Field vaccinated chickens with low antibody titres show equally insufficient protection against matching and non-matching genotypes of virulent Newcastle disease virus.

Newcastle disease (ND) is a severe threat to the poultry industry and is caused by virulent strains of Newcastle disease virus (NDV). Many countries maintain a vaccination policy, but NDV is rapidly evolving as shown by the discovery of several new genotypes in the last decades. We tested the efficacy of the currently used classical commercial ND vaccine based on the genotype II strain VG/GA, applied under standard field conditions, against outbreak strains. Field vaccinated broilers were challenged with four different viruses belonging to genotype II, V or VII. A large proportion of field vaccinated broilers showed suboptimal immunity and the protection level against early and recent NDV isolates was dramatically low. Furthermore, there were no significant differences in protection afforded by a genotype II vaccine against a genotype II virus challenge compared to a challenge with viruses belonging to the other genotypes. This study suggests that the susceptibility of vaccinated poultry to NDV infection is not the result of vaccine mismatch, but rather of poor vaccination practices.

Effect of natural and chimeric haemagglutinin genes on influenza A virus replication in baby hamster kidney cells.

Baby hamster kidney (BHK21) cells are used to produce vaccines against various viral veterinary diseases, including rabies and foot-and-mouth-disease. Although particular influenza virus strains replicate efficiently in BHK21 cells the general use of these cells for influenza vaccine production is prohibited by the poor replication of most strains, including model strain A/PR/8/34 [H1N1] (PR8). We now show that in contrast to PR8, the related strain A/WSN/33 [H1N1] (WSN) replicates efficiently in BHK21 cells. This difference is determined by the haemagglutinin (HA) protein since reciprocal reassortant viruses with swapped HAs behave similarly with respect to growth on BHK21 cells as the parental virus from which their HA gene is derived. The ability or inability of six other influenza virus strains to grow on BHK21 cells appears to be similarly dependent on the nature of the HA gene since reassortant PR8 viruses containing the HA of these strains grow to similar titres as the parental virus from which the HA gene was derived. However, the growth to low titres of a seventh influenza strain was not due to the nature of the HA gene since a reassortant PR8 virus containing this HA grew efficiently on BHK21 cells. Taken together, these results suggest that the HA gene often primarily determines influenza replication efficiency on BHK21 cells but that in some strains other genes are also involved. High virus titres could be obtained with reassortant PR8 strains that contained a chimeric HA consisting of the HA1 domain of PR8 and the HA2 domain of WSN. HA1 contains most antigenic sites and is therefore important for vaccine efficacy. This method of producing the HA1 domain as fusion to a heterologous HA2 domain could possibly also be used for the production of HA1 domains of other viruses to enable the use of BHK21 cells as a generic platform for veterinary influenza vaccine production.

Mutations in the M-gene segment can substantially increase replication efficiency of NS1 deletion influenza A virus in MDCK cells.

Influenza viruses unable to express NS1 protein (delNS1) replicate poorly and induce large amounts of interferon (IFN). They are therefore considered candidate viruses for live-attenuated influenza vaccines. Their attenuated replication is generally assumed to result from the inability to counter the antiviral host response, as delNS1 viruses replicate efficiently in Vero cells, which lack IFN expression. In this study, delNS1 virus was parallel passaged on IFN-competent MDCK cells, which resulted in two strains that were able to replicate to high virus titers in MDCK cells due to adaptive mutations especially in the M-gene segment but also in the NP and NS gene segments. Most notable were clustered U-to-C mutations in the M segment of both strains and clustered A-to-G mutations in the NS segment of one strain, which presumably resulted from host cell-mediated RNA editing. The M segment mutations in both strains changed the ratio of M1 to M2 expression, probably by affecting splicing efficiency. In one virus, 2 amino acid substitutions in M1 additionally enhanced virus replication, possibly through changes in the M1 distribution between the nucleus and the cytoplasm. Both adapted viruses induced levels of IFN equal to that of the original delNS1 virus. These results show that the increased replication of the adapted viruses is not primarily due to altered IFN induction but rather is related to changes in M1 expression or localization. The mutations identified in this paper may be used to enhance delNS1 virus replication for vaccine production.

MDCK cell line with inducible allele B NS1 expression propagates delNS1 influenza virus to high titres.

