Veterinary Microbiology 94 (2003)

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Genetic analysis of Newcastle disease virus strains

isolated in Bosnia-Herzegovina, Croatia, Slovenia

and Yugoslavia, reveals the presence of only a

single genotype, V, between 1979 and 2002

Enik

´

o

´

Wehmann

a

, Dorina Ujvári

a

, H. Mazija

b

, Maja Velhner

c

,

Irena Ciglar-Grozdani´c

b

, V. Savi´c

d

, Gordana Jermolenko

e

, Ž. ˇ

Caˇc

f

,

Estella Prukner-Radovˇci´c

b

, B. Lomniczi

a,∗ a_{Veterinary Medical Research Institute of the Hungarian Academy of Sciences,}

P.O. Box 18, 1581 Budapest, Hungary

b_{Department of Avian Pathology, Faculty of Veterinary Medicine, University of Zagreb,}

Heinzelova 55, 10000 Zagreb, Croatia

c_{Scientific Veterinary Institute, Rumenacki Put 6, 21000 Novi Sad, Yugoslavia} d_{Croatian Veterinary Institute, Poultry Centre, Heinzelova 55, 10000 Zagreb, Croatia}

e_{Scientific Veterinary Institute, Vojvode Toze 14, 11000 Beograd, Yugoslavia} f_{Department of Virology, Croatian Veterinary Institute, Savska 143, 10000 Zagreb, Croatia} Received 11 July 2002; received in revised form 12 November 2002; accepted 12 December 2002

Abstract

Newcastle disease (ND) epizootics in some European countries after the World War II were caused by ND virus (NDV) of multiple genotypes (IV–VIIa) occurring sequentially and/or simultaneously. This study was carried out to characterise the genetic composition of NDV strains during the outbreaks in the territory of the former Yugoslavia in order to enhance our understanding of the relationships of past epizootics in Europe. Sixty-eight NDV strains isolated between 1979 and 2002 were anal-ysed by restriction enzyme digestion and partial sequencing of the fusion protein gene. All isolates were placed in genotype V, an exotic type, that was introduced to western Europe in 1970. Residue substitution analysis has allowed the recognition of four genetic variants, Vb1–Vb4, and the tracing of their movements. Vb1, a dominant variant in Bulgaria from the late 1970s, was also wide spread in the former Yugoslavia throughout the period under investigation. Vb2, a variant occurring in the neighbouring countries in the early 1970s could be the founder of the epidemic in Yugoslavia and it was present up to the late 1980s. Variants Vb3 and Vb4 could be found only after 1987. In conclusion, the ND outbreaks in Yugoslavia were part of the epizootic wave due to genotype V viruses that started

∗_{Corresponding author. Tel.:}₊_{36-1-4674069; fax:}₊_{36-1-2521069.}

E-mail address: [email protected] (B. Lomniczi).

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270 E. Wehmann et al. / Veterinary Microbiology 94 (2003) 269–281

in western Europe in 1970 and became endemic in the region. Inter-country transmission occurred for all variants, and Vb3 and Vb4 might have evolved during the endemic period.

Keywords: Newcastle disease virus; F gene sequences; Epidemiology

1. Introduction

Newcastle disease (ND) is classified as a list A contagious disease of poultry by the Office International des Epizooties (OIE). It is caused by virulent strains of Newcastle disease virus (NDV), a member of avian paramyxovirus serotype 1. NDV belongs to the genus Avulavirus of the family Paramyxoviridae in the order of Mononegavirales (Mayo, 2002).

