Thrombocytopenia in Newcastle disease: haematological evaluation and histological study of bone marrow.

A Newcastle disease virus (NDV) isolated in Mexico and called Chimalhuacan strain was characterised by gene F restriction enzyme analysis and found to be a genotype II velogenic virus. Haematological evaluations and histological studies of bone marrow were conducted on chickens experimentally infected with the Chimalhuacan virus and on control chickens. Within 72 hours post infection (hpi), a 50% decrease in thrombocyte and monocyte counts and a complete cellular depletion in bone marrow islands were evident in the infected group. These findings suggest that the Chimalhuacan strain of NDV causes an early and severe damage of the haematopoietic cells including thrombocyte precursors, which might explain the marked thrombocytopenia detected in early stages of this disease.

Retrospective characterization of Newcastle Disease Virus Antrim '73 in relation to other epidemics, past and present.

In November 1973 Newcastle disease suddenly appeared in Northern Ireland, where the viscerotropic disease had not been seen in 3 1/2 years and the two Irelands had been regarded as largely disease free for 30 years. It was successfully controlled with only 36 confirmed affected layer flocks, plus 10 more slaughtered as 'dangerous contacts'. Contemporary investigations failed to reveal the source of the Irish epidemic. Using archival virus samples from most of the affected flocks, RT PCR was conducted with primers selected for all six NDV genes. Phylogenetic analyses of three genes, HN, M and F, confirmed vaccine as the cause of one of the outbreaks. The other six samples were identical and closely related to previous outbreaks in the United States and western Europe initiated by infected imported Latin American parrots. The probable cause of the epidemic followed from the importation from The Netherlands of bulk feed grains contaminated with infected pigeon faeces.

Antigenic and genetic diversity of early European isolates of Infectious bursal disease virus prior to the emergence of the very virulent viruses: early European epidemiology of Infectious bursal disease virus revisited?

Eleven Polish and Hungarian isolates of Infectious bursal disease virus (IBDVs) obtained in the 70/80s (early IBDV) and in the 90s (recent IBDV) were characterized in an Antigen-Capture-ELISA with a panel of neutralizing monoclonal antibodies (Mabs), and by nucleotide sequencing of the VP2 variable domain (vVP2). The viruses were compared with reference IBDV strains, among others with Faragher 52/70 (F52/70, classical, isolated 1970), 89163 (typical very virulent-vvIBDV, isolated 1989) and 91168 (antigenically modified vvIBDV, isolated 1991). Only one of the early isolates (Hungarian strain P1) proved antigenically and genetically similar to F52/70. Other early isolates exhibited no reactivity versus Mabs 3, 4, 5 and/or 8 and had a common previously unrecognized combination of amino acid changes in vVP2. The recent isolates all proved antigenically and genetically related to typical vvIBDV strain 89163, except the Polish isolate 93/35 which proved related to the 91168 strain although no epidemiological relationship had been documented between these viruses in the field. Phylogenetic analysis confirmed that the non-P1 early IBDVs represent a previously unrecognized group among serotype 1 IBDVs. It is discussed whether these early isolates are derivatives of the F52/70-like viruses that might still be present in the field, or whether they represent early IBDV strains that might have been present prior to and progressively replaced by the F52/70-like viruses, as the latter have been replaced by vvIBDVs in the late eighties.

Positive identification of Newcastle disease virus vaccine strains and detection of contamination in vaccine batches by restriction site analysis of the matrix protein gene.

Twelve vaccine batches prepared from avirulent vaccine strains of Newcastle disease virus produced by seven manufacturers were identified by analysis of the matrix (M) protein gene with restriction enzymes MboI and HinfI. The analyses have revealed the presence of the strain indicated by the manufacturers (namely B-1, LaSota or Ulster 2C), except in one case when the vaccine contained strain V4 Queensland instead of VGGA as indicated. In addition, several batches of both monovalent and combined vaccines containing strain LaSota of the same company consistently disclosed contamination with strain B-1. The mixed nature of the preparations was verified not only by the dual patterns of restriction fragments but also by separating the two components and identifying them individually. Restriction analysis of the M gene, by allowing positive identification of each of the lentogenic vaccine strains, should provide an improvement in controlling vaccine batches by revealing homologous contaminants or exchange of the vaccine strain.

On the origins and relationships of Newcastle disease virus vaccine strains Hertfordshire and Mukteswar, and virulent strain Herts'33.

