Angela Römer-Oberdörfer

Generation of recombinant lentogenic Newcastle disease virus from cDNA

Recombinant lentogenic Newcastle disease virus (NDV) of the vaccine strain Clone-30 was reproducibly generated after simultaneous expression of antigenome-sense NDV RNA and NDV nucleoprotein, phosphoprotein and RNA-dependent RNA polymerase from plasmids transfected into cells stably expressing T7 RNA polymerase. For this purpose, the genome of Clone-30, comprising 15186 nt, was cloned and sequenced prior to assembly into a full-length cDNA clone under control of a T7 RNA polymerase promoter. Recombinant virus was amplified by inoculation of transfection supernatant into the allantoic cavity of embryonated specific-pathogen-free (SPF) chicken eggs. Two marker restriction sites comprising a total of five nucleotide changes artificially introduced into noncoding regions were present in the progeny virus. The recombinant NDV was indistinguishable from the parental wild-type virus with respect to its growth characteristics in cell culture and in embryonated eggs. Moreover, an intracerebral pathogenicity index of 0.29 was obtained for both viruses as determined by intracerebral inoculation of day-old SPF chickens, proving that the recombinant NDV is a faithful copy of the parental vaccine strain of NDV.

A Recombinant Newcastle Disease Virus with Low-Level V Protein Expression Is Immunogenic and Lacks Pathogenicity for Chicken Embryos

ABSTRACT Newcastle disease virus (NDV) edits its P-gene mRNA by inserting a nontemplated G residue(s) at a conserved editing site (3′-UUUUUCCC-template strand). In the wild-type virus, three amino-coterminal P-gene-derived proteins, P, V, and W, are produced at frequencies of approximately 68, 29, and 2%, respectively. By applying the reverse genetics technique, editing-defective mutants were generated in cell culture. Compared to the wild-type virus, mutants lacking either six nucleotides of the conserved editing site or the unique C-terminal part of the V protein produced as much as 5,000-fold fewer infectious progeny in vitro or 200,000-fold fewer in 6-day-old embryonated chicken eggs. In addition, both mutants were unable to propagate in 9- to 11-day-old embryonated specific-pathogen-free (SPF) chicken eggs. In contrast, a mutant (NDV-P1) with one nucleotide substitution (UUCUUCCC) grew in eggs, albeit with a 100-fold-lower infectious titer than the parent virus. The modification in the first two mutants described above led to complete abolition of V expression, whereas in NDV-P1 the editing frequency was reduced to less than 2%, and as a result, V was expressed at a 20-fold-lower level. NDV-P1 showed markedly attenuated pathogenicity for SPF chicken embryos, unlike currently available ND vaccine strains. These findings indicate that the V protein of NDV has a dual function, playing a direct role in virus replication as well as serving as a virulence factor. Administration of NDV-P1 to 18-day-old embryonated chicken eggs hardly affected hatchability. Hatched chickens developed high levels of NDV-specific antibodies and were fully protected against lethal challenge, demonstrating the potential use of editing-defective recombinant NDV as a safe embryo vaccine.

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.

Newcastle Disease Virus (NDV) Marker Vaccine: an Immunodominant Epitope on the Nucleoprotein Gene of NDV Can Be Deleted or Replaced by a Foreign Epitope

ABSTRACT The nucleoprotein (NP) of Newcastle disease virus (NDV) functions primarily to encapsidate the virus genome for the purpose of RNA transcription, replication, and packaging. This conserved multifunctional protein is also efficient in inducing NDV-specific antibody in chickens. Here, we localized a conserved B-cell immunodominant epitope (IDE) spanning residues 447 to 455 and successfully generated a recombinant NDV lacking the IDE by reverse genetics. Despite deletion of NP residues 443 to 460 encompassing the NP-IDE, the mutant NDV propagated in embryonated specific-pathogen-free chicken eggs to a level comparable to that of the parent virus. In addition, a B-cell epitope of the S2 glycoprotein of murine hepatitis virus (MHV) was inserted in-frame to replace the NP-IDE. Recombinant viruses properly expressing the introduced MHV epitope were successfully generated, demonstrating that the NP-IDE not only is dispensable for virus replication but also can be replaced by foreign sequences. Chickens immunized with the hybrid recombinants produced specific antibodies against the S2 glycoprotein of MHV and completely lacked antibodies directed against the NP-IDE. These marked-NDV recombinants, in conjunction with a diagnostic test, enable serological differentiation of vaccinated animals from infected animals and may be useful tools in ND eradication programs. The identification of a mutation-permissive region on the NP gene allows a rational approach to the insertion of protective epitopes and may be relevant for the design of NDV-based cross-protective marker vaccines.

Generation of a velogenic Newcastle disease virus from cDNA and expression of the green fluorescent protein

SummaryNewcastle disease virus (NDV) is a pathogen that is important in the poultry industry worldwide. Specifically, the virulent (velogenic) NDV is aparticular threat because it has now occurred frequently worldwide. The outbreaks caused by highly virulent NDV in waterfowl and especially in goose flocks, have led to greater concern in recent years as aquatic birds were previously resistant to most virulent NDV strains from chickens. The molecular determinants of host tropism, virulence and emergence of NDV isolated from diseased goose flocks are poorly understood. In the present study, we rescued a highly virulent NDV isolated from a goose using the reverse genetics approach. Infectious virus was successfully generated by cotransfection of a full-length cDNA clone of the NDV strain ZJ1 with helper plasmids. The recombinant NDV was indistinguishable from the parental wild-type virus with respect to its growth kinetics in cell culture as well as its biological properties. A recombinant NDV expressing green fluorescent protein (GFP) was generated, and GFP was subsequently detected in cells and various organs from the infected chickens.

