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Virology

Arch Virol (1991) 118:151-161

© Springer-Verlag 1991 Printed in Austria

Detection of Newcastle disease virus RNA in infected allantoic fluids by in vitro enzymatic amplification (PCR) V6roniqueJestinI and A. Jestin 2 1UR Pathologic Aviaire and 2UR Station de Pathologie Porcine, CNEVA Laboratoire Central de Recherches Avicoleset Porcines, Ploufragan, France Accepted November 19, 1990

Summary. The Polymerase Chain Reaction (PCR) procedure was applied in order to identify the Newcastle disease virus (NDV), an avian paramyxovirus (A-PMV 1). The sequence selected for amplification consists of 238 bp lying in the gene encoding the fusion protein F. A pair of 19-mer and 18-mer oligonucleotides, flanking this sequence, were used as primers. Following RNA extraction by the proteinase K method, a cDNA was prepared using the previous 19-mer oligonucleotide as the primer. The amplification reaction product was analyzed by electrophoresis and ethidium bromide staining, using the restriction enzymes Hae III, Mbo II, and Nat I. The PCR was performed on cDNA prepared from 30 A-PMV 1 and 3 other strains (A-PMV 2, A-PMV 3, A-PMV 4). It was thereby demonstrated that the selected sequence was highly specific and constant. However, two of the PMV 1 strains isolated from feral ducks, are thought to present a deletion of about 25 bp inside this fragment as shown by the smaller length of the corresponding amplified product and the disappearance of the Nar I restriction site. The advantages of this technique, as a first step in evaluating virulence by means of molecular biology, is discussed.

Introduction The Newcastle disease virus, also called type 1 avian paramyxovirus (A-PMV 1), is the type species of the genus Paramyxovirus, family Paramyxoviridae. Its genome is a single strand of negative sense RNA of approximately 15 kb [-13] that codes for 6 gene products, one of them being the fusion protein-F. This protein is produced as a precursor molecule FO, which is cleaved into two fragments F 1 and F 2 under host-cell protease activity; there is a strict correlation between the cleavability of FO in tissue culture cells and the virulence of NDV isolates [20, 21]. In fact, a wide range of isolates (several hundred) have been described; these vary from the apathogenic to the extremely pathogenic, causing very severe

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V6ronique Jestin and A. Jestin

disease in many domestic avian species, resulting in nearly complete mortality [6]. Sequence analysis of the cleavage site (FO) of 13 virulent and avirulent purified NDV strains has revealed a correlation between pathogenicity and a high content of basic amino acid (Arg, Lys) residues at this site [-9, 24]: the pentapeptide preceding the new F 1 amino terminus, produced by cleavage, is Arg-Arg-Gln-Arg(Lys)-Arg in the virulent strains examined, whereas in the avirulent strains examined, a neutral amino acid is found in place of the basic arginine residues at positions 1 and 4 of the pentapeptide. The PCR technique was developed quite recently [-22] and it has permitted selective amplification of a short segment of genomic DNA more than 105-fold. It has been widely used in the diagnosis of human genetic disease I-4] as well as in the investigation of the mechanisms of mutagenesis involved in the development of malignancy [71, since it enables us to determine whether a specific change has occurred inside a given DNA segment. Very recently this method was applied, coupled with dideoxynucleotide sequencing, to the differentiation of vaccine and wild mumps viruses, which also belong to the genus Paramyxovirus [8]. Currently, the diagnosis of NDV is routinely performed by isolating the virus, then identifying it, using specific antisera or monoclonal antibodies, and subsequently characterizing its virulence by means of biological tests [3]. This takes at least two weeks, and requires about one hundred of SPF embryonated fowl eggs. To our knowledge, no application of PCR to the detection of NDV has been published. Our purpose was to develop a PCR test able to specifically detect a wide range of NDV isolates grown in allantoic fluids of embryonated eggs, in order to evaluate the specificity and the degree of conservation of the selected segment to be amplified. This is a preliminary step before carrying out further sequencing of the amplification products, or attempting PCR directly on organs from suspected animals. Materials and methods

Viruses

Purified NDV strain La Sota clone 30 was used to establish the PCR method. Then the avian paramyxoviruses listed in Table 1 were tested, They were grown in allantoic fluids of SPF eggs. Chemicals

