Avian Pathology ( 2001) 30, 465–469
Characterization of Newcastle disease viruses isolated in Italy in 2000 Giovanni Cattoli1, Ruth J. Manvell2, Ernesto Tisato1, Jill Banks2 & Ilaria Capua1* 1
National Reference Laboratory for Newcastle Disease and Avian Influenza, Istituto Zooprofilattico Sperimentale delle Venezie, Via Romea 14/A, 35020 Legnaro, Padova, Italy, and 2Avian Virology, VLA Weybridge, New Haw, Addlestone, KTI5 3NB, Surrey, UK
Thirty-two Newcastle disease virus isolates from the 2000 Italian epidemic were characterized by monoclonal antibody binding pattern and nucleotide sequencing of approximately 400 base pairs of the fusion gene. In addition, the pathogenicity of six of these isolates was assessed by means of the intracerebral pathogenicity test ( ICPI). The strains tested exhibited an ICPI ranging from 1.6 to 2.0. On the basis of the monoclonal antibody binding pattern, all isolates could be classified as belonging to group C1. Both monoclonal antibody and genomic analysis revealed a very high degree of homology, indicating a common source of infection. On the basis of the phylogenetic analysis, it appears that the Italian isolates are closely related to the recent isolates from the UK, Scandinavia and South East Europe, thus suggesting the circulation of this viral strain in Europe during the past 5 years.
Introduction Newcastle disease ( ND) is an infectious disease of birds caused by a virus belonging to the Rubulavirus genus of the Paramyxoviridae family. Avian paramyxoviruses are classified into nine serotypes ( APMV1 to APMV9) and Newcastle disease virus ( NDV) is synonymous to avian paramyxovirus serotype 1 ( Rima et al., 1995) . Since April 2000, Italy has been facing a ND epidemic, which initially involved intensively reared poultry and, subsequently, dealers and backyard flocks, primarily located in the northern and central regions of the country. To date, 254 outbreaks have been notified, affecting a variety of avian species, with a total of 17 outbreaks in intensively reared poultry, 17 in dealers or “svezzatori”, one in an ostrich farm, and 219 in backyard flocks ( Capua et al., 2001). The epidemiological investigation, performed as indicated by EU Directive 92/66/EC ( Council Directive, 1992), highlighted the crucial role of a broiler hatchery as the primary source of infection of the epidemic. This hatchery had imported 1-dayold chicks and hatching eggs from several EU Member states and third countries.
Nucleotide sequencing has proved to be a useful tool for epidemiological studies together with antigenic characterization using monoclonal antibodies ( Alexander et al., 1997, 1999a; Lomniczi et al., 1998). In this paper, we report the results of the molecular and serologic characterization of the isolates in an attempt to establish the origin of the infection. Materials and methods NDV isolates were obtained and identified as indicated by Directive 92/66/EC, and their virulence was assessed by means of the intracerebral pathogenicity test ( ICPI), performed as described by EU directive 92/66/EC. Isolates were further classified using a panel of 25 monoclonal antibodies as described by Alexander et al. ( 1997). Nucleotide sequence of the viral fusion ( F) gene was obtained from 32 selected isolates of different origin, extracting viral RNA from infective allantoic fluid, according to the instructions of the manufacturer ( High Pure RNA Isolation Kit; Roche Diagnostics GmbH, Germany). Amplification of approximately 400 base pairs of the F gene was accomplished by reverse transcription-polymerase chain reaction as reported by Alexander et al. ( 1999a). To detect any differences between isolates, the variable F gene signal sequence was included in the amplicon. The cDNA was sequenced by using the BigDye Terminator cycle sequencing ready reaction kit ( PE Applied Biosystems, USA). Sequences were then aligned together with F gene sequences available on GenBank, and phylogenetic analysis was
* To whom correspondence should be addressed. E-mail:
[email protected] Received 30 January 2001. Accepted 6 April 2001. ISSN 0307-9457 ( print)/ISSN 1465-3338 ( online)/01/050465-05 © 2001 Taylor & Francis Ltd DOI: 10.1080/03079450120078644
466 G. Cattoli et al. performed for 305 nucleotides using the Fitch-Margoliash and Least Squares method ( KITSCH) from the PHYLIP phylogenetic inference package, version 3.5c ( Felsenstein, 1993). The nucleotide sequence of the representative strain 3015/V00 has been submitted to GenBank ( accession number AY024333).
