Ortrud Werner

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.

Epidemiological and ornithological aspects of outbreaks of highly pathogenic avian influenza virus H5N1 of Asian lineage in wild birds in Germany, 2006 and 2007.

In Germany, two distinct episodes of outbreaks of highly pathogenic avian influenza virus of subtype H5N1 (HPAIV H5N1) in wild birds occurred at the beginning of 2006, and in summer 2007. High local densities of wild bird populations apparently sparked clinically detectable outbreaks. However, these remained restricted in (i) number of birds, (ii) species found to be affected, (iii) time, and (iv) location despite the presence of several hundred thousands of susceptible wild birds and further stressors (food shortage, harsh weather conditions and moulting). Northern and southern subpopulations of several migratory anseriform species can be distinguished with respect to their preference for wintering grounds in Germany. This corroborates viral genetic data by Starick et al. (2008) demonstrating the introduction of two geographically restricted virus subpopulations of Qinghai-like lineage (cluster 2.2.A and 2.2.B) into northern and southern Germany, respectively, in 2006. The incursion of virus emerging in 2007, found to be distinct from the clusters detected in 2006 (Starick et al., 2008), may have been associated with moulting movements. Intensive past-outbreak investigations with negative results of live and dead wild birds and of terrestrial scavengers excluded continued circulation of virus on a larger scale. However, persistence of virus in small pockets of local wild bird populations could not be ruled out resiliently. 1.5% of investigated sera originating from cats sampled at the epicentres of the Ruegen 2006-outbreak contained H5-antibodies. Passive monitoring was found to be highly superior to live bird surveillance when aiming at the detection of HPAIV H5N1 in wild birds (P < 0.0001).

Rapid and Highly Sensitive Pathotyping of Avian Influenza A H5N1 Virus by Using Real-Time Reverse Transcription-PCR

ABSTRACT Rapid typing of the pathogenicity of avian influenza A viruses (AIV) of subtypes H5 and H7 is crucial to initiate adequate protective measures preventing the spread of highly pathogenic AIV (HPAIV). Here, a new real-time reverse transcription-PCR assay which enables sensitive and specific detection and cleavage site analysis of HPAIV H5N1 of the Qinghai lineage is described.

Protection of chickens from lethal avian influenza A virus infection by live-virus vaccination with infectious laryngotracheitis virus recombinants expressing the hemagglutinin (H5) gene.

The H5 hemagglutinin (HA) gene of a highly pathogenic avian influenza virus (AIV) isolate (A/chicken/Italy/8/98) was cloned and sequenced, and inserted at the non-essential UL50 (dUTPase) gene locus of a virulent strain of infectious laryngotracheitis virus (ILTV). Northern and Western blot analyses of the obtained ILTV recombinants demonstrated stable expression of the HA gene under control of the human cytomegalovirus immediate-early gene promoter. In vitro replication of the HA-expressing ILTV mutants was not affected, and infection of chickens revealed a reduced but still considerable virulence, similar to that of a UL50 gene deletion mutant without foreign gene insertion. The immunized animals produced specific antibodies against ILTV and AIV HA, and were protected against challenge infections with either virulent ILTV, or two different highly pathogenic AIV strains (A/chicken/Italy/8/98, A/chicken/Scotland/59). After challenge, no ILTV could be reisolated from protected animals, and shedding of AIV was considerably reduced. Thus, although attenuation remains to be improved, genetically engineered ILTV live-virus vaccines might be used as vectors to protect chickens also against other pathogens.