The sources of the chemicals used for RNA extraction were proteinase K (Boehringer Mannheim) and phenol ultra pure (Gibco BRL). For making cDNA, reverse transcriptase M-MLV (Gibco-BRL), methylmercuric hydroxyde (Serva), mercapto-ethanol (Sigma) were used. Primers were synthetized by Appligene. The choice of which primer was made with the assistance of a microcomputer: PC/gene program (Intelligenetics Inc and Genofit SA), along the F gene sequences [5, 16, 19, 23]. Primer 1 was a 19-mer oiigonucleotide, 5' CTTTGCTCACCCCCCTTGG 3'

Detection of Newcastle disease virus by PCR

153

Table 1. Viral strains and isolates Type 1 avian paramyxoviruses International reference strains B1 Beaudette C (Bean) Essex (Esx) F* GB Texas (GBT) H* Herts Komarov* (Kom) Kuwait 256* (Kuw) La Sota (LS) LS Clone 30b ( e l 30) Queensland V 4* (QV4) Roakin (Roak) Ulster 2 C* (Uls)

Virulence a

Year of isolation

Host

1983 1983 1983 1988 t990 1983 1983 1984 1984 1986 1989 1977 1977 1980 1982

Fowl Fowl Fowl Fowl Fowl Pigeon Pigeon Pigeon Pigeon Pigeon Pigeon Feral duck Feral duck Feral duck Feral duck

L

M V L V M V M V L L L M L

French reference challenge strain Plou~agan (Plou) V French isolates 83299 83309 84182 88072 90015 83230 83333 84135 84392 86164 89350 MC 110"* MJ 8/1"* ML 66/1"* MY lI6/4**

(299) (309) (182) (072) (015) (230) (333) (135) (392) (164) (350) (Mc) (Mj) (M1) (My)

M M L L-M L M M M L? M L L M? L? L?

Other types of paramyxoviruses PMV 2/chicken/California/Yucaipa/56* PMV 3~parakeet~Netherlands/449~75* PMV 4/duck/Hong Kong/D 3/75*

(PMV 2) (PMV3) (PMV 4)

a Velogenic (V), mesogenic (M), lentogenic (L), respectively b Including the prototype strain and a variant strain obtained at the laboratory * Gift of D. J. Alexander (Central Veterinary Laboratory, Weybridge, U.K.) ** Isolated by Pr. Hannoun (Institut Pasteur Paris, France) ? Uncomplete determination of pathotype

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V6ronique Jestin and A. Jestin

localized at position 315 to 333 of the cDNA; primer 2 was a 18-mer oligonucleotide, 5' CTTCCCAACTGCCACTGC 3' localized at position 572 to 589 of the r cDNA. The kit Amplitaq (Perkin-Elmer Cetus) was used for DNA amplification; the dnTP of this kit were also employed for cDNA preparation. To identify PCR products the following restriction enzymes were used: Hae III, Mob II, NarI (Biolabs). DNA molecular-weight marker V (pBR 322 Hae III digested DNA) (Boehringer Mannheim) was used for reference.

RNA extraction and cDNA preparation RNA extraction was performed according to the method previously described [17], with some modifications. Briefly 200 gt (either 200 gg purified ND virus or unpurified infected allantoic fluids exhibiting an average haemagglutinating activity of 160 HA U with lower and upper limits ranging from 16 to 1280, respectively) were made to 2% SDS, 0.01 M EDTA, 0.1 M Tris, 60 gg proteinase K, then incubated at 37°C for 30min. Then 1/2 vol of phenol was added and mixed vigorously. The resulting upper aqueous phase was extracted once more with phenol and twice more with ether. The final aqueous phase was precipitated with 5 vol of ethanol and 1/20 vol NaC1 5 M at - 70 °C overnight. Just before carrying out cDNA preparation, the RNA was spun down at 12,000 g for 30 rain and dried in vacuo. For the preparation of cDNA, a method was adapted from that described by Maniatis [15]. Briefly, the RNA pellet was redissolved in 1 ~tg primer 1, and incubated with methyl mercuric hydroxyde 3.75 mM for 10 rain at room temperature, then with mercapto-ethanol for an additional 5 min. Finally dNTPmix, plus the optimized buffer and reverse transcriptase, were added according to the protocol supplied with the enzyme, and incubated for 1 h at 37 °C. The cDNA obtained was stored at - 70 °C before processing enzymatic amplification.