Influenza A viruses lacking the gene encoding the non-structural NS1 protein (delNS1) have potential use as live attenuated vaccines. However, due to the lack of NS1, virus replication in cell culture is considerably reduced, prohibiting commercial vaccine production. We therefore established two stable MDCK cell lines that show inducible expression of the allele B NS1 protein. Upon induction, both cell lines expressed NS1 to about 1000-fold lower levels than influenza virus-infected cells. Nevertheless, expression of NS1 increased delNS1 virus titres to levels comparable to those obtained with an isogenic virus strain containing an intact NS1 gene. Recombinant NS1 expression increased the infectious virus titres 244 to 544-fold and inhibited virus induced apoptosis. However, NS1 expression resulted in only slightly, statistically not significant, reduced levels of interferon-ß production. Thus, the low amount of recombinant NS1 is sufficient to restore delNS1 virus replication in MDCK cells, but it remains unclear whether this occurs in an interferon dependent manner. In contrast to previous findings, recombinant NS1 expression did not induce apoptosis, nor did it affect cell growth. These cell lines thus show potential to improve the yield of delNS1 virus for vaccine production.

A comparative infection study of pigeon and avian paramyxovirus type 1 viruses in pigeons: evaluation of clinical signs, virus shedding and seroconversion.

The pathogenesis of pigeon paramyxovirus type 1 (PPMV-1) isolate AV324/96 and of its recombinant derivative, rgAV324, was studied in pigeons. For comparison, the virulent chicken virus FL-Herts, which is a recombinant derivative of strain Herts/33, was also included. After inoculation by the combined intraocular, intranasal and intratracheal route, clinical signs, virus shedding and serological responses were examined. Clinical signs were observed only in the FL-Herts-infected group. All virus-inoculated pigeons had positive tracheal swabs until 5 days post infection. However, only the AV324/96-infected and rgAV324-infected birds, and not the FL-Herts-infected birds, shed virus in the cloaca. The AV324/96-infected pigeons showed higher mean antibody titres than the rgAV324-infected birds, whereas the antibody titres of the FL-Herts-infected group were rather low. The results show that the pigeon strain AV324 is not virulent for pigeons, but underlines the potential risk of poultry becoming infected by PPMV-1 shed by non-symptomatic pigeons.

Passaging of a Newcastle disease virus pigeon variant in chickens results in selection of viruses with mutations in the polymerase complex enhancing virus replication and virulence.

Some Newcastle disease virus (NDV) variants isolated from pigeons (pigeon paramyxovirus type 1; PPMV-1) do not show their full virulence potential for domestic chickens but may become virulent upon spread in these animals. In this study we examined the molecular changes responsible for this gain of virulence by passaging a low-pathogenic PPMV-1 isolate in chickens. Complete genome sequencing of virus obtained after 1, 3 and 5 passages showed the increase in virulence was not accompanied by changes in the fusion protein--a well known virulence determinant of NDV--but by mutations in the L and P replication proteins. The effect of these mutations on virulence was confirmed by means of reverse genetics using an infectious cDNA clone. Acquisition of three amino acid mutations, two in the L protein and one in the P protein, significantly increased virulence as determined by intracerebral pathogenicity index tests in day-old chickens. The mutations enhanced virus replication in vitro and in vivo and increased the plaque size in infected cell culture monolayers. Furthermore, they increased the activity of the viral replication complex as determined by an in vitro minigenome replication assay. Our data demonstrate that PPMV-1 replication in chickens results in mutations in the polymerase complex rather than the viral fusion protein, and that the virulence level of pigeon paramyxoviruses is directly related to the activity of the viral replication complex.

The viral replication complex is associated with the virulence of Newcastle disease virus.

Virulent strains of Newcastle disease virus ([NDV] also known as avian paramyxovirus type 1) can be discriminated from low-virulence strains by the presence of multiple basic amino acid residues at the proteolytic cleavage site of the fusion (F) protein. However, some NDV variants isolated from pigeons (pigeon paramyxovirus type 1 [PPMV-1]) have low levels of virulence, despite the fact that their F protein cleavage sites contain a multibasic amino acid sequence and have the same functionality as that of virulent strains. To determine the molecular basis of this discrepancy, we examined the role of the internal proteins in NDV virulence. Using reverse genetics, the genes encoding the nucleoprotein (NP), phosphoprotein (P), matrix protein (M), and large polymerase protein (L) were exchanged between the nonvirulent PPMV-1 strain AV324 and the highly virulent NDV strain Herts. Recombinant viruses were evaluated for their pathogenicities and replication levels in day-old chickens, and viral genome replication and plaque sizes were examined in cell culture monolayers. We also tested the contributions of the individual NP, P, and L proteins to the activity of the viral replication complex in an in vitro replication assay. The results showed that the replication proteins of Herts are more active than those of AV324 and that the activity of the viral replication complex is directly related to virulence. Although the M protein affected viral replication in vitro, it had only a minor effect on virulence.