The global situation of ND is characterised by enzootic infection in many developing countries where village chickens are considered to be the main reservoir of the virus (Awan et al., 1994). These populations of chickens serve as the source of infection for ND epi-zootics occurring in disease-free areas, even in other continents. The first reported wave of ND in Europe, at around the beginning of the World War II, was part of the world-wide dis-semination of the disease also referred to as the first panzootic (Hanson, 1974; Alexander, 1988). Genetic analysis of NDV strains isolated before the 1960s, however, revealed the occurrence of at least three genetic lineages (genotypes II–IV) that also reflected geograph-ical associations (Toyoda et al., 1989; Ballagi-Pordány et al., 1996). Consistent with the above findings, longitudinal analysis of NDV strains from Italy and Bulgaria has revealed that genotype IV viruses were prevalent before the 1970s, but thereafter ND epizootics were characterised by the presence of multiple genotypes (IV–VIIa and VIIb) occurring sequentially and/or simultaneously (Herczeg et al., 2001; Czeglédi et al., 2002). A devas-tating wave of outbreaks commenced in Europe in 1970 supposedly caused by the same virus that was transmitted by imported psittacine birds to California in 1971 (Walker et al., 1973; Francis and Efren-Rivelli, 1972; Russell and Alexender, 1983). The causative agent of this epizootic in some European countries was classified into genotype V (Ballagi-Pordány et al., 1996; Herczeg et al., 2001; Czeglédi et al., 2002). Between 1992 and 1995, a less severe wave of infection occurred in western Europe, and was caused by VIIa viruses which were prevalent in the Far East (Lomniczi et al., 1998; Yang et al., 1999). Sporadic cases due to viruses belonging to genotypes VI and VIIb were also identified in some European countries (Jørgensen et al., 1998; Alexander et al., 1999; Cattoli et al., 2001; Herczeg et al., 2001; Wehmann et al., 2003).

ND was first reported in the territory of the former Yugoslavia in 1941 at Savski Marof, near Zagreb, in connection with the movement of Italian and German troops (Hupbauer and Topolnik, 1944). The severe epizootic that followed was partly brought under control in the mid-1950s by using mesogenic vaccines (Fiolic, 1957). Notwithstanding, the number of reported cases of ND outbreaks fluctuated between approximately 500 and 1500 per year throughout the 1960s. This picture was rather typical for the ND situation of a number of other European countries. However, while in other areas a significant reduction of ND cases was achieved by the second half of the 1960s, in Yugoslavia the incidence diminished to 200–300 cases per year only a decade later (Anonymous, 1961–1985). No reliable data

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are available on the incidence of ND in the succession states, Bosnia-Herzegovina (BH), Croatia (HR), Slovenia (SL) and Yugoslavia, but in HR the number of reported cases was in the range of 10–100 in the period between 1987 and 1992 (Mazija and Rukavina, 1992). In an effort to enhance our understanding on the history and events of ND epizootics in Europe, genetic analysis of 68 NDV strains isolated in the territory of the former Yugoslavia after 1979 was performed. The main questions addressed were: What genotypes of NDV were responsible for outbreaks during the past decades? Was there any parallel between the prevalence of genotypes here and in other countries in Europe?

2. Materials and methods

2.1. Viruses

All NDV strains were isolated from chicken flocks. Isolates received from the laboratories in the territory of the former Yugoslavia were analysed in the Veterinary Medical Research Institute, Budapest. Viruses were plaque purified before propagating in embryonated eggs and RNA extraction. In order to indicate the geographical origin of the viruses, they were labelled with a two-letter country code for the succession states of Yugoslavia, BH, HR, SL as well as abbreviations Sr (Serbia) and Vo (Vojvodina) were used for provinces of Yugoslavia (YU). The designation and origin of viruses are listed inTable 1. Designations were kept for already published sequences (Lomniczi et al., 1998).

2.2. Preparation of viral RNA, reverse transcription (RT), polymerase chain reaction (PCR) and restriction enzyme cleavage site analysis

These procedures were performed as described (Ballagi-Pordány et al., 1996; Lomniczi et al., 1998). Briefly, a 1349 bp portion (approximately 75%) of the fusion (F) protein gene (nucleotides 334 and 1682) was amplified by RT-PCR and products digested with restriction enzymes HinfI, BstOI or RsaI. DNA fragments were separated by agarose gel electrophoresis and physical maps of the fragments and restriction sites were constructed. Group specific cleavage sites or combinations of sites were described in detail in previous papers (Ballagi-Pordány et al., 1996; Lomniczi et al., 1998; Herczeg et al., 1999).