cDNA amplification using the polymerase chain reaction (PCR) A protocol was adapted from that supplied with the Gene Amp Kit (Perkin-Elmer Cetus) and those previously tested using a microtechnique [10, 11]. Briefly, the test was calibrated in such a way that dNTP were 1.25 mM each, PCR primers were 100 ng each, Taq polymerase was adjusted to 1 U in a total volume of 20 gl. Several dilutions of each cDNA obtained (previously denatured at 100°C for 10min) were tested as detailed below, cDNA was amplified with a programmable thermal cycler (Techne PHC-1). The PCR programm was defined as denaturation at 94°C for 1.5 min, annealing at 51 °C for 2.5min, elongation at 75 °C for 1 rain for 35 cycles. After the 35th cycle, the time of extension at 75 °C was 2rain.

Analysis of PCR products Amplified cDNA were electrophoresed on ethidium bromide agarose 2% gel at 200 V. A 275-bp fragment identified with M 5 marker was sought. The products of digestions by restriction enzymes were analyzed as well. These were chosen according to the restriction site analysis given by the PC gene program mentioned above, as illustrated in Fig. 1. Before enzymatic digestion, DNA from the PCR product was extracted by a simplified procedure in comparison with the one described above. Briefly, phenol was directly added to the PCR product, and following two phenol extractions, the resulting aqueous phase was directly precipitated. The dried DNA pellet obtained was resuspended in dd H 2 0 , aliquoted in four tubes. The first three were digested with each enzyme Hae III, Mbo II, NarI adjusted respectively to 20, 10, and 8 U and incubated for 1 h 30min at 37 °C in the

Detection of Newcastle disease virus by PCR

Primer

155

cDNA gene coding f o r the F p r o t e i n of N e w c a s t l e D i s e a s e virus

1 )

315

572

333

589

Primer

2

275 bp fragment

131

144 b p

bp

,~i I

!

" !

Hae

III

458

77 b p , 8 8

bp

187,

ii b p

,

198 b p

41.

' Nar I

!

|

. . . . . . . . . . . . . . . . .

391

402

$ Mbo I I

A T 396

T - T-A C-G

- 401

C-G

391

hairpin

a strong

C-G

dG

free

(25°C)

loop exhibiting

energy value

:

= -13,8 kCal

- G-C G-C

/\ GGGAGACAGG

Potential

G-C

- 406

ATTATTGGCG

Fig. l. Characteristicsofthesegmentamplifiedandrepresentation oftherestriction maps appropriate buffer recommended by the supplier. The fourth tube was the undigested control. Digestion fragments were analyzed on ethidium bromide agarose gel, prepared as mentioned above. Results

Amplification of purified ND V La Sota clone 30 strain The results are shown in Fig. 2. The 275-bp fragment predicted was visualized. It was digested by Hae III in two fragments localized at the same place as the predicted fragments 131 bp and 144 bp. Digestion of the amplified fragment by Nar I exhibited two groups of fragments localized at positions 184-192 bp and 80-89 bp which were in agreement with the theoretical fragments 187 bp, 198 bp and 77 bp, 88 bp. Digestion by Mbo II was inefficient as predicted.

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V6ronique Jestin and A. Jestin

Fig. 2. Characterization of amplification product using restriction endonucleases. A Amplification product digested by Nar I. B Amplification product digested by Mbo II. C Amplification product digested by Hae III. T Undigested amplification product, 200 V 2 h. m DNA molecular-weight marker