Results The virulence of six of the 32 NDV isolates was assessed by the ICPI test, and the index values obtained ranged from 1.6 to 2.0 ( Table 1). On the basis of the monoclonal antibody binding pattern, all 32 viruses could be classified as belonging to group C1 ( Alexander et al., 1997) ( Tables 1 & 2). Both monoclonal antibody and genomic analyses of the NDV strains involved in the recent Italian epidemic revealed a very high degree of homology regardless of the geographical origin and species of isolation, thus indicating a common source of infection. Particularly, nucleotide sequence analysis showed that 31 of the 32 strains examined were
identical. Strain 3013/00 presented a single nucleotide difference, with a similarity of 99.7% compared with the other 31 isolates. On the basis of the phylogenetic analysis, it appears that the Italian isolates are closely related to recent isolates from the UK and Scandinavia obtained during 1996 and 1997 ( Alexander et al., 1999a) , and isolates obtained from south-eastern Europe ( Figure 1, strain BG431/96 and FIVi 1001/96/1; nucleotide similarity, 99.0%), thus suggesting spread of this virus strain in the European continent throughout the past 5 years. Discussion The monoclonal antibody binding pattern and the phylogenetic analysis indicate that the NDV strains responsible for the Italian 2000 epidemic are very closely related to the viruses that caused the outbreaks in the UK and in Scandinavia in recent years ( Alexander et al., 1998a,b, 1999a). Therefore,
Table 1. NDV strains from Italian outbreaks in 2000 that are included in the present study
Strain 3013/V00 3014/V00 3015/V00 3021/V00 3022/V00 3232/V00 3270/V00 3272/V00 3274/V00 3280/V00 3282/V00 3286/V00 3287/V00 3308/V00 3309/V00 3310–1/V00 3310–2/V00 3311/V00 3348/V00 3372/V00 3466/V00 3467/V00 3468/V00 3469/V00 3501/V00 3505/V00 3506/V00 3507/V00 3508/V00 3509/V00 3536/V00 3543/V00 a
Nd, Not determined.
Date of isolation May May May May May May May May May June June June June June June June June June June June June June June June June June June June June June June June
2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000
Region
Species
ICPI
MAb group
Piemonte Piemonte Veneto Piemonte Friuli V.G. Veneto Veneto Marche Umbria Marche Toscana Toscana Toscana Emilia R. Emilia R. Marche Marche Umbria Trento Umbria Lombardia Lombardia Lombardia Trento Emilia R. Emilia R. Emilia R. Emilia R. Umbria Umbria Marche Friuli V.G.
Broiler Broiler Broiler Broiler Broiler Broiler Broiler Turkey Broiler Turkey Broiler Broiler Broiler Broiler Broiler Layer Layer Broiler Broiler Broiler Layer Broiler Broiler Broiler Broiler Broiler Broiler Layer Broiler Broiler Broiler Broiler
Nda Nd 1.8 1.6 2.0 Nd Nd Nd Nd 1.8 Nd Nd 1.8 Nd 2.0 Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd
C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1
Newcastle disease viruse characterization 467 Table 2. Monoclonal antibody binding pattern Monoclonal
14 424 445 479 481 688 A E F G H I
11 23 32 45 49 55 57 67 68 69 70 79 85 J K L M N O P Q R S T U V
3 38 43 54 161 165 W X Y Z £ $
ND isolate + Ulster 2C Pigeon/Eng/617/83 –
+
+
+ +
+
+
+ +
–
–
+
+
+
+ +
–
–
+ +
–
–
+ +
–
–
+ +
+
–
+ +
+
–
+ +
+
–
+ +
+
–
+ +
+
–
+
+ +
C1
+
+
–
+
+
–
–
+
–
+
+
+
+
+
+
+
+
+
+
–
–
–
+
–
+
+ Italy 3015/00 Denmark 96/75317 + Great Britain 97/1 +
+ + +
– – –
+ + +
+ + +
– – –
– – –
+ + +
– – –
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
– – –
– – –
– – –
+ + +
– – –
+ + +
The mAbs used were: A to I, 14, 424, 445, 479, 481, 688 prepared against NDV Ulster 2C; J to V, U11, U23, U32, U45, U49, U55, U57, U67, U68, U69, U70, U79 and U85 prepared against Ulster 2C; W to Z, £ and $, 3/617, 38/617, 43/617, 54/617, 161/617 and 165/617 prepared against isolate PMV 1/ pigeon /England/ 617/83.