2.3. Sequence analysis

A portion of the F gene (between nucleotides 47 and 420) was sequenced as described (Lomniczi et al., 1998) and phylogenetic analysis was performed by the program TREECON for Windows 1.3b that created a distance tree by the neighbour-joining algorithm (Van de Peer and de Wachter, 1997). Distance values were taken from a distance matrix (not shown) prepared by CLUSTAL W of the MEGALIGN program. Nucleotide sequences of NDV strains were deposited in the GenBank and accession numbers are shown inTable 1. Accession numbers of further strains are: AY117015 (HU-27/79), AY117016 (HU-457/81), AY117017 (HU-22/82), AY117018 (HU-232/83), AY117019 (HU-242/84) and AY117020 (HU-432/86).

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Table 1

Designation, origin and grouping of NDV strains isolated in BH, HR and Yugoslavia (Sr and Vo) listed by year of isolations

Designation of strains Place of isolation Genetic grouping based on Accession number

This paper Original RSPa N/AaSPb

HR-448/79 448 Slavonija Vb Vb2 AY116971

HR-567/81 123 Virovitica Vb/1 Vb1 AY116972

BH-568/81 897 Brˇcko Vb Vb2 AY116973

HR-123/81 123 Novi Marof –c Vb2 AY116974

HR-897/81 897 Virovitica Vb –

HR-569/82 1 Varaždin Vb/1 Vb1 AY116975

HR-571/83 147 Slavonski Brod Vb/2 Vb2 AY116976

HR-572/83 188 Darda Vb/1 Vb1 AY116977

HR-116/83 116 Osijek area Vb Vb2 AY116978

HR-233/83 233 Osijek area Vb – HR-853/83 853 Osijek area Vb – HR-268/84 V-616 Križevci Vb/2 Vb2 AY116979 HR-270/84 V-815 Varaždin – Vb2 AY116980 HR-271/84 V-816 Djakovo Vb – HR-272/84 V-847 Cakovecˇ Vb/2 Vb2 AY116981 HR-273/84 V-1023 Samobor Vb/2 Vb2 AY116982 HR-275/84 V-1247 Vrbovec Vb/2 – HR-433/84 433 Darda Vb Vb2 AY116983

YU(Vo)-251/84 539 Stara Pazova – Vb2 AY116984

YU(Sr)-291/84 n.a.d _Karlovci _Vb _–

YU(Sr)-292/84 n.a. Selevac Vb/1 –

YU(Sr)-293/84 n.a. Velika Plana Vb Vb2 AY116985

YU(Sr)-295/84 n.a. Zajeˇcar Vb –

HR-579/86 697 Darda Vb Vb3 AY116988

HR-207/87 207 n.a. Vb Vb2 AY116989

HR-580/87 5 Zagreb area Vb –

HR-581/87 40 Zagreb area Vb/2 –

HR-582/87 92 Split Vb Vb4a AY116990

HR-583/87 137 Darda Vb Vb4a AY116991

HR-585/87 390 Slavonski Brod Vb/1 –

BH-587/87 521 Bosanski Brod Vb/1 Vb1 AY116992

BH-590/87 635 Bosanski Brod Vb/1 –

HR-591/88 318 Zagreb area Vb Vb4a AY116993

HR-596/88 1064 Varaždin Vb –

SL-592/88 444 Sabota Vb Vb4a AY116994

YU(Vo)-593/88 6503 Baˇcka Topola Vb/1 Vb1 AY116995

BH-594/88 605 Bosanski Brod – Vb1 AY116996

YU(Vo)-A/89 7401 Novi Sad Vb Vb3 AY116997

YU(Vo)-B/89 8259 Mol Vb Vb3 AY116998

HR-40/90 40 Varaždin Vb/1 Vb1 AY116999

HR-41/90 41 Darda Vb Vb4a AY117000

HR-42/90 42 Vinkovci Vb/1 –

HR-44/90 44 Darda – Vb1 AY117001

HR-388/90 388 Varaždin Vb Vb4a AY117002

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Table 1 (Continued )