Amplification of ND V infected allantoic fluids Amplification of La Sota infected allantoic fluids gave the same results (data not shown). It should be noted that the intensity of the positive signal did not correlate with the cDNA dilution. The NDV strains previously listed, prepared in allantoic fluids, were tested as shown in Table 2. A positive 275 bp signal was obtained for at least one of the cDNA dilutions of the 27/30 NDV strain after one or two cDNA preparations were made. Four of these strains (84135, Beaudette C, 83230, and 83299) exhibited additional fragments which disappeared once new preparations of primers were used (Table 2). On four occasions three additional strains, referenced as Mj 8/1, Mc 110, and F, showed fragments that were smaller than expected, at about 230-250 bp (Fig. 3). The first two strains, which gave a more intense signal, were analyzed with restriction enzymes. It appeared that they had lost the Nar I site, in comparison with NDV strains exhibiting the 275 bp signal, as shown in Fig. 4. The Hae III restriction site appeared unmodified (data not shown). At the same time, the other avian paramyxoviruses tested gave no signal. Discussion

Although a positive signal exhibiting the expected number of nucleotides (275 bp) was shown, following NDV La Sota clone 30 strain amplification, the identity of the amplified product was proven using restriction endonucleases. These were chosen according to the restriction map given by the PC/gene program. The Nar I and Hae III endonucleases were shown to cut at the expected

Detection of Newcastle disease virus by PCR

157

Table 2. Identification of Newcastle disease strains using PCR Strain

cDNA

Beaudette C (Beau)

1a

F

PCR

Result

1b

_

2

c 1/2d, 1/20, 1/200 -1/10

2

3 4

+ ?e 1/2, 1/10, 1/50 + f 1/50, 1/250, 1/1250

1

1 2 3 4

-1/2, 1/20, 1/200 250 bp signal 1/2g very weak 250 bp signal 1/10g - 1/2

2 Queensland V4 (QV4)

1 2

1 2

-1/2, 1/20, 1/200 + 1/2, 1/10, 1/50

83299 (299)

1 2

1 2

+ ? 1/2, 1/20, 1/200 + 1/5000

84182 (182)

1 2

1 2

-1/2, 1/20, 1/200 + 1/2, 1/10, 1/50

83230

1 2

1 2

+ ? 1/2g + 1/2g

83333 (333)

1 2

1 2

-1/2, 1/20, 1/200 + 1/10g

84135 (135)

I 2

1 2

+ 71/2, 1/10, 1/50 + 1/250, 1/1250

Mc 110 (Mc)

1

1 2 3 4

250 bp 250bp 250 bp 250 bp

1 2 3 4

230-250bp signal 1/2, 1/208 230-250bp signal 1/2, 1/10g 250 bp signal 1/2g 250 bp signal 1/2

2 Mj 8/1 (Mj)

1 2

signal signal signal signal

1/2, 1/20g 1/2, 1/10, 1/50 1/2 1/2

M1 66/1 (M 1)

1 2

1 2

-1/2, 1/20, 1/200 + 1/10, 1/50

My 116/4 (My)

1 2

1 2

250 bp signal 1/2, 1/20 + 1/2, 1/10, 1/50

18 other PMV 1 strains listed in Table 1

1

1

+ at leasth one of the cDNA dilutions tested

a Range of cDNA preparations required in order to obtain the result described in the next column b Range of PCR test required in order to obtain the result described in the next column c Absence of any signal for the cDNA dilutions tested cDNA dilutions showing the result enounced e+ ? Presence of a 275 bp signal plus extraneous signal(s) at the dilutions listed f+ Presence of a 275 bp signal without any extraneous signals at the dilutions listed gAbsence of signal for other cDNA dilutions hFor 13/18 cDNA tested: 2 or 3 dilutions giving a positive signal

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V~ronique Jestin and A. Jestin

Fig. 3. Amplification by PCR of cDNA from avian paramyxovirus strains, a, b, c cDNA dilutions tested respectively: 1/2, 1/10, 1/50. + cDNA diluted 1/200 (prepared from 164 strain), 200 V 1 h. m DNA molecular-weight marker

Fig. 4. Analysis of amplification product from strain Mc 1t0 and Mj 8/1 using Nar I as restriction endonuclease. + Digested by NarI; undigested, m DNA molecular-weight marker. Amplification product from Komarov cDNA (Kom) is used as a positive control sites, while M b o II did n o t cut as predicted. The m i g r a t i o n o f the P C R p r o d u c t incubated with M b o II was slightly different w h e n c o m p a r e d to the undigested product, due to the different buffer.

