Immune response after oral immunization of goats and foxes with an NDV vectored rabies vaccine candidate.

Vaccination of the reservoir species is a key component in the global fight against rabies. For wildlife reservoir species and hard to reach spillover species (e. g. ruminant farm animals), oral vaccination is the only solution. In search for a novel potent and safe oral rabies vaccine, we generated a recombinant vector virus based on lentogenic Newcastle disease virus (NDV) strain Clone 30 that expresses the glycoprotein G of rabies virus (RABV) vaccine strain SAD L16 (rNDV_GRABV). Transgene expression and virus replication was verified in avian and mammalian cells. To test immunogenicity and viral shedding, in a proof-of-concept study six goats and foxes, representing herbivore and carnivore species susceptible to rabies, each received a single dose of rNDV_GRABV (108.5 TCID50/animal) by direct oral application. For comparison, three animals received the similar dose of the empty viral vector (rNDV). All animals remained clinically inconspicuous during the trial. Viral RNA could be isolated from oral and nasal swabs until four (goats) or seven days (foxes) post vaccination, while infectious NDV could not be re-isolated. After four weeks, three out of six rNDV_GRABV vaccinated foxes developed RABV binding and virus neutralizing antibodies. Five out of six rNDV_GRABV vaccinated goats displayed RABV G specific antibodies either detected by ELISA or RFFIT. Additionally, NDV and RABV specific T cell activity was demonstrated in some of the vaccinated animals by detecting antigen specific interferon γ secretion in lymphocytes isolated from pharyngeal lymph nodes. In conclusion, the NDV vectored rabies vaccine rNDV_GRABV was safe and immunogenic after a single oral application in goats and foxes, and highlight the potential of NDV as vector for oral vaccines in mammals.

New Insights into Lymphocystis Disease Virus Genome Diversity.

Lymphocystis disease viruses (LCDVs) are viruses that infect bony fish which has been found in different locations across the globe. Four virus species have been classified by the International Committee on Taxonomy of Viruses (ICTV), despite remarkable discrepancies in genome size. Whole genome sequencing and phylogenetic analysis of LCDVs from wild fish from the North Sea and partial sequences from gilthead sea bream of an aquafarm located in the Aegean Sea in Turkey confirm that the LCDV1 genome at 100 kb is approximately half the size of the genomes of LCDV2-4. Since the fish species, of which LCDV1 was isolated, differ taxonomically at the order level, co-speciation can be excluded as the driver of the adaptation of the genome of this nucleocytoplasmic large DNA virus, but may represent an adaptation to the lifestyle of this demersal fish in the northeast Atlantic.

Protection of Chickens with Maternal Immunity Against Avian Influenza Virus (AIV) by Vaccination with a Novel Recombinant Newcastle Disease Virus Vector.

Newcastle disease virus (NDV) vectors expressing avian influenza virus (AIV) hemagglutinin of subtype H5 protect specific pathogen-free chickens from Newcastle disease and avian influenza. However, maternal AIV antibodies (AIV-MDA+) are known to interfere with active immunization by influencing vaccine virus replication and gene expression, resulting in inefficient protection. To overcome this disadvantage, we inserted a transgene encoding a truncated soluble hemagglutinin (HA) in addition to the gene encoding membrane-bound HA from highly pathogenic avian influenza virus (HPAIV) H5N1 into lentogenic NDV Clone 30 genome (rNDVsolH5_H5) to overexpress H5 antigen. Vaccination of 3-wk-old AIV-MDA+ chickens with rNDVsolH5_H5 and subsequent challenge infection with HPAIV H5N1 3 wk later resulted in 100% protection. Vaccination of younger chickens with higher AIV-MDA levels 1 and 2 wk after hatch resulted in protection rates of 40% and 85%, respectively. However, all vaccinated chickens showed strongly reduced shedding of challenge virus compared with age-matched, nonvaccinated control chickens. All control chickens succumbed to the HPAIV infection with a grading in disease progression between the three groups, indicating the influence of AIV-MDAs even at a low level. Furthermore, the shedding and serologic data gathered after immunization indicate sufficient replication of the vaccine virus, which leads to the assumption that lower protection rates in younger AIV-MDA+ chickens are caused by an H5 antigen-specific block and not by the interference of the AIV-MDA and the vaccine virus itself. In summary, solid protective efficacy and reduced virus transmission were achieved in 3-wk-old AIV-MDA+ chickens, which is relevant especially in regions endemically infected with HPAIV H5N1.