Designation of strains Place of isolation Genetic grouping based on Accession number

This paper Original RSPa _N/AaSPb

YU(Vo)-D/92 17431 Baˇc Vb –

YU(Vo)-E/92 17119 Beˇcej Vb Vb4b AY117004

YU(Sr)-4/92 4 Zajeˇcar Vb/4 Vb4b AY117005

YU(Sr)-9/92 9 Požarevac Vb/4 –

YU(Sr)-11/92 11 Kovaˇcevac – Vb4b AY117006

YU(Sr)-7/93 7 Šabac – Vb1 AY117007

YU(Vo)-1/94 787 Temerin Vb/4 Vb4b AY117008

YU(Vo)-4/94 738 Beˇcej Vb/4 Vb4b AY117009

YU(Vo)-5/94 786 Durdevo Vb –

YU(Vo)-8/94 763 Petrovac Vb/4 –

YU(Vo)-10/94 680 Beˇcej Vb –

YU(Vo)-14/94 909 Baˇcko Petrovo Selo Vb –

HR-Zel/94 155 Zelina Vb Vb4a AY117010

YU(Vo)-6/95 261 Baˇcka Topola Vb/4 –

YU(Vo)-7/95 314 Kanjiža Vb Vb4a AY117011

YU(Vo)-11/95 691 Beˇcej Vb –

YU(Vo)-12/95 857 Baˇcka Palanka Vb –

YU(Vo)-13/95 891 Titel Vb Vb4b AY117012

YU(Sr)-3/95 3 Šabac Vb/4 Vb4b AY117013

YU(Sr)-6/95 6 Požarevac Vb Vb4b AY117014

YU(Vo)-1744/02 1744 Melenci Vb Vb4b AF525396

a_{Restriction site pattern (see}_{Fig. 2}_). b_{Nucleotide and amino acid signature pattern.} c_{Not done.}

d_{Not available.}

3. Results

Sixty-eight NDV strains isolated in the territory of the former Yugoslavia between 1979 and 2002 were subjected to genetic analyses. Viruses were screened by restriction enzyme cleavage site distribution followed by sequence analysis of representative isolates. Data of NDV strains and the results of their genetic analyses are summarised inTable 1. Surprisingly, all isolates belonged to genotype V that comprised viruses derived during the 1970–1974 epi-zootic in Europe and California (Ballagi-Pordány et al., 1996; Herczeg et al., 2001; Czeglédi et al., 2002). Generally, genotype V viruses can be assigned into two subtypes exhibiting restriction site patterns Va or Vb based on the lack or the presence of an RsaI cleavage site at nucleotide position 540 (Fig. 1). It is interesting that while strains isolated in the region all displayed pattern Vb, those derived from western Europe and during the early 1970s (with the exception of Northants’72) showed pattern Va (Wehmann, 2000; Czeglédi et al., 2002). Sequence analysis has also confirmed the placement of the local NDV strains in genotype V (Fig. 2). As shown by the dendrogram, lineage V contained closely related isolates (variation in nucleotide sequence is within 3%), and only two clusters, Vb1 and Vb3, were resolved clearly in the branch. In order to reveal the finer genetic composition of subgroup Vb, residue substitution analysis was also performed (Fig. 3). This has revealed the existence

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274 E. W ehmann et al. /V eterinary Micr obiolo gy 94 (2003) 269–281

Fig. 1. Physical maps of restriction fragments and cleavage sites of the F genes of NDV variants in subtypes Va and Vb. Normal numerals above the line designate the last nucleotide of a fragment except 334 that is the starting position of the amplified region. Italics below the line mark fragment length in base pairs.