Intramuscular inoculation of calves with an experimental Newcastle disease virus-based vector vaccine elicits neutralizing antibodies against Rift Valley fever virus.

In the past decade, the use of Newcastle disease virus (NDV) as a vaccine vector for the prevention of economically important livestock diseases as well as for human diseases has been extensively explored. In this study, we have constructed a recombinant NDV vaccine virus, named NDFL-Gn, that produces the Rift Valley fever virus (RVFV) Gn glycoprotein. Calves were immunized via either the intranasal route or the intramuscular route. Delivery via the intranasal route elicited no detectable antibody responses, whereas delivery via the intramuscular route elicited antibodies against both NDV and the Gn protein. The RVFV-neutralizing activity of the antisera from intramuscularly vaccinated calves was demonstrated, suggesting that NDV is a promising vaccine vector for the prevention of RVF in calves.

Virulence of pigeon paramyxovirus type 1 does not always correlate with the cleavability of its fusion protein.

Some pigeon paramyxovirus type 1 (PPMV-1) strains exhibit low virulence in chickens, despite their fusion (F) protein's multi-basic cleavage site. To elucidate the molecular basis of the low pathogenicity of these strains, we constructed an infectious full-length cDNA clone of PPMV-1 strain AV324. This strain is non-virulent for chickens, although its F protein contains the typical virulence motif (112)RRKKRF(117). By using reverse genetics, we exchanged the F genes of AV324 and a virulent Newcastle disease virus (NDV) strain (Herts) and evaluated the recovered chimeric viruses for their pathogenicity in 1-day-old chickens and in embryonated eggs. Our results show that the F protein of AV324, and probably those of similar PPMV-1 strains, are functionally not different from those of virulent NDV strains and that the difference in pathogenicity must be determined by other factors.

VP1, the RNA-dependent RNA polymerase and genome-linked protein of infectious bursal disease virus, interacts with the carboxy-terminal domain of translational eukaryotic initiation factor 4AII.

Infectious bursal disease virus (IBDV), a member of the family Birnaviridae, is a non-enveloped, double-stranded RNA virus. Viral protein 1 (VP1), the putative RNA-dependent RNA polymerase, occurs in virions both as a free polypeptide and as a genome-linked protein, called VPg. To gain more insight in its function, we initiated a yeast two-hybrid screen. With this approach we identified the carboxy-terminal domain of eukaryotic translation initiation factor 4AII (eIF4AII) as an interactor for VP1. The association between these molecules was confirmed by co-immunoprecipitation analyses. eIF4A plays an essential role in the initiation of translation of both capped and uncapped mRNAs. Its association with IBDV VP1 suggests an involvement of this viral protein in IBDV mRNA translation. An interaction between VP1 and full-length eIF4AII was, however, not observed. In view of the known two-domain structure of eIF4AII it is conceivable that the interaction of VP1 with full-length eIF4AII requires collaborating proteins that open up its structure and expose the VP1-binding site in the carboxy-terminal domain. The biological relevance of the potential VP1-eIF4AII interaction is discussed.

Tissue tropism in the chicken embryo of non-virulent and virulent Newcastle diseases strains that express green fluorescence protein.

The tissue tropism of non-virulent and virulent Newcastle disease virus (NDV) was investigated using 8-day-old and 14-day-old embryonating chicken eggs (ECE), inoculated with an infectious clone of the non-virulent La Sota strain (NDFL-GFP) or its virulent derivative (NDFLtag-GFP). Both strains expressed the gene encoding jellyfish green fluorescence protein (GFP) as a marker. The GFP was readily expressed in chicken embryo cells infected with the NDV strains indicating virus replication. Whereas both strains replicated in the chorioallantoic membrane (CAM) and infected the skin of 8-day-old ECE, only the virulent strain (NDFLtag-GFP) spread to internal organs (pleura/peritoneum). In 14-day-old ECE, the initial target organs appeared to be the CAM and the lungs for both strains. At 48 h after inoculation, the virulent strain (NDFLtag-GFP) had also spread to the spleen and heart and was detected in a wide-range of embryonic cell types. The kinetics of virus replication and spread in the CAM closely resembled each other in both the 8-day-old and 14-day-old ECE. Infection of 8-day-old and 14-day-old ECE forms a convenient model to investigate tissue tropism of NDV, as well as the kinetics of viral infection. The advantage of using GFP is that samples can be easily screened by direct fluorescence microscopy without any pre-treatment.