A Novel Recombinant Newcastle Disease Virus Vectored DIVA Vaccine against Peste des Petits Ruminants in Goats.

Peste des petits ruminants virus (PPRV, species: small ruminant morbillivirus) is the causative agent of the eponymous notifiable disease, the peste des petits ruminants (PPR) in wild and domestic sheep and goats. Mortality rates vary between 50% and 100%, causing significant losses of estimated 1.5 to 2 billion US Dollars per year. Live-attenuated PPRV vaccine strains are used in the field for disease prevention, but the application of a more thermostable vaccine enabling differentiation between infected and vaccinated animals (DIVA) would be highly desirable to achieve the goal of global disease eradication. We generated a recombinant Newcastle disease virus (rNDV) based on the live-attenuated NDV Clone 30 that expresses the surface protein hemagglutinin (H) of PPRV strain Kurdistan/11 (rNDV_HKur). In vitro analyses confirmed transgene expression as well as virus replication in avian, caprine, and ovine cells. Two consecutive subcutaneous vaccinations of German domestic goats with rNDV_HKur prevented clinical signs and hematogenic dissemination after an intranasal challenge with virulent PPRV Kurdistan/11. Virus shedding by different routes was reduced to a similar extent as after vaccination with the live-attenuated PPRV strain Nigeria 75/1. Goats that were either not vaccinated or inoculated with parental rNDV were used as controls. In summary, we demonstrate in a proof-of-concept study that an NDV vectored vaccine can protect against PPR. Furthermore, it provides DIVA-applicability and a high thermal tolerance.

Coexpression of soluble and membrane-bound avian influenza virus H5 by recombinant Newcastle disease virus leads to an increase in antigen levels.

Newcastle disease virus (NDV) vectors expressing avian influenza virus (AIV) haemagglutinin (HA) of subtype H5 simultaneously protect chickens from Newcastle disease (ND) as well as avian influenza (AI). The expressed, membrane-bound surface protein HA is incorporated into virions while soluble HA has been described as a potent antigen. We tested whether co-expression of both HA variants from the same NDV vector increased the overall level of HA, which could be important for optimal immunogenicity. Recombinant NDVsolH5_H5 co-expressed membrane-bound H5 of highly pathogenic (HP) AIV H5N1, detectable in infected cells, and soluble H5, which was secreted into the supernatant. This virus was compared to recombinant NDV that express either membrane-bound (rNDVH5) or soluble H5 (rNDVsolH5). Replication in embryonated chicken eggs (ECEs) and in cell culture, as well as pathogenicity in ECEs, was not influenced by the second heterologous transcriptional unit. However, the co-expression of soluble H5 with membrane-bound H5 increased total protein level about 5.25-fold as detected by MS quantification. Hence, this virus is very interesting as a vaccine virus in chickens against HPAIV infections in situations in which previous H5-expressing NDVs have reached their limit, such as in the face of pre-existing AIV maternal immunity.

W protein expression by Newcastle disease virus.

Differential editing of transcripts from the Newcastle disease virus (NDV) phosphoprotein gene results in mRNAs capable of encoding the phosphoprotein (P), the V protein, and the W protein which share a common N-terminus but specify different C-termini. Whereas the expression and viral incorporation of the P - and V proteins by NDV has been documented, evidence for the existence of a W protein was lacking. To analyze expression of the NDV W protein, two peptides encompassing predicted antigenic sites of the unique C-terminal W protein amino acid sequence of NDV Clone 30 were used for the generation of W-specific rabbit antisera. One of them detected plasmid-expressed W protein and identified W protein after infection by indirect immunofluorescence and Western blot analyses. W protein was absent in cells infected by a newly generated recombinant NDV lacking W protein expression. Furthermore, Western blot and mass spectrometric analyses indicated the incorporation of W protein into viral particles. Confocal microscopic analyses of infected cells revealed nuclear accumulation of W protein that could be attributed to a bipartite nuclear localization sequence (NLS) within its unique C-terminal part. Redistribution of the W protein to the cytoplasm within transfected cells confirmed functionality of the NLS after mutation of its two basic clusters. This finding was additionally corroborated in cells infected with a recombinant virus expressing the mutated W protein.