Figure 1. Phylogenetic tree: sequences of NDV strains of the 2000 Italian epidemic are compared with sequences of NDV strains previously isolated in Europe and in other countries, mainly in the past 10 years. The latter are labelled according to Alexander et al. ( 1999a) and Herczeg et al. ( 1999). GenBank accession numbers are in italics. The scale bar indicates the expected number of nucleotide substitutions. FIVi 1001/96/1 is representative of viruses isolated in Scandinavia and the UK during 1996 to 1997 ( Alexander et al., 1999a). Italy 3015/00 is representative of the 31 viruses with identical sequences.
468 G. Cattoli et al.
C1 strains are still circulating in Europe despite the limited number of notified outbreaks in the years 1998 and 1999 ( Alexander et al., 1998a, 1999b; Sander 1998; Pittman, 1999) . These findings are supported by the data of Herczeg et al. ( 1999, 2001), who reported NDV strains with a novel genotype in the Republic of South Africa and in Mozambique in 1993 to 1995, that subsequently spread to south-eastern Europe, and then to northern Europe. In fact, the Italian and north-European strains cluster in the VIIb group ( Lomniczi et al., 1998) , together with strains from South Africa ( ZA26/93, ZA360/95) and Bulgaria ( BG431/96) ( nucleotide similarity, 98.0, 96.8 and 99.0%, respectively) . As clearly illustrated in Figure 1, the Italian strains are not closely related genetically to the NDV strains isolated previously in Italy. Owing to the virulence of the strain, it appears rather unlikely that the disease could be misdiagnosed in a na¨õ ve population, and therefore infection may well be circulating in vaccinated birds in circumstances similar to those reported by Capua et al. ( 1993). This is in keeping with the suspected introduction into Italy with imported hatching eggs. Specific surveillance programmes aiming to identify reservoirs of NDV are recommended in the European Union and in Third Countries. In conclusion, the results presented herein support the thesis that velogenic C1 strains have been circulating throughout Europe since 1995–1996, and probably still are. This should be a serious warning for public and company veterinarians, and for farmers who should improve biosecurity and control measures for ND. Acknowledgements The authors are grateful to the diagnostic laboratories and field veterinarians who submitted pathological samples and viral strains, making this study possible. References Alexander, D.J., Manvell, R.J., Lowings, J.P., Frost, K.M., Collins, M.S., Russell, P.H. & Smith, J.E. ( 1997). Antigenic diversity and similarities detected in avian paramyxovirus type 1 ( Newcastle disease virus) isolates using monoclonal antibodies. Avian Pathology, 26, 399–418. Alexander, D.J., Manvell, R.J. & Frost, K.M. ( 1998a). Report of the European Union Reference Laboratories for Avian Influenza and Newcastle Disease 1998. In Proceedings of the Joint Fifth Annual Meetings of the National Newcastle Disease and Avian Influenza Laboratories of Countries of the European Union, 9–10 November 1998 ( pp. 61–66). Vienna, Austria. Alexander, D.J., Morris, H.T., Pollitt, W.J., Sharpe, C.E., Eckford, RL., Sainsbury, R.M.Q., Mansley, L.M., Gough, R.E. & Parsons, G. ( 1998b). Newcastle disease outbreaks in domestic fowl and turkeys in Great Britain during 1997. The Veterinary Record, 143, 209–212. Alexander, D.J., Banks, J., Collins, M.S., Manvell, R.J., Frost, K.M., Speidel, E.C. & Aldous, E.W. ( 1999a). Antigenic and genetic characterisation of Newcastle disease viruses isolated from outbreaks in domestic fowl and turkeys in Great Britain during 1997. The Veterinary Record, 145, 417–421.