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Fig. 2. Distance tree of the nucleotide sequences of NDV strains based on nucleotides between 47 and 420 of the F gene. Accession number of strains in bold types are shown inTable 1. Sequences of strains with normal letters were taken from the GenBank (see also inHerczeg et al., 2001; Czegl´edi et al., 2002). Groupings including subtypes and variants in genotype V are indicated on the right.

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Fig. 3. Nucleotide and amino acid signature sequences of the F genes of NDV variants Vb1–Vb4 derived in the territory of the former Yugoslavia and in some of the neighbouring countries.

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Fig. 4. Geographical distribution of genotype V variants of NDV in the territory of the former Yugoslavia and in some of the neighbouring countries: (䊏) Vb1 (<1981–1993); (䊉) Vb2 (<1979–1987); ( ) Vb3 (1986–1998); () Vb4a (1987–1995); (䉱) Vb4b (1991–2002).

of four discernible genetic variants, Vb1–Vb4, each with characteristic nucleotide and amino acid signature patterns. The clusters comprising these variants are marked in the phylogenetic tree. The geographical distribution of the NDV variants are shown inTable 1 andFig. 4. Strains belonging to the most diverged cluster, Vb1, were already described in Bulgaria (Czeglédi et al., 2002). These viruses can be identified by restriction site markers alone: the extra HinfI site at position 1648 and the lack of the BstOI site at nucleotide 1116 (see patterns Vb/1 inFig. 1). Their distinctiveness is also indicated by nucleotide exchanges at three positions and a Lys4 → Arg substitution (Fig. 3) (even the 46 nu-cleotides of the non-coding region of the F gene displayed four exchanges, in addition to those at the coding region (data not shown)). The earliest known Vb1 isolate, BG-25/78, derived from Bulgaria where this was the most prevalent variant between 1978 and 1988 (Czeglédi et al., 2002). The Vb1 viruses appeared to be continuously present in the territory of the former Yugoslavia (mainly in HR) between 1981 and 1993. Cluster Vb2 contained strains that apparently derived from the period prior to 1987. By this time, the variant was scattered throughout the region: in HR, northern Sr (Vo), mid-Sr and BH. A restriction site variant, designated as Vb/2, of the cluster Vb2 was detected in different locations in 1983–1984 (seeFig. 1). Furthermore, members of Vb2 were already present in Hungary (HU-10/72) and Italy (IT-47/74), but apparently not in Bulgaria, at the early 1970s, shortly after the time when group V viruses entered these countries. Cluster Vb3 comprised vir-tually identical strains deriving from the eastern part of HR and Vo in 1986 and 1989. An identical isolate from Hungary (HU-432/86) indicated epidemiological link. This vari-ant is characterised by a Leu28 → Pro substitution and t → c transitions at nucleotide positions 83, 141 and 237. Finally, cluster Vb4 is characterised by a Gly104 → Glu sub-stitution and a number of nucleotide changes. Three exchanges (g45 → a, a195 → g and c294 → t) further divide this group into Vb4a and Vb4b (Fig. 3). The first com-prised strains from HR, SL and Vo between 1987 and 1995, whereas members of Vb4b

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were exclusively isolated between 1991 and 2002 in Sr and Vo. The majority of Vb4b viruses shared restriction site patterns, designated as Vb/4 (Fig. 1). This clade also com-prised a much earlier isolate from Bulgaria (BG-90/77) indicating a possible source of these viruses.

In previous work, we have reported the use of shared derived amino acids of the different genotypes and found that Val105 → Ala and Ile118 → Val substitutions were specific to a limited number of group V isolates (Lomniczi et al., 1998; Herczeg et al., 1999). Now this observation has been extended to over 50 strains, implying that these positions can be utilised in the identification of genotype V viruses (Fig. 3). The amino acid motif at the F1/F2 cleavage site was112RRQKR/FVG119. (The sole exception was strain Northants’72 from England that had residue R115.) It is interesting that the amino acid replacements K4→R, L28→P and G104→E, specifying clusters Vb1, Vb3 and Vb4, respectively, are apparently ancestral type residues in that they are characteristic of genotypes II–IV viruses that were responsible for epizootics before the 1970s (Lomniczi et al., 1998).