A genome-wide siRNA screen identifies a druggable host pathway essential for the Ebola virus life cycle.

BACKGROUND: The 2014-2016 Ebola virus (EBOV) outbreak in West Africa highlighted the need for improved therapeutic options against this virus. Approaches targeting host factors/pathways essential for the virus are advantageous because they can potentially target a wide range of viruses, including newly emerging ones and because the development of resistance is less likely than when targeting the virus directly. However, systematic approaches for screening host factors important for EBOV have been hampered by the necessity to work with this virus at biosafety level 4 (BSL4). METHODS: In order to identify host factors involved in the EBOV life cycle, we performed a genome-wide siRNA screen comprising 64,755 individual siRNAs against 21,566 human genes to assess their activity in EBOV genome replication and transcription. As a screening platform, we used reverse genetics-based life cycle modelling systems that recapitulate these processes without the need for a BSL4 laboratory. RESULTS: Among others, we identified the de novo pyrimidine synthesis pathway as an essential host pathway for EBOV genome replication and transcription, and confirmed this using infectious EBOV under BSL4 conditions. An FDA-approved drug targeting this pathway showed antiviral activity against infectious EBOV, as well as other non-segmented negative-sense RNA viruses. CONCLUSIONS: This study provides a minable data set for every human gene regarding its role in EBOV genome replication and transcription, shows that an FDA-approved drug targeting one of the identified pathways is highly efficacious in vitro, and demonstrates the power of life cycle modelling systems for conducting genome-wide host factor screens for BSL4 viruses.

Viral vector vaccines protect cockatiels from inflammatory lesions after heterologous parrot bornavirus 2 challenge infection.

Avian bornaviruses are causative agents of proventricular dilatation disease (PDD), a chronic neurologic and often fatal disorder of psittacines including endangered species. To date no causative therapy or immunoprophylaxis is available. Our previous work has shown that viral vector vaccines can delay the course of homologous bornavirus challenge infections but failed to protect against PDD when persistent infection was not prevented. The goal of this study was to refine our avian bornavirus vaccination and infection model to better represent natural bornavirus infections in order to achieve full protection against a heterologous challenge infection. We observed that parrot bornavirus 2 (PaBV-2) readily infected cockatiels (Nymphicus hollandicus) by combined intramuscular and subcutaneous injection with as little as 102.7foci-forming units (ffu) per bird, whereas a 500-fold higher dose of the same virus administered via peroral and oculonasal route did not result in persistent infection. These results indicated that experimental bornavirus challenge infections with this virus should be performed via the parenteral route. Prime-boost vaccination of cockatiels with Newcastle disease virus (NDV) and modified vaccinia virus Ankara (MVA) vectors expressing the nucleoprotein and phosphoprotein genes of PaBV-4 substantially blocked bornavirus replication following parenteral challenge infection with 103.5ffu of heterologous PaBV-2. Only two out of six vaccinated birds had very low viral levels detectable in a few organs. As a consequence, only one vaccinated bird developed mild PDD-associated microscopic lesions, while mock-vaccinated controls were not protected against PaBV-2 infection and inflammation. Our results demonstrate that NDV and MVA vector vaccines can protect against invasive heterologous bornavirus challenge infections and subsequent PDD. These vector vaccines represent a promising tool to combat avian bornaviruses in psittacine populations.

Viral vector vaccines expressing nucleoprotein and phosphoprotein genes of avian bornaviruses ameliorate homologous challenge infections in cockatiels and common canaries.