Alexander, D.J., Manvell, R.J. & Frost, K.M. ( 1999b). Report of the European Union Reference Laboratories for Avian Influenza and Newcastle Disease 1999. In Proceedings of the Joint Sixth Annual Meetings of the National Newcastle Disease and Avian Influenza Laboratories of Countries of the European Union, 29–30 November 1999 ( pp. 72–77). Brussels, Belgium. Capua, I., Scacchia, M., Toscani, T. & Caporale, V. ( 1993). Unexpected isolation of virulent Newcastle disease virus from commercial embryonated fowls’ eggs. Journal of Veterinary Medicine B, 40, 609–612. Capua, I., Mutinelli, F., Cattoli, G. & Pozzato, N. ( 2001). An overview on the avian influenza and Newcastle disease epidemics in Italy during 1999 and 2000. In Proceedings of the Fiftieth Western Poultry Disease Conference, 24–26 March 2001 ( pp. 8–11). Davis, CA, USA. Council Directive ( 1992). Council Directive 92/66/EEC of 14 July 1992 introducing Community measures for the control of Newcastle disease. Official Journal of the European Communities, L260, 1–20. Felsenstein, J. ( 1993). PHYLIP: phylogenetic inference package version 3.5c. Seattle, WA: University of Washington. Herczeg, J., Wehmann, E., Bragg, R.R., Travassos Dias, P.M., Hadjiev, G., Werner, O. & Lomniczi, B. ( 1999). Two novel genetic groups ( VIIb and VIII) responsible for recent Newcastle disease outbreaks in Southern Africa, one ( VIIb) of which reached Southern Europe. Archives of Virology, 144, 2087–2099. Herczeg, J., Pascucci, S., Massi, P., Luini, M., Selli, L., Capua, I. & Lomniczi, B. ( 2001). A longitudinal study of velogenic Newcastle disease virus genotypes isolated in Italy between 1960 and 2000. Avian Pathology, 30, 163–168. Lomniczi, B., Wehmann, E., Herczeg, J., Ballagy-Pordany, A., Kaleta, E.F., Werner, O., Meulemans, G., Jorgensen, P.H., Mant´e, A.P., Gielkens, A.L.J., Capua, I. & Damoser, J. ( 1998). Newcastle disease outbreaks in recent years in Western Europe were caused by an old ( VI) and a novel genotype ( VII). Archives of Virology, 143, 49–64. Pittman, M., ( 1999). Avian influenza and Newcastle disease in the European community: legislative aspects ( Doc.XXIV/2913/99). In Proceedings of the Joint Sixth Annual Meetings of the National Newcastle Disease and Avian Influenza Laboratories of Countries of the European Union, 29–30 November 1999 ( pp. 83–88). Brussels, Belgium. Rima, B., Alexander, D.J., Billeter, M.A., Collins, P.L., Kingsbury, D.W., Lipkind, M.A., Nagai, Y., Orvell, C., Pringle, C.R. & ter Meulen, V. ( 1995). Paramyxoviridae. In F.A. Murphy, C.M. Fauquet, D.H.L. Bishop, S.A. Ghabrial, A.W. Jarvis, G.P. Martelli, M.A. Mayo & M.D. Summers ( Eds.), Virus Taxonomy. Sixth Report of the International Committee on Taxonomy of Viruses ( pp. 268–274). Vienna: Springer-Verlag. Sander, K. ( 1998). Avian Influenza and Newcastle disease in the European Community: sanitary situation and legislative developments in 1998 to date. In Proceedings of the Joint Fifth Annual Meetings of the National Newcastle Disease and Avian Influenza Laboratories of Countries of the European Union, 9–10 November 1998 ( pp. 67–70). Vienna, Austria.