The origins and relationships of Newcastle disease virus (NDV) vaccine strains Hertfordshire (H) and Mukteswar, and the virulent Herts'33 were studied using partial sequence analysis of the fusion protein gene. The mesogenic strain H was obtained by egg passages of a field virus isolated in England in 1933 (later known as Herts'33). Different lines of the strain Herts'33, however, divided into two distinct groups: genotype IV, and a hitherto undescribed lineage, which comprised the Weybridge line (Herts'33/56). Vaccine strain H and the two clusters comprising viruses designated Herts'33 displayed 6.5 to 6.8% and 15.6 to 16.3% mutational distances, respectively, which precluded parent-offspring relationships with either of them. In contrast, the different lines of the vaccine strain Mukteswar, which was reportedly derived from an Indian field isolate in the mid-1940s, showed 98.9 to 100% sequence similarity to strain H. It is therefore probable that the two vaccines were derived from the same virus stock.

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.

Occurrence of genotypes IV, V, VI and VIIa in Newcastle disease outbreaks in Germany between 1939 and 1995.

Forty-five velogenic Newcastle disease virus strains isolated in Germany between 1939 and 1995 were analysed by restriction enzyme digestion and sequencing to shed light on the relationships of past epizootics. Viruses derived from the period prior to 1970 belonged to a clade (IVea) of genotype IV comprising the earliest isolates from Europe, and could be isolated until the late seventies from poultry. Essex'70-like viruses, the prototype of genotype V, were already present at the beginning of the 1970-74 epizootic and in sporadic cases thereafter, indicating that these Newcastle disease outbreaks started in Western Europe. A genotype VI (subtype VIc) isolate was obtained in the early 1980s from a single outbreak in poultry. Outbreaks between 1993-95 were again part of a Western European epizootic caused by a genotype VIIa virus that was prevalent in the Far East.

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).

Pseudorabies virus expressing bovine herpesvirus 1 glycoprotein B exhibits altered neurotropism and increased neurovirulence.

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 of Pseudorabies virus (PrV), an alphaherpesvirus which causes Aujeszky's 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 PrV's natural host, swine, 4-week-old piglets were intranasally infected with 10(6) 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.

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

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.

Lentogenic field isolates of Newcastle disease virus isolated in Canada and Hungary are identical with the vaccine type used in the region.

Lentogenic field isolates of Newcastle disease virus were examined by restriction enzyme analysis of RT-PCR products generated from the matrix protein gene that discriminates between strains LaSota and B-1, the two most widely used lentogenic vaccine viruses. Isolates were derived from regions where, exclusively or predominantly, only one type of vaccine was employed. Viruses collected in Hungary for two decades were exclusively of LaSota-type while the Canadian collection predominantly included B-1, which corresponded to the vaccine types used in the regions. Isolation of vaccine type lentogenic viruses from unvaccinated flocks supports the occurrence of area spread of these lentogenic viruses.

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

Newcastle 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.

Isolation and characterization of avian paramyxovirus type 1 (Newcastle disease) viruses from a flock of ostriches (Struthio camelus) and emus (Dromaius novaehollandiae) in Europe with inconsistent serology.

During a 95-day study period in 1995 in Denmark, 18 ostriches in a flock of 77 ostriches and four emus held in quarantine died. Clinical and pathological observations did not indicate the presence of transmissible infectious disease in the flock. Management failures and indoor housing were believed to have contributed significantly to the number of deaths. Samples from 17 of the dead ostriches were examined virologically. Three isolates of avian paramyxovirus serotype 1 (APMV-1) were obtained from intestines and intestinal contents of dead ostriches submitted for laboratory investigations. In ICPI tests in day-old chicks values for the three APMV-1 isolates were in the range 1.63-1.69. Characterization by means of mouse monoclonal antibodies and by restriction site analysis revealed that the three isolates were indistinguishable and similar to APMV-1 viruses present in a simultaneous epizootic of Newcastle disease in back yard poultry in Denmark. Blood samples were taken from all live birds in the flock after 25 and 95 days of quarantine and all were negative for antibodies to APMV-1 in haemagglutination inhibition tests. All samples taken after 95 days of quarantine were also negative for antibodies to APMV-1 in serum neutralization tests performed in chicken embryo cells. Blood samples taken after 95 days of quarantine were tested in a commercial ELISA for antibodies to APMV-1. In this test 35% of the samples were positive, 35% were border line and 30% were negative.

Rapid identification of Newcastle disease virus vaccine strains LaSota and B-1 by restriction site analysis of their matrix gene.

A region constituting 88% of the matrix gene of Newcastle disease virus vaccine strains LaSota and B-1 was amplified by reverse transcription-polymerase chain reaction. Amplified products of LaSota and B-1 strains derived from vaccine serials of different companies were digested with restriction enzymes MboI and HinfI. Strain characteristic cleavage site maps were obtained that allowed for a reliable and rapid differentiation between strains LaSota and B-1.

