B. Lomniczi

Newcastle disease outbreaks in recent years in Western Europe were caused by an old (VI) and a novel genotype (VII)

SummaryNewcastle disease virus (NDV) strains, isolated from outbreaks during epizootics between 1992 and 1996 in Western European countries, were compared by restriction enzyme cleavage site mapping of the fusion (F) protein gene between nucleotides 334 and 1682 and by sequence analysis between nucleotides 47 and 435. Both methods revealed that NDV strains responsible for these epizootics belong to two distinct genotypes. Strains derived from sporadic cases in Denmark, Sweden, Switzerland and Austria were classified into genotype VI [6], the same group which caused outbreaks in the Middle East and Greece in the late 1960’s and in Hungary in the early 1980’s. In contrast, viruses that caused epizootics in Germany, Belgium, The Netherlands, Spain and Italy could be classified into a novel genotype (provisionally termed VII), hitherto undetected in Europe. It is possible that the genotype VII viruses originated in the Far East because they showed a high genetic similarity (97%) to NDV strains isolated from Indonesia in the late 1980’s.

Identification and grouping of Newcastle disease virus strains by restriction site analysis of a region from the F gene

SummaryA 75% region of the F gene (between nucleotides 334 and 1682) of Newcastle disease virus (NDV) RNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR). PCR products were cleaved by three restriction endonucleases and the positions of thirty cleavage sites were mapped in more than 200 NDV strains. Restriction site analysis established six major groups of NDV isolates and unique fingerprints of vaccine strains. Group I comprised lentogenic strains isolated mainly from waterfowl with some from chickens. “Old” (prior to 1960s) North American isolates of varying virulence including lentogenic and mesogenic vaccine strains belonged to group II. Group III included two early isolates from the Far East. Early European strains (Herts 33 and Italien) of the first panzootic (starting in the late 1920s) and their descendants with some modifications were placed into group IV. NDV strains isolated during the second panzootic of chickens (starting in the early 1960s) were classified into two groups. Group V included strains originating in imported psittacines and in epizootics of chickens at the early 1970s. Group VI comprised strains from the Middle East in the late 1960s and later isolates from Asia and Europe. Pigeon paramyxovirus-1 strains that were responsible for the third panzootic formed a distinct subgroup in group VI. Our grouping of NDV strains has confirmed group differences established by monoclonal antibodies. It is concluded that restriction site analysis of F gene PCR amplicons is a relatively fast, simple and reliable method for the differentiation and identification of NDV strains.

Two novel genetic groups (VIIb and VIII) responsible for recent Newcastle disease outbreaks in Southern Africa, one (VIIb) of which reached Southern Europe

Summary 34 strains of Newcastle disease virus (NDV) isolated during epizootics in the Republic of South Africa and in Mozambique between 1990 and 1995, and in Bulgaria and Turkey in 1995–1997 were identified by restriction enzyme and partial sequence analysis of the fusion (F) protein gene. The majority of isolates in southern Africa and those from Bulgaria and Turkey were placed into a novel group which has been termed VIIb. Group VIIb is part of a larger genetic cluster (VII) that also includes NDV strains from the Far East and some western European countries (VIIa). The genetic distance of 7–8, 5% between genotype VIIa and VIIb viruses excludes the existence of a direct epidemiological link between recent southern African epizootics and outbreaks in either western Europe in the 1990’s or those of the Far East. Another hitherto unrecorded genotype (VIII) was also found in South Africa with descendants of putative ancestral members isolated in the 1960’s. The genetic distance of recent group VIII strains from the major epizootic genotype (VIIb) is over 11%, therefore outbreaks caused by them were epidemiologically unrelated. Genotype VIII viruses must have been maintained in South Africa by endemic infections during the past decades while group VIIb appears to be introduced more recently.