V6ronique Jestin and A. Jestin

159

As the purpose of this experiment was to find a method for characterizing the isolates grown in allantoic fluids which are sent to our national reference laboratory, identified NDV strains and isolates grown in allantoic fluids were tested by PCR. By testing several dilutions (from 1/2 to 1/1250) of two independent preparations of cDNA, from two different RNA extractions, the PCR appeared quite successful for the 27/30 PMV 1 (NDV) strains. In the future, it would be advisable to systematically apply this method to identify new isolates. A positive signal was also obtained for the three other NDV strains (Mc 110, Mj 8/1 and F), but the amplified segment was smaller than expected. Using restriction endonucleases, it was demonstrated that for two of these strains, Mc 110 and Mj 8/1 (isolated from feral ducks), the corresponding segment had lost the Nar I restriction site. The latter was localized at the place where the RNA was supposed to form a very strong hairpin, as shown by computer analysis. This hairpin was in fact demonstrated experimentally (data not shown), since amplification of cDNA prepared without methyl mercuric hydroxyde, exhibited a major lower band corresponding to about 250 bp, which was not digested by Nar I. This suggests that Mc 110 and Mj 8/1 might have a deletion in their F genome since the length of the amplified segment for these two strains is the normal length (275 bp) less the size of the hairpin (25 bp). Moreover, by using monoclonal antibodies [2, 12, 18], these two strains have been shown to be quite different from other NDV strains. More surprising is the result given by strain F, since anti-fusion protein monoclonal antibodies did not reveal differences with other PMV 1 strains [18]. However, using a panel of different monoclonal antibodies, this strain shows similar characteristics to strains isolated from feral ducks [2]. Since the signal given as a result of the amplification of this strain was very weak, despite four attempts, it was not possible to analyse the amplified product by restriction endonucleases. In that particular situation, the use of a synthetic labelled probe (for hybridization following blot transfer) might offer a method for identifying the amplified segment more accurately. The PCR test was PMV 1 specific, since it was not possible to detect other avian paramyxoviruses such as A-PMV 2, A-PMV 3, A-PMV4, which were grown in the same way. These strains were employed preferentially because they were known to bear common epitopes to NDV [1]. Within the limitations described, the PCR method appeared to detect a very specific and constant segment. To our knowledge this is the first mention of PCR used to identify the Newcastle disease virus. We are currently investigating whether it is possible to apply this method to detect NDV in organs. Moreover, the amplified segment includes that portion which encodes the cleavage site, and is of great interest in evaluating virulence. The sequencing of the amplified products will be the next step of this study.