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

A 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. Restrictions 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 in the early 1970s. Group V1 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 V1. 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.

Mutations affecting the UL21 gene contribute to avirulence of pseudorabies virus vaccine strain Bartha.

Analysis of the live attenuated pseudorabies virus (PrV) vaccine strain Bartha indicated location of a major determinant for PrV neurovirulence within the genomic BamHI fragment 4 (B. Lomniczi et al., 1984, J. Virol. 52, 198-205). To more precisely localize the defect, marker rescue experiments were performed using cloned subfragments of BamHI-4. Rescuants were analyzed after intracerebral infection for their virulence in chicken, as well as after intranasal infection for virulence in pigs. We show that the defect associated with attenuation in strain Bartha is located in a 3.8-kb subfragment of BamHI-4 which encompasses the PrV UL20 and UL21 genes and a putative origin of replication (B. Klupp, H. Kern, and T. C. Mettenleiter, 1992, Virology 191, 900-908). Sequence analysis of this region of the strain Bartha genome and comparison with the corresponding region in wild-type PrV strain Ka revealed the presence of eight point mutations. Four nucleotide exchanges reside within the UL21 gene with three of them leading to amino acid substitutions; one is located in the intergenic region between the UL20 and UL21 genes and three are localized downstream from the UL21 gene. Neither the UL20 gene nor the putative origin sequence was affected. Insertional inactivation of the UL21 gene in wild-type PrV strain Ka led to a marked attenuation of the virus for pigs infected by the intranasal route. In summary, our data show that the PrV UL21 gene is a major determinant of PrV virulence and that point mutations affecting the UL21 gene of live vaccine strain Bartha contribute to its attenuated phenotype.

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

Examples 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.

Round table on control of Aujeszky's disease and vaccine development based on molecular biology.

A summary is given on the 4 topics which were discussed during the round table and which represent current knowledge on the molecular biology of Aujeszky's disease (pseudorabies) virus. They include a review on 1. the genome and gene products of the virus; 2. the viral genes associated with virulence; 3. the immunological role of the viral gene products and 4. studies intended to compare the efficacy of several commercially available vaccines and to establish a possible correlation between antibodies against individual structural viral proteins and degree of protection. It was concluded that gI deleted vaccines appear to be the best choice for use in intensive vaccination programmes towards eradication of Aujeszky's disease virus. However, there remains a need for development of more potent vaccines which induce strong humoral and cell mediated immune responses and afford complete protection, virological protection included. It is often observed that live vaccine strains which are completely avirulent lose much capacity to replicate and spread within the vaccinated animal. It is, however, not excluded that a certain degree of dissemination may be needed to be fully efficacious. Loss of virulence may thus be accompanied by too much loss of immunogenicity. An improved genetic stability of live vaccine strains when they are obtained by genetic manipulation, possibly justifies a more widespread dissemination of the vaccine strain in the body compared to that with conventionally developed strains or compared to what is presently allowed.

The ability of pseudorabies virus to grow in different hosts is affected by the duplication and translocation of sequences from the left end of the genome to the UL-US junction.

Pseudorabies virus is a herpesvirus which has a class 2 genome. However, under certain growth conditions it acquires a genome with class 3-like characteristics. In these variants, the leftmost sequences of the long (L) component of the viral genome have been duplicated and translocated to the right of the L component next to the short (S) component, resulting in an L component that is bracketed by inverted repeats. Consequently, the L component can invert and is found in two orientations relative to the S component. The translocation is accompanied invariably by a deletion of sequences that are normally present in the wild-type genome at the right end of the L component. The virion variants with an invertible L component have a growth advantage over wild-type virus in chicken embryo fibroblasts and chickens; they also have a growth disadvantage in mice or rabbit kidney cells. The changed growth characteristics of the variants reside entirely in the changed structure of the junction between the S and L components. Replacement of that region of the DNA with wild-type sequences restores the wild-type phenotype. To determine whether the modified growth characteristics of the variants are related to the translocation or to the deletion, mutants that have a deletion or that have a deletion as well as a translocation similar to those observed in the variants were constructed, and the growth characteristics of these mutants were determined. We show that the modified growth characteristics of the mutants with an invertible L component can be attributed to the translocation of the leftmost terminal sequences of the genome next to the inverted repeat; they are not related to the deletion of the sequences normally present at the right end of the L component. The translocation of the leftmost 325 bp of the genome is sufficient to confer upon the virus the modified cell-type-specific growth characteristics. Furthermore, the modified growth characteristics are contingent upon the presence of 68 bp spanning the internal junction between the L and S components.