Phylogenetic analysis reveals extensive evolution of avian paramyxovirus type 1 strains of pigeons (Columba livia) and suggests multiple species transmission

Partial sequence and residue substitution analyses of the fusion protein gene were performed for 68 strains of avian paramyxovirus type 1 of pigeons (PPMV-1), an antigenic variant of Newcastle disease virus (NDV) of chickens, derived from 16 countries between 1978 and 2002. The majority of isolates clustered into a single genetic lineage, termed VIb/1, within genotype VI of NDV strains of chickens, whereas a small number of isolates that originated in Croatia after 1995, grouped in a highly diverged lineage, termed VIb/2, indicating a separate host-switching event from that of VIb/1 strains. Four distinct subgroups of lineage VIb/1, Iraqi (IQ), early European (EU/ea), North American (NA) and recent European (EU/re) have emerged and circulated in the past decades. Subgroup EU/ea and NA strains were responsible for the main streams of infection in the 1980s, while EU/re viruses for infections in the 1990s. The degree of genetic diversity of viruses in the early phase of the epizootic suggested a prolonged infection period of the pigeon-type viruses prior to the emergence of the disease in the early 1980s. Shared derived character analysis showed a close genetic relationship to Sudanese viruses from the mid-1970, suggesting that PPMV-1 viruses could be of African origin.

The occurrence of five major Newcastle disease virus genotypes (II, IV, V, VI and VIIb) in Bulgaria between 1959 and 1996.

Partial sequence and restriction enzyme cleavage site analyses of the fusion protein gene were used to genotype 47 Newcastle disease virus strains isolated between 1959 and 1996 in Bulgaria. Viruses belonged to five major genotypes that appeared to be associated with epizootics characterized by temporal and/or geographical restrictions. Genotype IV viruses (responsible for the European branch of the first panzootic) dominated the scene up to the early 1980s, interspersed with sporadic outbreaks caused by genotype II (US strains causing pneumoencephalitis) viruses. Genotype V viruses (transmitted by psittacines from South America) were first shown in 1973 and persisted until the late 1980s. Genotype VI (earliest members from the Middle-East 1968/70 outbreaks) was represented by scattered isolations between 1974 and 1996. A genotype VIIb (recent Middle East epizootic) virus was isolated as early as in 1984. Newcastle disease epizootics in Bulgaria were highlighted by multiple infection with more than one genotype at any one time.

A longitudinal study of velogenic Newcastle disease virus genotypes isolated in Italy between 1960 and 2000

Thirty-six representative velogenic strains of Newcastle disease virus isolated in Italy since 1960 were characterized by restriction site and partial sequence analyses of the fusion protein gene. Viruses belonging to the six known genotypes of Lomniczi et al . were found. Genotype IV, which was most probably the main epizootic group in Europe before the war, was responsible for outbreaks in the 1960s and persisted until the late 1980s in Italy. An epizootic peak in 1972 to 1974 coincided with the appearance of genotype V viruses that were present for more than a decade. Outbreaks in 1992 were caused by genotype VIIa viruses and were part of a contemporaneous epizootic of Far East origin that affected Western European countries. The Newcastle disease epizootic that commenced in Italy in May 2000 was due to a genotype VIIb virus that is indistinguishable from those causing sporadic outbreaks in Great Britain and Northern Europe in the late 1990s. Isolated cases yielded a variant of genotype VI (reference epizootic: Middle East in the late 1960s) and a group VIII virus (enzootic in South Africa).

Multiple defects in the genome of pseudorabies virus can affect virulence without detectably affecting replication in cell culture