Distance values of strains with known isolation times have allowed to estimate the ap-proximate rate of nucleotide exchange under field conditions (values were taken from a distance matrix; not shown). The intracluster variations in Vb1 were in the range of 0–1.1% in a 15-year period (e.g. 1.1% between BG-25/78 and YU(Sr)-7/93), 0.3–1.4% per 16 years for Vb2 (e.g. 1.4% between HU-10/72 and IT-129/88 or 0.5% between HU-10/72 and HR-207/87) and 0–1.9% in a 25-year time span for Vb4 (e.g. 0.8% between BG-90/77 and YU(Vo)-1/94, or 1.1% between BG-90/77 and YU(Vo)-1744/02, or 0.3% between HU-457/81 and YU(Vo)-7/95). Therefore an approximately 1% per decade rate of change in nucleotide sequences can be inferred under field conditions. Variations between variants Vb2 and Vb3 or Vb4 are around or within 1% again whereas divergence between Vb1 and the others are in the range of 1–3%.

4. Discussion

In the previous studies, it has been shown that in the neighbouring countries of the former Yugoslavia, as well as Bulgaria, Italy and Hungary, genotype IV viruses were dominant in the period prior to 1970 and they persisted until the 1980s (Ballagi-Pordány et al., 1996; Wehmann, 2000; Herczeg et al., 2001; Czeglédi et al., 2002). Consistent with these find-ings, group IV viruses also disclosed area specific divergence indicating long-term local evolution due to endemic infection (Fig. 2). Although these data support the notion that the European branch of the first panzootic was most likely maintained by genotype IV viruses they apparently did not survive in Yugoslavia, even if had been present before as in Italy and Hungary, until the 1980s.

The occurrence of genotype V viruses from the early 1970s in other European coun-tries, such as Bulgaria, England, Germany, Hungary and Italy, was reported previously (Ballagi-Pordány et al., 1996; Herczeg et al., 2001; Czeglédi et al., 2002). The relatively narrow overall range of nucleotide sequence variation of genotype V strains (<3%) is typical for viruses deriving from an epizootic type of infection (Holmes et al., 1995). Consequently, the results of the current study have demonstrated that the outbreaks in Yugoslavia were part of the European epizootic wave that started in western Europe in the middle of 1970,

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and spread south- and eastward reaching other countries in the subsequent years (Kelly, 1973; Anonymous, 1973).

A notable feature of the ND situation in the territory of the former Yugoslavia was that genotype V viruses, exotic in Europe, have survived in the area over two decades as opposed to some other countries where the infection was shortly eradicated. The high proportion of extensively reared poultry in Yugoslavia contributed to the inability to eradicate ND which led to the unusually long endemic period (Djunin et al., 1982; Mazija et al., 1985). Since Va strains seemed to be more frequent in the early 1970s in other countries, the question arose whether the two subgroups, Va and Vb, were introduced independently, or viruses with Va patterns mutated to Vb during the epizootic period in Europe, and this is why Vb strains were over-represented in the later phase. The latter possibility is less likely because there is evidence that the two varieties were introduced to Europe at around the same time. For instance, in addition to isolate Northants’72, other Vb strains (HU-10/72 and IT-47/74) were already present at the beginning of the epizootic in Hungary and Italy (Wehmann, 2000; Herczeg et al., 2001). In the absence of early isolates, however, it is a matter of speculation whether subtype Va was introduced into the former Yugoslavia at all. If it was, it seems that it disappeared by the 1980s, as happened in Italy and Bulgaria (Herczeg et al., 2001; Czeglédi et al., 2002).