´ RESUM E´ Caract´erisation des virus de la maladie de Newcastle isol´es en Italie en 2000 Trente deux virus de la maladie de Newcastle ( NDV), isol´es lors de l’´epizootie italienne de 2000, ont e´ t´e caract´eris´es en e´ tudiant leur r´eactivit´e avec un panel d’anticorps monoclonaux et le s´equençage nucl´eotidique du g‘ene de fusion ( approximativement 400 bp). De plus, la pathog´enicit´e de six de ces isolats a e´ t´e e´valu´ee par le test de pathog´enicit´e intrac´er´ebrale ( ICPI). Les souches e´tudi´ees ont pr´esent´e un ICPI variant de 1,6 ‘a 2,0. La r´eactivit´e de tous les isolats avec les anticorps monoclonaux a permis de les classer dans le groupe C1. Les deux tests, r´eactivit´e avec les anticorps monoclonaux et l’analyse g´enomique ont mis en e´ vidence un degr´e e´ lev´e d’homologie entre les souches, indiquant une source d’infection commune. L’analyse phylog´en´etique a mis en e´ vidence que les isolats italiens sont proches des
Newcastle disease viruse characterization 469 souches isol´ees r´ecemment en Grande Bretagne, Scandinavie et Sud Est de l’Europe, sugg´erant ainsi la circulation de cette souche virale en Europe depuis les cinq derni‘eres ann´ees.
die Zirkulation dieses Virusstammes in Europa im Laufe der vergangenen 5 Jahre schließen l¨asst. RESUMEN
ZUSAMMENFASSUNG Charakterisierung von Newcastle-Disease-Virusst a¨ mmen, die im Jahr 2000 in Italien isoliert wurden Zweiunddreißig Newcastle-Disease-Virus ( NDV)-Isolate aus der italienischen Epidemie im Jahr 2000 wurden durch die Bindungsmuster monoklonaler Antik¨o rper und die Nukleotid-Sequenzierung von etwa 400 bp des Fusionsgens charakterisiert. Außerdem wurde die Pathogenit¨at von sechs dieser Isolate mittels intrazerebralem Pathogenit¨atsindex ( ICPI) beurteilt. Die untersuchten St¨amme hatten einen ICPI von 1,6 bis 2,0. Aufgrund der Bindungsmuster der monoklonalen Antik¨orper konnten alle Isolate als Angeh¨orige der Gruppe C1 klassifiziert werden. Sowohl die Untersuchungen mit monoklonalen Antik¨orpern als auch die Genom-Analysen zeigten eine sehr hochgradige Homologie, was auf eine gemeinsame Infektionsquelle hindeutete. Aufgrund der phylogenetischen Analyse hat es den Anschein, dass die italienischen Isolate mit den j¨u ngsten Isolaten aus Großbritannien, Skandinavien und S¨u dosteuropa eng verwandt sind, was folglich auf
Caracterizaci´on de virus de enfermedad de Newcastle aislados en Italia en el 2000 Treinta y dos cepas de virus de Newcastle de una epidemia localizada en Italia durante el an˜ o 2000 fueron caracterizadas mediante el patr´on de uni´o n de anticuerpos monoclonales y la secuencia de nucle´otidos de aproximadamente 400 pares de bases pertenecientes al gen de fusi´on. Adem´as, la patogenicidad de seis de estas cepas fue probada mediante el test de patogenicidad intracerebral ( ICPI). Las cepas probadas exhib´õ an un ICPI de 1.6 a 2.0. Seg´un el patr´on de uni´on de los anticuerpos todas las cepas fueron clasificadas dentro del grupo C1. Tanto los anticuerpos monoclonales como el an´alisis gen´omico revelaron un alto grado de homolog´õ a, indicando una fuente com´un de infecci´o n. Segu´ n el an´alisis filogen´etico, parece que las cepas italianas se encuentran muy relacionadas con las cepas recientemente aisladas en el Reino Unido, Escandinavia y el sudeste de Europa, lo que sugiere que ha existido circulaci o´ n de estas cepas v´õ ricas en Europa en los u´ ltimos cinco an˜ os.