Rescue of infectious bursal disease virus from mosaic full-length clones composed of serotype I and II cDNA.

Infectious Bursal Disease Virus (IBDV) is the causative agent of one of the most important and wide-spread infectious diseases among commercial chicken flocks. IBDV causes a depletion of B-lymphoid cells in the bursa of Fabricius, inducing immunosuppression, morbidity, or even acute mortality. Because currently used live IBDV vaccines are derivatives from field isolates no serologic discrimination between field isolates and live vaccines can be made. The recently developed reverse genetics techniques for IBDV allows one to generate genetically modified IBDVs which might have altered biological and antigenic properties. Here, we describe the rescue of mosaic serotype I IBDVs, of which the polyprotein encoding region was partly replaced by the corresponding region of a serotype II strain. A mosaic virus, containing the C-terminal part of serotype II VP3 showed only a slightly delayed release of progeny virus compared to unmodified serotype I virus, while maximum viral titers at 25 h post infection were equal. Since serotype specific epitope(s) are present in the C-terminal part of VP3, we were able to discriminate this rescued virus from serotype I and II IBDV strains. These findings make the use of a chimeric VP3 a promising approach to develop an IBDV marker vaccine.

Comparison of RNA and cDNA transfection methods for rescue of infectious bursal disease virus.

Specific alterations in the genetic material of RNA viruses rely on a technique known as reverse genetics. Transfection of cells with the altered generic material is a critical step of this procedure. In this report we have compared RNA and cDNA transfection methods for the efficiency of transient protein expression and rescue of (recombinant) infectious bursal disease virus (IBDV). Quantitative expression analysis of the secreted alkaline phosphatase reporter protein, and qualitative expression levels of an IBDV protein showed both that cDNA transfection results in a much higher level of protein expression than RNA transfection. Because the rescue of a crippled variant of IBDV was achieved consistently using the cDNA transfection method, but failed when we used the RNA transfection method, we favor the cDNA transfection method for the rescue of (recombinant) IBDV from cloned cDNA.

Identical amyloid precursor proteins in two breeds of chickens which differ in susceptibility to develop amyloid arthropathy.

Chickens (Gallus gallus domesticus) can suffer from AA amyloidosis featuring the joints as major targets of amyloid accumulation. Analysis of post-mortem recordings from commercial chickens revealed that amyloid arthropathy frequently occurred in brown layer chickens, but never in white layers. The suspected higher susceptibility of brown layers was confirmed experimentally by inducing amyloidosis with an arthropathic and amyloidogenic strain of E. faecalis. Sequence analysis of cDNA coding for SAA, the amyloid precursor protein, revealed that the SAA proteins are identical in both breeds, although some nucleotide differences existed in the untranslated regions of the mRNAs. The chicken SAA gene was found to be a single copy gene which comprises 4 exons. The first of these exons apparently occupies a conserved position and is not translated. Investigation of the affected joints using in situ hybridization demonstrated local SAA gene expression. It is concluded that the likelihood of an E. faecalis induced arthritis to progress to amyloidosis is breed-dependent, but is not a consequence of a more amyloidogenic SAA.

Generation of a recombinant chimeric Newcastle disease virus vaccine that allows serological differentiation between vaccinated and infected animals.

Using a recently developed reverse genetics system, we have generated a recombinant Newcastle disease virus (NDV) vaccine in which the gene encoding the hemagglutinin-neuraminidase (HN) has been replaced by a hybrid HN gene consisting of the cytoplasmic domain, transmembrane region, and stalk region of HN of NDV, and the immunogenic globular domain of HN of avian paramyxovirus type 4 (APMV4). The objective was to generate a chimeric live vaccine that induces a protective immune response against NDV by eliciting neutralizing antibodies against the fusion (F) protein, but which can be differentiated from wild-type NDV on the basis of different antibodies elicited by their HN proteins. Pathogenicity tests in day-old chickens showed that the recombinant was non-virulent (intracerebral pathogenicity index [ICPI]=0.00). A vaccination-challenge experiment in 4-week-old specific pathogen free chickens demonstrated that the recombinant was completely safe and was able to protect chickens from challenge with a lethal dose of virulent NDV. By using a secreted form of HN produced in Pichia pastoris, a test was developed that allowed serological differentiation between animals vaccinated with the recombinant vaccine and animals infected with NDV. These results demonstrate that genetically modified marker vaccines can be generated from small RNA viruses that lack non-essential genes.