Avian bornaviruses are causative agents of proventricular dilatation disease (PDD), an often fatal disease of parrots and related species (order Psittaciformes) which is widely distributed in captive psittacine populations and may affect endangered species. Here, we established a vaccination strategy employing two different well described viral vectors, namely recombinant Newcastle disease virus (NDV) and modified vaccinia virus Ankara (MVA) that were engineered to express the phosphoprotein and nucleoprotein genes of two avian bornaviruses, parrot bornavirus 4 (PaBV-4) and canary bornavirus 2 (CnBV-2). When combined in a heterologous prime/boost vaccination regime, NDV and MVA vaccine viruses established self-limiting infections and induced a bornavirus-specific humoral immune response in cockatiels (Nymphicus hollandicus) and common canaries (Serinus canaria forma domestica). After challenge infection with a homologous bornavirus, shedding of bornavirus RNA and viral loads in tissue samples were significantly reduced in immunized birds, indicating that vaccination markedly delayed the course of infection. However, cockatiels still developed signs of PDD if the vaccine failed to prevent viral persistence. Our work demonstrates that avian bornavirus infections can be repressed by vaccine-induced immunity. It represents a first crucial step towards a protective vaccination strategy to combat PDD in psittacine birds.

Different regions of the newcastle disease virus fusion protein modulate pathogenicity.

Newcastle disease virus (NDV), also designated as Avian paramyxovirus type 1 (APMV-1), is the causative agent of a notifiable disease of poultry but it exhibits different pathogenicity dependent on the virus strain. The molecular basis for this variability is not fully understood. The efficiency of activation of the fusion protein (F) is determined by presence or absence of a polybasic amino acid sequence at an internal proteolytic cleavage site which is a major determinant of NDV virulence. However, other determinants of pathogenicity must exist since APMV-1 of high (velogenic), intermediate (mesogenic) and low (lentogenic) virulence specify a polybasic F cleavage site. We aimed at elucidation of additional virulence determinants by constructing a recombinant virus that consists of a lentogenic NDV Clone 30 backbone and the F protein gene from a mesogenic pigeon paramyxovirus-1 (PPMV-1) isolate with an intracerebral pathogenicity index (ICPI) of 1.1 specifying the polybasic sequence R-R-K-K-R*F motif at the cleavage site. The resulting virus was characterized by an ICPI of 0.6, indicating a lentogenic pathotype. In contrast, alteration of the cleavage site G-R-Q-G-R*L of the lentogenic Clone 30 to R-R-K-K-R*F resulted in a recombinant virus with an ICPI of 1.36 which was higher than that of parental PPMV-1. Substitution of different regions of the F protein of Clone 30 by those of PPMV-1, while maintaining the polybasic amino acid sequence at the F cleavage site, resulted in recombinant viruses with ICPIs ranging from 0.59 to 1.36 suggesting that virulence is modulated by regions of the F protein other than the polybasic cleavage site.

Avian paramyoxvirus-8 immunization reduces viral shedding after homologous APMV-8 challenge but fails to protect against Newcastle disease.

BACKGROUND: Protection against infection by Newcastle disease virus (NDV), also designated as avian paramyxovirus subtype-1 (APMV-1), is mediated by immune responses to the two surface glycoproteins, hemagglutinin-neuraminidase (HN) and fusion (F) protein. Thus, a chimeric APMV-1 based vaccine that encodes APMV-8 HN- and F-proteins and expresses the hemagglutinin of avian influenza virus (AIV) H5N1, is able to protect against HPAIV H5N1 but fails to protect against NDV [PLoS One8:e72530, 2013]. However, it is unclear whether avirulent APMV-subtypes, like APMV-8 can induce subtype-specific immunity and protect from a homologous challenge. FINDINGS: APMV-8 infections of 3- and 6-weeks-old specific pathogen free (SPF)-chickens did not induce any clinical signs but was associated with virus shedding for up to 6 days. Viral replication was only detected in oropharyngeal- and never in cloacal swabs. Upon reinfection with homologous APMV-8, viral shedding was restricted to day 2 and in contrast to naive SPF-chickens, only RNA but no infectious virus was recovered. No protection was induced against virulent NDV challenge, although morbidity and mortality was delayed in APMV-8 primed chickens. This lack of protection is in line with a lack of reactivity of APMV-8 specific sera to APMV-1 HN-protein: Neither by hemagglutin-inhibition (HI) test nor immunoblot analyses, cross-reactivity was detected, despite reactivity to internal proteins. CONCLUSIONS: Immune responses mounted during asymptomatic APMV-8 infection limit secondary infection against homologues reinfection and facilitates a delay in the onset of disease in a subtype independent manner but is unable to protect against Newcastle disease, a heterologous APMV-subtype.