Neurotropism of highly pathogenic avian influenza virus A/chicken/Indonesia/2003 (H5N1) in experimentally infected pigeons (Columbia livia f. domestica)

This investigation assessed the susceptibility of experimentally infected pigeons to the highly pathogenic avian influenza virus (HPAIV) H5N1 that caused recent outbreaks of avian influenza in birds and humans in several countries of Asia. For this purpose 14 pigeons were infected ocularly and nasally with 108 EID50 and clinical signs were recorded and compared with five chickens infected simultaneously as positive controls. The chickens demonstrated anorexia, depression, and 100% mortality within 2 days postinoculation. Three of the pigeons died after a history of depression and severe neurological signs consisting of paresis to paralysis, mild enteric hemorrhage, resulting in a mortality of 21%. Gross lesions in these pigeons were mild and inconsistent. Occasionally subcutaneous hyperemia and hemorrhage and cerebral malacia were observed. Microscopic lesions and detection of viral antigen were confined to the central nervous system of these pigeons. In the cerebrum and to a minor extent in the brain stem a lymphohistiocytic meningoencephalitis with disseminated neuronal and glial cell necrosis, perivascular cuffing, glial nodules, and in one bird focally extensive liquefactive necrosis could be observed. The remaining nine pigeons showed neither clinical signs nor gross or histological lesions associated with avian influenza, although seroconversion against H5 indicated that they had been infected. These results confirm that pigeons are susceptible to HPAIV A/chicken/Indonesia/2003 (H5N1) and that the disease is associated with the neurotropism of this virus. Although sentinel chickens and most pigeons did not develop disease, further experiments have to elucidate whether or not Columbiformes are involved in transmission and spread of highly pathogenic avian influenza.

Newcastle disease virus: Detection and characterization by PCR of recent German isolates differing in pathogenicity

The fusion (F) protein plays an important role in determining the virulence of Newcastle disease virus (NDV) strains. A reverse transcriptase-polymerase chain reaction (RT-PCR) is described which amplifies a 362 bp fragment encompassing the region of the F protein most important for pathogenicity. A specific PCR product was obtained independent of strain, pathogenicity and host of origin. Sequencing of the region specifying the F protein cleavage site confirmed the correlation between deduced amino acid sequence and pathogenicity. Oligonucleotides corresponding to the sequence of the pathospecific region were designed for recent German NDV isolates and labelled with digoxigenin. Hybridization of PCR fragments of different isolates with pathotype-specific oligonucleotides allowed an estimation of the pathogenicity of most isolates. Results were in good agreement with experimentally determined ICPI values.

Characterization of avian paramyxovirus type 1 strains isolated in Germany during 1992 to 1996

In Germany all avian paramyxoviruses (APMV) isolated in regional laboratories are collected and characterized by the National Reference Laboratory. From 1992 until 1996, 635 APMV-1 virus isolates were submitted from almost all regions. Of these viruses, 371 were isolated from chickens, 39 from other poultry, 171 from pigeons and 54 from exotic birds. All isolates were examined for virulence in intracerebral pathogenicity index (ICPI) tests, for their ability to react with a panel of monoclonal antibodies (mAb) and their thermostability. In addition, the nucleotide sequences of the cleavage site of the fusion protein of a few virus isolates were determined. Most isolates from chickens and other poultry were of the velogenic pathotype. This virus was responsible for the epizootic in 1993 to 1995 in many small flocks. The same virus was obtained from some pigeons and some exotic birds. The pathogenicity of the velogenic/epizootic virus was high with most viruses giving ICPI values of 1.8 to 1.9, and the sequences of the cleavage site of all velogenic isolates tested were closely related. However, viruses isolated at the beginning of the epizootic period differed from viruses isolated towards the end in their reaction with some mAbs. 149 virus isolates were identified as pigeon variant PMV-1 (PPMV-1). Most of these were obtained from pigeons but a few were isolated from chickens and other birds. Most lentogenic isolates proved to be vaccine virus strains.