The significance of the recognition of clusters comprising very closely related viruses lies in their power to confer direct evidence for the extent of epidemiological links between outbreaks at different locations and/or time points. In the early 1980s, at least two genetic variants, Vb1 and Vb2, circulated in HR and Sr. Vb1 was shown to be the dominant variant in Bulgaria from the late 1970s onwards (first isolate: BG-25/78), then also persisted in Yugoslavia between 1981 and 1993 (Czeglédi et al., 2002). It is assumed that Vb2 viruses could be the founders of the NDV infection in the wider area as attested by the presence of this variant in the neighbouring countries (HU-10/72 and IT-47/74) and the lack of early (<1975) isolates among the other Vb variants. Consequently, the movement of Vb2 viruses, too, between the former Yugoslavia, Hungary and Italy, must have been taking place. Variant Vb2 was replaced by Vb4a later in the decade, and the latter could be detected not only in the different regions of HR between 1987 and 1995 but in Vo (YU(Vo)-7/95), as well. Circumstantial evidence supports the notion that variant Vb4b (occurring between 1991 and 2002) also came from Bulgaria. Firstly, a much earlier isolate, BG-90/77, is only less than 0.5% mutational distance away from the Yugoslavian isolates a decade later. Secondly, these variants were confined to Yugoslavia, which is the immediate neighbour of Bulgaria and were absent from HR. It appears that separation of the countries resulted in the segregation of the genetic variants as well. Although such findings do confirm epidemiological connections between adjacent areas, in the absence of a more complete range of isolates the direction of spread cannot always be determined, as in the case of variant Vb1. The complex aetiology of the epizootic can also be revealed by reference to specific locations. For instance, in Darda, an intensive farming area in eastern HR, all four variants, Vb1 (1983), Vb2 (1984), Vb3 (1986) and Vb4a (1987) were involved in maintaining infection.

As for the origin of genetic variants, an important question is whether the viruses were introduced independently to Europe, or they emerged de novo during the epizootic started in the early 1970s. Knowing the rate of change of nucleotides helps to address this problem. In the current work several examples supported an approximately 1% rate of nucleotide change

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per decade, and this has confirmed our earlier findings concerning the field evolution of isolates derived from Bulgaria (Czeglédi et al., 2002). Working with this value, it is possible that Va, the dominant variant in western Europe, as well as Vb1 and Vb2 were introduced separately, while variants Vb3 and Vb4 emerged during the epizootic period in Europe or even locally. It has to be noted, that the situation of poultry management in Yugoslavia was rather favourable for the evolution of NDV. The unusually long endemic period (at least 23 years) allowed viruses to pass through innumerable generations and novel variants to emerge. The lack of isolates in Yugoslavia prior to 1979 does not preclude the possibility that genotype V viruses were introduced early in the decade as in other countries. Another interesting feature of the epidemiology of ND in the former Yugoslavia was that apparently no old European (group IV) or exotic genotypes could be detected here, such as genetic groups VI and VIIb in Bulgaria and Italy, or VIIa, in western Europe (Lomniczi et al., 1998; Alexander et al., 1999; Cattoli et al., 2001; Herczeg et al., 2001; Czeglédi et al., 2002).

It can be concluded that the genetic composition of NDV strains in the territory of the former Yugoslavia was unique when compared to that in other ND epizootics in Europe. Viruses prevailing in the past two decades belonged exclusively to genotype V (restriction pattern Vb). Fine genetic analyses, such as nucleotide and amino acid signature pattern determination combined with restriction site evaluation revealed several genetic variants and their movement in the region. It appears that some of these variants might have emerged during the genotype V European epizootic or locally, and still persist in the region.

Acknowledgements

Part of this work was supported by grants from the National Science Fund (OTKA, Nos. T 025297 and T 034917), Hungary. The authors thank Dr. L. Tekes, for the generous support by the Central Veterinary Institute, Budapest, Hungary; Dr. N.J. Dimmock, Depart-ment of Biological Sciences, University of Warwick, Coventry, United Kingdom, Dr. Éva Nagy, Department of Pathobiology, University of Guelph, Ontario, Canada, for reading the manuscript and Dr. T. Szamkó, for preparing the map.

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