Anterograde glycoprotein-dependent transport of newly generated rabies virus in dorsal root ganglion neurons.

UNLABELLED: Rabies virus (RABV) spread is widely accepted to occur only by retrograde axonal transport. However, examples of anterograde RABV spread in peripheral neurons such as dorsal root ganglion (DRG) neurons indicated a possible bidirectional transport by an uncharacterized mechanism. Here, we analyzed the axonal transport of fluorescence-labeled RABV in DRG neurons by live-cell microscopy. Both entry-related retrograde transport of RABV after infection at axon endings and postreplicative transport of newly formed virus were visualized in compartmentalized DRG neuron cultures. Whereas entry-related transport at 1.5 µm/s occurred only retrogradely, after 2 days of infection, multiple particles were observed in axons moving in both the anterograde and retrograde directions. The dynamics of postreplicative retrograde transport (1.6 µm/s) were similar to those of entry-related retrograde transport. In contrast, anterograde particle transport at 3.4 µm/s was faster, indicating active particle transport. Interestingly, RABV missing the glycoproteins did not move anterogradely within the axon. Thus, anterograde RABV particle transport depended on the RABV glycoprotein. Moreover, colocalization of green fluorescent protein (GFP)-labeled ribonucleoproteins (RNPs) and glycoprotein in distal axonal regions as well as cotransport of labeled RNPs with membrane-anchored mCherry reporter confirmed that either complete enveloped virus particles or vesicle associated RNPs were transported. Our data show that anterograde RABV movement in peripheral DRG neurons occurs by active motor protein-dependent transport. We propose two models for postreplicative long-distance transport in peripheral neurons: either transport of complete virus particles or cotransport of RNPs and G-containing vesicles through axons to release virus at distal sites of infected DRG neurons. IMPORTANCE: Rabies virus retrograde axonal transport by dynein motors supports virus spread over long distances and lethal infection of the central nervous system. Though active rabies virus transport has been widely accepted to be unidirectional, evidence for anterograde spread in peripheral neurons supports the hypothesis that in some neurons RABV also enters the anterograde pathway by so-far unknown mechanisms. By live microscopy we visualized fast anterograde axonal transport of rabies virus. The velocities exceeded those of retrograde movements, suggesting that active, most likely kinesin-dependent transport machineries are involved. Dependency of anterograde transport on the expression of virus glycoprotein G and cotransport with vesicles further suggest that complete enveloped virus particles or cotransport of virus ribonucleoprotein and G-containing vesicles occurred. These data provide the first insight in the mechanism of anterograde rabies virus transport and substantially contribute to the understanding of RABV replication and spread of newly formed virus in peripheral neurons.

Pigeon paramyxovirus type 1 variants with polybasic F protein cleavage site but strikingly different pathogenicity.

Newcastle disease viruses (NDV) isolated from pigeons (pigeon paramyxovirus type 1; PPMV-1) are mostly of mesogenic pathotype and characterized by a polybasic amino acid sequence motif at the fusion protein (F) cleavage site. This feature also applies to isolate R75/98 from Germany. Its genome consists of 15,192 nucleotides and it specifies an intracerebral pathogenicity index (ICPI) of 1.1, as is typical for mesogenic NDV. Recombinant R75/98 (rR75/98) derived by reverse genetics also possesses a polybasic F protein cleavage site but exhibits ICPI of 0.28, indicating a lentogenic virus. While ten virus passages of rR75/98 on embryonated chicken eggs did not result in any alteration of virus characteristics, virus which had been re-isolated from the brain of an intracerebrally inoculated chicken showed an increase in virulence, characterized by an ICPI of 0.93. Comparison of whole genome sequences of rR75/98 and re-isolated rR75/98 (RrR75/98) demonstrated only two amino acid differences, one in the F protein (N472 K) and one in the polymerase protein (K2168R). This result indicates that only very few amino acid alterations are sufficient to modulate virus virulence in the presence of a polybasic amino acid sequence at the proteolytic F protein cleavage site.

