipulations such as

autoradiography.

The method presented here is a simple, economical and relatively rapid procedure

for the differentiation of large numbers of NDV and other paramyxovirus strains. The

method is simple since it requires only one ultracentrifugation step of the virus, one

electrophoretic step and stainin g. A more extensive purification than that performed

in the present procedure is superfluous. The results presented in Fig. 1 show that

virions obtained after one purification step contain little contaminating protein. The

233

HN

NP

M

Fig. 5. Peptide mapping of different ‘lines’ of the strain LaSota after digestion with 5’. aureus V8 (A) and

chymotrypsin (B). The origin of LaSota ‘lines’ maintained in different laboratories: lane 1, Clone-30

(Intervet); lane 2. LaSota, this laboratory since 1965; lane 3, LaSota (‘Biol’); lane 4, LaSota (Dessau); U:

undigested strain LaSota.

slight contamination present in the preparations, i.e. actin, below the NP apparently

does not influence the interpretation of the patterns because at least eight peptides are

generated with V8 enzyme from actin (Lamb and Choppin, 1978; Lomniczi and

Morser, 1981) and these do not become visible. In addition, none of the peptides

obtained by partial V8 digestion of the coo- _a propagated Sandai virus, a murine

paramyxovirus (Fig. 2) seem to corn&ate with NDV peptides. This method also

rendered unnecessary separation and separate analysis of the three major structural

proteins of NDV since any difference between the peptide patterns of two strains

indicates their non-identity even without knowing which polypeptides are different.

Obviously identical peptide patterns indicate identical polypeptides and identical

virus strains.

The method is economical since one 38-ml ultracentrifuge tube yields enough

purified virus for up to 5 analyses. This means that the allantoic fluid of one egg is

enough for the analysis of a strain. Labelling of virus polypeptides with isotopes is also

234

- HN

Fig. 6. One-dimensional V8 generated peptide patterns of the strain Texas GB digested and electrophores-

ed on different days.

unnecessary as the peptide bands can be localized by staining with Coomassie blue.

The method is rapid since 18 strains can be purified in 1 day analysed on the following

day and only one gel run is needed.

The differentiating capacity of the method presented here exceeds that of the

procedures used earlier for identification of lentogenic strains, e.g. heat-stability of

haemagglutinin, because its use can be extended to strains of other virulence grades.

Five of the six laboratory strains studied by us were also analysed by Russell and

Alexander (1983) who found that, with the exception of strain H and Herts 33, the

strains could be differentiated by using monoclonal antibodies. Strain tiertfordshire

(H) is a mesogenic strain attenuated from the virulent Herts 33 (cit. Russell and

Alexander, 1983). The latter two strains are the oldest NDV isolates and have been

maintained independently durin g the last 50 yr. Presumably because of this, the

distance has grown between them so that their peptide patterns have become some-

235

what dissimilar. In any case, strain H itself is heterogenous on the basis of the

biological properties of its subpopulations (Lomniczi, 1976).

Further data on the value of peptide analysis are needed particularly to evaluate its

applications for epidemiological studies of NDV strains. According to our prelimi-

nary results, most of the fifteen strains isolated from the same outbreak have identical

patterns, while strains isolated from outbreaks occurring at different times and

locations can be distinguished (diva Nagy and B. Lomniczi, manuscript in prep,).

At present we do not know whether or not the method can also be used for positive

identification of strains, i.e. for the unambiguous recognition of a particular strain

among others. Perhaps the discriminating power of the technique could be augmented

by digestion with different enzymes,

ACKNOWLEDGEMENTS

We thank Dr. N.J. Dimmock for critical reading of the manuscript. The excellent

technical assistance of Mrs. J. SzCcsy is appreciated.

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