Several independently isolated vaccine strains of pseudorabies virus were studied to identify the functions that play a role in the expression of virulence of this virus. All the strains that were studied grew well in three different cell types. No differences that could be correlated with avirulence could be detected either in the virus yield produced by the cells or in the length of the eclipse phases. All the attenuated strains, however, had lost their ability to replicate efficiently in the brains of day-old chickens. The defects leading to the decrease in the virulence for day-old chickens varied in the different vaccine strains. The Tatarov vaccine strain is defective in the thymidine kinase (TK) gene; restoration of a functional TK gene restores to this strain its virulence for day-old chickens and for pigs. Three out of four different, independently isolated avirulent strains were found to be defective in different loci, as determined by their ability to generate virulent recombinants. Two strains, Bartha and Buk Z300, however, yielded few virulent recombinants, indicating that they were defective in at least one closely linked function. Furthermore, all the virulent recombinants obtained from cells coinfected with different pairwise combinations of the vaccine strains had higher LD50 values than virulent wild-type virus, indicating that the recombinants had not acquired all the functions necessary for optimum expression of virulence. Partial virulence was also restored to Buk Z900 by marker rescue with sequences originating from three different regions of the wild-type pseudorabies virus genome. All three of these regions were different from those that had previously been shown to rescue virulence of the Bartha strain (B. Lomniczi, S. Watanabe, T. Ben-Porat, and A. S. Kaplan, 1987, J. Virol. 61, 796-801). Our results thus show that (1) defects in several different loci of the pseudorabies virus genome can affect virulence without detectably affecting growth in cell culture and (2) most vaccine strains have multiple defects contributing to their lack of virulence.

Pseudorabies Virus Expressing Bovine Herpesvirus 1 Glycoprotein B Exhibits Altered Neurotropism and Increased Neurovirulence

ABSTRACT Herpesvirus glycoproteins play dominant roles in the initiation of infection of target cells in culture and thus may also influence viral tropism in vivo. Whereas the relative contribution of several nonessential glycoproteins to neurovirulence and neurotropism ofPseudorabies virus (PrV), an alphaherpesvirus which causes Aujeszkys disease in pigs, has recently been uncovered in studies using viral deletion mutants, the importance of essential glycoproteins is more difficult to assess. We isolated an infectious PrV mutant, PrV-9112C2, which lacks the gene encoding the essential PrV glycoprotein B (gB) but stably carries in its genome and expresses the homologous gene of bovine herpesvirus 1 (BHV-1) (A. Kopp and T. C. Mettenleiter, J. Virol. 66:2754–2762, 1992). Apart from exhibiting a slight delay in penetration kinetics, PrV-9112C2 was similar in its growth characteristics in cell culture to wild-type PrV. To analyze the effect of the exchange of these homologous glycoproteins in PrVs natural host, swine, 4-week-old piglets were intranasally infected with 106 PFU of either wild-type PrV strain Kaplan (PrV-Ka), PrV-9112C2, or PrV-9112C2R, in which the PrV gB gene was reinserted instead of the BHV-1 gB gene. Animals infected with PrV-Ka and PrV-9112C2R showed a similar course of disease, i.e., high fever, marked respiratory symptoms but minimal neurological disorders, and excretion of high amounts of virus. All animals survived the infection. In contrast, animals infected with PrV-9112C2 showed no respiratory symptoms and developed only mild fever. However, on day 5 after infection, all piglets developed severe central nervous system (CNS) symptoms leading to death within 48 to 72 h. Detailed histological analyses showed that PrV-9112C2R infected all regions of the nasal mucosa and subsequently spread to the CNS preferentially by the trigeminal route. In contrast, PrV-9112C2 primarily infected the olfactory epithelium and spread via the olfactory route. In the CNS, more viral antigen and significantly more pronounced histological changes resulting in more severe encephalitis were found after PrV-9112C2 infection. Thus, our results demonstrate that replacement of PrV gB by the homologous BHV-1 glycoprotein resulted in a dramatic increase in neurovirulence combined with an alteration in the route of neuroinvasion, indicating that the essential gB is involved in determining neurotropism and neurovirulence of PrV.

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.

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 analysed 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 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.

High frequency intergenomic recombination of suid herpesvirus 1 (SHV-1, Aujeszky's disease virus).

SummaryExamples are given of observations made with field isolates of suid herpesvirus 1 (SHV-1) which indicate that intergenomic recombination is a common phenomenon associated with the virus. This was further confirmed by experimental co-infection of a pig with 2 virus strains with different, stable and easily identifiable genomic markers, followed by natural transmission to a group of contact pigs. A variety of recombinants was subsequently isolated, while none of the parental strains were re-isolated from any of the pigs. It is suggested that co-invasion of cells and recombination between viral genomes play a role in the life cycle of the virus.