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.

Pathogenicity and immunogenicity of different recombinant Newcastle disease virus clone 30 variants after in ovo vaccination.

Even though Newcastle disease virus (NDV) live vaccine strains can be applied to 1-day-old chickens, they are pathogenic to chicken embryos when given in ovo 3 days before hatch. Based on the reverse genetics system, we modified recombinant NDV (rNDV) established from lentogenic vaccine strain Clone 30 by introducing specific mutations within the fusion (F) and hemagglutinin-neuraminidase (HN) proteins, which have recently been suggested as being responsible for attenuation of selected vaccine variants (Mast et al. Vaccine 24:1756-1765, 2006) resulting in rNDV49. Another recombinant (rNDVGu) was generated to correct sequence differences between rNDV and vaccine strain NDV Clone 30. Recombinant viruses rNDV, rNDV49, and rNDVGu have reduced virulence compared with NDV Clone 30, represented by lower intracerebral pathogenicity indices and elevated mean death time. After in ovo inoculation, hatchability was comparable for all infected groups. However, only one chicken from the NDV Clone 30 group survived a 21-day observation period; whereas, the survival rate of hatched chicks from groups receiving recombinant NDV was between 40% and 80%, with rNDVGu being the most pathogenic virus. Furthermore, recombinant viruses induced protection against challenge infection with virulent NDV 21 days post hatch. Differences in antibody response of recombinant viruses indicate that immunogenicity is correlated to virulence. In summary, our data show that point mutations can reduce virulence of NDV. However, alteration of specific amino acids in F and HN proteins of rNDV did not lead to further attenuation as indicated by their pathogenicity for chicken after in ovo inoculation.

Coexpression of avian influenza virus H5 and N1 by recombinant Newcastle disease virus and the impact on immune response in chickens.

To analyze the contribution of neuraminidase (NA) toward protection against avian influenza virus (AIV) infection, three different recombinant Newcastle disease viruses (NDVs) expressing hemagglutinin (HA) or NA, or both, of highly pathogenic avian influenza virus (HPAIV) were generated. The lentogenic NDV Clone 30 was used as backbone for the insertion of HA of HPAIV strain A/chicken/Vietnam/P41/05 (H5N1) and NA of HPAIV strain A/duck/Vietnam/TG24-01/05 (H5N1). The HA was inserted between the genes encoding NDV phosphoprotein (P) and matrixprotein (M), and the NA was inserted between the fusion (F) and hemagglutinin-neuraminidase protein (HN) genes, resulting in NDVH5VmPMN1FHN. Two additional recombinants were constructed carrying the HA gene between the NDV P and M genes (NDVH5VmPM) or the NA between F and HN (NDVN1FHN). All recombinants replicated well and stably expressed the HA gene, the NA gene, or both. Chickens immunized with NDVH5VmPMN1FHN or NDVH5VmPM were protected against two different HPAIV H5N1 and also against HPAIV H5N2. In contrast, immunization of chickens with NDVN1FHN induced NDV- and AIV N1-specific antibodies but did not protect the animals against a lethal dose of HPAIV H5N1. Furthermore, expression of AIV N1, in addition to AIV H5 by NDV, did not increase protection against HPAIV H5N1.

Efficacy of Newcastle disease virus recombinant expressing avian influenza virus H6 hemagglutinin against Newcastle disease and low pathogenic avian influenza in chickens and turkeys.