Avian influenza virus monitoring in wintering waterbirds in Iran, 2003-2007

BackgroundVirological, molecular and serological studies were carried out to determine the status of infections with avian influenza viruses (AIV) in different species of wild waterbirds in Iran during 2003-2007. Samples were collected from 1146 birds representing 45 different species with the majority of samples originating from ducks, coots and shorebirds. Samples originated from 6 different provinces representative for the 15 most important wintering sites of migratory waterbirds in Iran.ResultsOverall, AIV were detected in approximately 3.4% of the samples. However, prevalence was higher (up to 8.3%) at selected locations and for certain species. No highly pathogenic avian influenza, including H5N1 was detected. A total of 35 AIVs were detected from cloacal or oropharyngeal swab samples. These positive samples originated mainly from Mallards and Common Teals.Of 711 serum samples tested for AIV antibodies, 345 (48.5%) were positive by using a nucleoprotein-specific competitive ELISA (NP-C-ELISA). Ducks including Mallard, Common Teal, Common Pochard, Northern Shoveler and Eurasian Wigeon revealed the highest antibody prevalence ranging from 44 to 75%.ConclusionResults of these investigations provide important information about the prevalence of LPAIV in wild birds in Iran, especially wetlands around the Caspian Sea which represent an important wintering site for migratory water birds. Mallard and Common Teal exhibited the highest number of positives in virological and serological investigations: 43% and 26% virological positive cases and 24% and 46% serological positive reactions, respectively. These two species may play an important role in the ecology and perpetuation of influenza viruses in this region. In addition, it could be shown that both oropharyngeal and cloacal swab samples contribute to the detection of positive birds, and neither should be neglected.

The non-essential UL50 gene of avian infectious laryngotracheitis virus encodes a functional dUTPase which is not a virulence factor

The DNA sequence of the infectious laryngotracheitis virus (ILTV) UL50, UL51 and UL52 gene homologues was determined. Although the deduced UL50 protein lacks the first of five conserved domains of the corresponding proteins of mammalian alphaherpesviruses, the ILTV gene product was also shown to possess dUTPase activity. The generation of UL50-negative ILTV mutants was facilitated by recombination plasmids encoding green fluorescent protein (GFP), and expression constructs of predicted transactivator proteins of ILTV (alphaTIF, ICP4) were successfully used to increase the infectivity of viral genomic DNA. A GFP-expressing UL50-deletion mutant of ILTV showed reduced cell-to-cell spread in vitro, and was attenuated in vivo. A similar deletion mutant without the foreign gene, however, propagated like wild-type ILTV in cell culture and was pathogenic in chickens. We conclude that the viral dUTPase is not required for efficient replication of ILTV in the respiratory tract of infected animals. The replication defect of the GFP-expressing ILTV recombinant is most likely caused by toxic effects of the reporter gene product, since spontaneously occurring inactivation mutants exhibited wild-type-like growth.

Characterization of a recombinant Newcastle disease virus expressing the green fluorescent protein

A recombinant Newcastle disease virus (NDV) expressing the green fluorescent protein (GFP) was generated by applying reverse genetics techniques. The GFP open reading frame flanked by NDV transcription start and stop sequences was inserted between the fusion (F)- and hemagglutinin-neuraminidase genes in a full-length cDNA clone of NDV. This plasmid transcribing antigenome RNA was cotransfected with helper plasmids expressing viral nucleoprotein, phosphoprotein and large protein into cells stably expressing T7 RNA polymerase. The rescued virus was first propagated in embryonated eggs and the allantoic fluid was used to infect cells. Northern blot analysis of RNA isolated from infected cells demonstrated the proper transcription of the introduced GFP-mRNA. The appearance of GFP in live infected cells confirmed further the recovery of a recombinant NDV (rNDVGFP1) expressing the reporter gene. The expression of the heterologous gene was maintained stably for at least five passages in embryonated eggs. The replication kinetics in embryonated eggs and pathogenicity in chickens of rNDVGFP1 did not differ significantly from that of the parent virus. Using GFP autofluorescence, virus infected cells could be tracked easily in native preparations, organ explants and primary tracheal cell cultures. Taken together, these data demonstrate the use of GFP-expressing recombinant NDV for analysis of NDV dissemination and pathogenesis and indicate the potential usefulness of NDV as a vaccine vector.