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.

Vaccination with Newcastle Disease Virus Vectored Vaccine Protects Chickens Against Highly Pathogenic H7 Avian Influenza Virus

Abstract A recombinant Newcastle disease virus (NDV) was engineered to express the hemagglutinin (HA) gene of avian influenza virus (AIV) subtype H7. The HA gene was inserted between the genes encoding NDV fusion and hemagglutinin–neuraminidase proteins. Within the H7 open reading frame, an NDV gene end-like sequence was eliminated by silent mutation. The expression of H7 protein was detected by western blot analysis and indirect immunofluorescence. The existence of H7 protein in the envelope of recombinant Newcastle disease virions was shown by immunoelectron microscopy. The protective efficacy of recombinant NDVH7m against virulent NDV, as well as against highly pathogenic avian influenza virus (HPAIV), was evaluated in specific-pathogen-free chickens. After a single immunization, all chickens developed NDV-specific, as well as AIV H7-specific, antibodies and were completely protected from clinical disease after infection with a lethal dose of virulent NDV or the homologous H7N1 HPAIV, while all control animals died within four days. Shedding of AIV challenge virus was strongly reduced compared to nonvaccinated control birds. Furthermore, the immunized birds developed antibodies against the AIV nucleoprotein after challenge infection. Thus, NDVH7m could be used as a marker vaccine against subtype H7 avian influenza.

Enhancement of Pathogenicity of Newcastle Disease Virus by Alteration of Specific Amino Acid Residues in the Surface Glycoproteins F and HN

Abstract Recombinant viruses were rescued after site-specific mutagenesis of a full-length clone of the lentogenic Newcastle disease virus (NDV) strain Clone 30. To assess the contribution of different amino acids to virulence, specific alterations were introduced into the fusion (F) protein and in the hemagglutinin-neuraminidase (HN) protein based on sequence comparison between NDV strains of different virulence. Modification of the proteolytic cleavage site in the F protein to a polybasic motif increased the intracerebral pathogenicity index (ICPI) from 0.0 to 1.28. Moreover, the additional exchange of amino acid 123 of the HN protein from tryptophan to cysteine in combination with alteration of amino acid 27 of the F protein from cysteine to arginine increased the ICPI to 1.5. The HN mutation visibly altered conformation of the protein, resulting in the formation of disulfide-linked HN dimers that may indicate that this HN conformation is beneficial for the virulent phenotype.

Identification of a Mutation in Editing of Defective Newcastle Disease Virus Recombinants That Modulates P-Gene mRNA Editing and Restores Virus Replication and Pathogenicity in Chicken Embryos

ABSTRACT Editing of P-gene mRNA of Newcastle disease virus (NDV) enables the formation of two additional proteins (V and W) by inserting one or two nontemplated G residues at a conserved editing site (5′-AAAAAGGG). The V protein of NDV plays an important role in virus replication and is also a virulence factor presumably due to its ability to counteract the antiviral effects of interferon. A recombinant virus possessing a nucleotide substitution within the A-stretch (5′-AAgAAGGG) produced 20-fold-less V protein and, in consequence, was impaired in replication capacity and completely attenuated in pathogenicity for chicken embryos. However, in a total of seven serial passages, restoration of replication and pathogenic capacity in 9- to 11-day-old chicken embryos was noticed. Determining the sequence around the editing site of the virus at passage 7 revealed a C-to-U mutation at the second nucleotide immediately upstream of the 5′-A5 stretch (5′-GuUAAgAAGGG). The V mRNA increased from an undetectable level at passage 5 to ca. 1 and 5% at passages 6 and 7, respectively. In addition, similar defects in another mutant possessing a different substitution mutation (5′-AAAcAGGG) were restored in an identical manner within a total of seven serial passages. Introduction of the above C-to-U mutation into the parent virus (5′-GuUAAAAAGGG) altered the frequency of P, V, and W mRNAs from 68, 28, and 4% to 15, 44, and 41%, respectively, demonstrating that the U at this position is a key determinant in modulating P-gene mRNA editing. The results indicate that this second-site mutation is required to compensate for the drop in edited mRNAs and consequently to restore the replication capacity, as well as the pathogenic potential, of editing-defective NDV recombinants.

Chimeric Newcastle Disease Virus Protects Chickens against Avian Influenza in the Presence of Maternally Derived NDV Immunity

Newcastle disease virus (NDV), an avian paramyxovirus type 1, is a promising vector for expression of heterologous proteins from a variety of unrelated viruses including highly pathogenic avian influenza virus (HPAIV). However, pre-existing NDV antibodies may impair vector virus replication, resulting in an inefficient immune response against the foreign antigen. A chimeric NDV-based vector with functional surface glycoproteins unrelated to NDV could overcome this problem. Therefore, an NDV vector was constructed which carries the fusion (F) and hemagglutinin-neuraminidase (HN) proteins of avian paramyxovirus type 8 (APMV-8) instead of the corresponding NDV proteins in an NDV backbone derived from the lentogenic NDV Clone 30 and a gene expressing HPAIV H5 inserted between the F and HN genes. After successful virus rescue by reverse genetics, the resulting chNDVFHN PMV8H5 was characterized in vitro and in vivo. Expression and virion incorporation of the heterologous proteins was verified by Western blot and electron microscopy. Replication of the newly generated recombinant virus was comparable to parental NDV in embryonated chicken eggs. Immunization with chNDVFHN PMV8H5 stimulated full protection against lethal HPAIV infection in chickens without as well as with maternally derived NDV antibodies. Thus, tailored NDV vector vaccines can be provided for use in the presence or absence of routine NDV vaccination.