A recombinant Newcastle disease virus (NDV) expressing H6 hemagglutinin (HA) of a low pathogenic avian influenza virus (LPAIV) was generated by reverse genetics (NDVH6). The H6 open reading frame was inserted as an additional transcription unit between the fusion and hemagglutinin-neuraminidase (HN) gene of lentogenic NDV clone 30. Expression of the foreign gene was demonstrated by northern blot, western blot, and indirect immunofluorescence analyses. The protective efficacy against Newcastle disease and avian influenza of subtype H6 was evaluated in 3-wk-old chickens and turkeys. A single vaccination protected specific-pathogen-free (SPF) chickens against a subsequent lethal NDV infection and prevented shedding of AIV after homologous H6 LPAIV infection. Furthermore, vaccinated and AIV-infected animals could be differentiated by detection of AIV nucleoprotein-specific antibodies. Three-week-old commercial turkeys, exhibiting NDV-specific maternal antibodies, were partially protected against a lethal NDV challenge infection. The mortality rate of NDVH6-immunized turkeys was reduced to 40% compared to 90% in unvaccinated birds. After H6 LPAIV infection, shedding in NDVH6-immunized turkeys was only marginally reduced compared to NDV-immunized control birds. We previously described HA-expressing NDV recombinants as potent bivalent vaccines against Newcastle disease and highly pathogenic avian influenza of subtype H5 or H7. The results presented here are in contrast to the high protective efficacy in SPF chickens, as a single vaccination with NDVH6 was insufficient in turkeys in the presence of maternal antibodies against NDV. Therefore, the vector virus has to be improved to overcome these limitations.

Influence of insertion site of the avian influenza virus haemagglutinin (HA) gene within the Newcastle disease virus genome on HA expression.

Members of the order Mononegavirales express their genes in a transcription gradient from 3' to 5'. To assess how this impacts on expression of a foreign transgene, the haemagglutinin (HA) of highly pathogenic avian influenza virus (HPAIV) A/chicken/Vietnam/P41/05 (subtype H5N1) was inserted between the phosphoprotein (P) and matrix protein (M), M and fusion protein (F), or F and haemagglutinin-neuraminidase protein (HN) genes of attenuated Newcastle disease virus (NDV) Clone 30. In addition, the gene encoding the neuraminidase of HPAIV A/duck/Vietnam/TG24-01/05 (subtype H5N1) was inserted into the NDV genome either alone or in combination with the HA gene. All recombinants replicated well in embryonated chicken eggs. The expression levels of HA-specific mRNA and protein were quantified by Northern blot analysis and mass spectrometry, with good correlation. HA expression levels differed only moderately and were highest in the recombinant carrying the HA insertion between the F and HN genes of NDV.

Vaccination with Newcastle disease virus vectored vaccine protects chickens against highly pathogenic H7 avian influenza virus.

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.

Level of protection of chickens against highly pathogenic H5 avian influenza virus with Newcastle disease virus based live attenuated vector vaccine depends on homology of H5 sequence between vaccine and challenge virus.

Vaccination of poultry against avian influenza is of high priority, in particular after the dramatic spread of subtype H5N1 in Asia, Africa and Europe. Newcastle disease virus (NDV) has been developed as a vector for the expression of the main immunogen of avian influenza virus, hemagglutinin (HA). An NDV vector based vaccine has several advantages. It allows easy serological differentiation between infected and vaccinated animals by the detection of antibodies against non-HA influenza proteins. Moreover, it can be administered easily to large numbers of animals by spray or drinking water. We recently showed that chickens could be protected against infection with highly pathogenic avian influenza virus (HPAIV) A/chicken/Italy/8/98 (H5N2) after immunization with a recombinant Newcastle disease virus, NDVH5m, which expresses the homologous hemagglutinin. Here, we describe that immunization with NDVH5m conferred only partial protection against lethal infection with heterologous HPAIV A/duck/Vietnam/TG24-01/05 (H5N1). Comparison of amino acid sequences of both H5 proteins showed only 93.6% amino acid identity. Therefore, a new NDV recombinant (NDVH5Vm) was generated which expresses the H5 protein of HPAIV A/chicken/Vietnam/P41/05 (H5N1). This recombinant virus protected chickens against lethal infection with HPAIV H5N1 (Vietnam) already after one immunization. Our data thus show that application of a vector-based vaccine in the control of influenza may require adaptation of the vaccine to currently circulating viruses.