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References 1. Abenes G, Kida H, Yanagawa R (1986) Antigenic mapping and functional analysis of the F protein of Newcastle disease virus using monoclonal antibodies. Arch Virol 90:97-110 2. Alexander D J, Manvell RJ, Kemp PA, Parsons G, Collins MS, Brockman S, Russel PH, Lister SA (1987) Use of monoclonal antibodies in the characterization of avian paramyxovirus type 1 (Newcastle disease virus) isolates submitted to an international reference laboratory. Avian Pathol 16:553-565 3. Alexander DJ (1988) Newcastle disease diagnosis. In: Alexander DJ (ed) Newcastle disease. Kluwer, Boston, pp 147-160 4. Attree O, Vidaud D, Vidaud M, Anselem S, Lavergne JM, Goossens M (1989) Mutations in the catalytic domain of human coagulation factor IX: rapid characterization by direct genome sequencing of DNA fragments displaying altered melting behaviour. Genomics 4:266-272 5. Bingham RW, Chambers P, Emmerson PT (1987) Newcastle disease virus gene clones. European Patent application 0227 414 A2 6. Erdei J, Erdei J, Bachir K, Kaleta EF, Shortridge KF, Lomniczi B (1987) Newcastle disease vaccine (La Sota) strain specific monoclonal antibody. Arch Virol 96:265-269 7. Farr CJ, Saiki RK, Erlich HA, Mc Cormick F, Marshall CJ (1988) Analysis of RAS gene mutations in acute myeloid leukemia by polymerase chain reaction and oligonucleotide probes. Proc Natl Acad Sci USA 85:1629-1633 8. Forsey T, Mawn JA, Yates PJ, Bentley ML, Minor PD (1990) Differentiation of vaccine and wild mumps viruses using the polymerase chain reaction and dideoxynucleotide sequencing. J Gen Virol 71:987-990 9. Glickamn RL, Syddall RJ, Lorio RM, Sheehan JP, Bratt MA (1988) Quantitative basic residue requirements in the cleavage activation site of the fusion glycoprotein as a determinant of virulence for Newcastle disease virus. J Virol 62:354-356 10. JestinA, FoulonT, Blanchard P (1989) Detection ofpseudorabies virus DNA in infected cells by in vitro enzymatic amplification. In: Contribution of molecular biology to veterinary virology. Proceedings of the First Congress of the European Society for Veterinary Virology, Liege, 5-7 April, 1989, p 51 11. Jestin A, Foulon T, Pertuisset B, Blanchard P, Labourdet M (1990) Rapid detection of pseudorabies virus genomic sequences in biological samples from infected pigs using polymerase chain reaction DNA amplification. Vet Microbiol 23:317-328 12. Jestin V, Cherbonnel M, Morin M, Guittet M, Bennejean G (1989) Characterization of French avian paramyxovirus type 1 (PMV 1) isolates with a panel of monoclonal antibodies to the Ploufragan strain of Newcastle virus. Arch Virol 105:189-198 13. Kolakofsky D, Boy de la Tour E, Delius H (1974) Molecular weight determination of Sendai and Newcastle disease virus RNA. J Virol 13:261-268 14. Long Le, Brasseur R, Wemers C, Meulemans G, Burny A (1988) Fusion (F) protein gene of Newcastle disease virus: sequence and hydrophobicity. Comparative analysis between virulent and avirulent strains. Virus Genes 1:333-350 15. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, New York, pp 230-234 16. Mc Ginnes LW, Morrison TG (1986) Nucleotide sequence of the gene encoding the Newcastle disease virus fusion protein and comparisons of paramyxovirus fusion protein sequences. Virus Res 5:343-356 17. Mc Gookin R (1984) RNA extraction by the proteinase K method. In: Walker JM (ed) Methods in molecular biology, vol 2, nucleic acids. Humana Press, Clifton, NJ, pp 109-112

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18. Meulemans G, Gonze M, Carlier MC, Petit P, Burny A, Le Long (1987) Evaluation of the use of monoclonal antibodies to haemagglutinin and fusion glycoproteins of Newcastle disease virus for virus identification and strain differentiation purposes. Arch Virol 92:55-62 19. Millar NS, Chambers P, Emmerson PT (1988) Nucleotide sequence of the fusion and haemagglutinin-neuraminidase glycoprotein genes of Newcastle disease virus strain Ulster: molecular basis for variations in pathogenicity between strains. J Gen Virol 69: 613-620 20. Nagai Y, Klenk HD, Rott R (1976) Proteolytic cleavage of the viral gylcoproteins and its significance for the virulence of Newcastle disease virus. Virology 72:484-508 21. Nagai Y, Klenk HD (t977) Activation of precursors to both gtycoproteins of Newcastle disease virus by proteolytic cleavage. Virology 77:125-134 22. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (1985) Enzymatic amplification of D-globulin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350-1354 23. Schaper UM, Fuller FJ, Ward MDW, Mehrotra Y, Stone HO, Stripp BR, De Buysscher EV (i988) Nucleotide sequence of the envelope protein genes of a highly virulent neurotropic strain of Newcastle disease virus. Virology 165:291-295 24. Toyoda T, Sakaguchi T, Imai K, Inocencio NM, Gotoh B, Hamaguchi M, Nagai Y (1987) Structural comparison of the cleavage activation site of the fusion glycoprotein between virulent and avirulent Strains of Newcastle disease virus. Virology 158:242-247 Authors' address: Veronique Jestin, UR Pathologic Aviaire, CNEVA Laboratoire Central de Recherches Avicoles et Porcines, B.P. 53, F-22440 Ploufragan, France. Received August 22, 1990

Detection of Newcastle disease virus RNA in infected allantoic fluids by in vitro enzymatic amplification (PCR).

The Polymerase Chain Reaction (PCR) procedure was applied in order to identify the Newcastle disease virus (NDV), an avian paramyxovirus (A-PMV 1). Th...
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