Genome replication of Newcastle disease virus: involvement of the rule-of-six.

We examined replication of Newcastle disease virus (NDV) by using minigenomes consisting of the 3' leader and 5' trailer regions of NDV flanking a reporter gene encoding secreted placental alkaline phosphatase (SEAP). Negative-sense minigenome RNA was generated from transfected plasmid DNA by means of in vivo transcription. Subsequent replication of minigenome RNA was determined either after infection with NDV helpervirus or after contransfection with helperplasmids that expressed the essential viral replication proteins NP, P, and L. In both systems, efficient replication of minigenome RNA was observed only if the genome size was a multiple of six nucleotides. Hence, in these systems, replication of NDV minigenome RNA's is strictly dependent on the rule-of-six. When the supernatant from helpervirus-infected, transfected cells was used to infect fresh monolayers, efficient transfer of SEAP activity by virus-like particles was observed only if the size of the minigenome RNA obeyed the rule-of-six. However, after several serial passages, we also observed efficient transfer of SEAP activity by virus-like particles derived from minigenome RNA's that did not obey the rule-of-six. Evidence was obtained which indicated that successful replication of these minigenomes was not due to a change in genome size.

Rescue of very virulent and mosaic infectious bursal disease virus from cloned cDNA: VP2 is not the sole determinant of the very virulent phenotype.

Many recent outbreaks of infectious bursal disease in commercial chicken flocks worldwide are due to the spread of very virulent strains of infectious bursal disease virus (vvIBDV). The molecular determinants for the enhanced virulence of vvIBDV compared to classical IBDV are unknown. The lack of a reverse genetics system to rescue vvIBDV from its cloned cDNA hampers the identification and study of these determinants. In this report we describe, for the first time, the rescue of vvIBDV from its cloned cDNA. Two plasmids containing a T7 promoter and either the full-length A- or B-segment cDNA of vvIBDV (D6948) were cotransfected into QM5 cells expressing T7 polymerase. The presence of vvIBDV could be detected after passage of the transfection supernatant in either primary bursa cells (in vitro) or embryonated eggs (in vivo), but not QM5 cells. Rescued vvIBDV (rD6948) appeared to have the same virulence as the parental isolate, D6948. Segment-reassorted IBDV, in which one of the two genomic segments originated from cDNA of classical attenuated IBDV CEF94 and the other from D6948, could also be rescued by using this system. Segment-reassorted virus containing the A segment of the classical attenuated isolate (CEF94) and the B segment of the very virulent isolate (D6948) is not released until 15 h after an in vitro infection. This indicates a slightly retarded replication, as the first release of CEF94 is already found at 10 h after infection. Next to segment reassortants, we generated and analyzed mosaic IBDVs (mIBDVs). In these mIBDVs we replaced the region of CEF94 encoding one of the viral proteins (pVP2, VP3, or VP4) by the corresponding region of D6948. Analysis of these mIBDV isolates showed that tropism for non-B-lymphoid cells was exclusively determined by the viral capsid protein VP2. However, the very virulent phenotype was not solely determined by this protein, since mosaic virus containing VP2 of vvIBDV induced neither morbidity nor mortality in young chickens.

Generation of full-length cDNA of the two genomic dsRNA segments of infectious bursal disease virus.

To determine the complete nucleotide sequence of Infectious Bursal Disease virus (IBDV) isolates, an efficient method was developed to generate full-length cDNA of both the genomic A- and B-segments. Reverse transcription was carried out at the highest possible temperature (50 degrees C) for the reverse transcriptase enzyme, and the single stranded cDNA was subsequently amplified by using an optimized PCR. The double stranded, full-length cDNA was efficiently cloned into a high copy number plasmid. Our results show that the entire cDNA of both the A- and B-segment of a classical attenuated isolate (CEF94), and a very virulent field isolate (D6948), can be cloned. The method will simplify greatly the procedure to generate full-length cDNA and determine the nucleotide sequence of the entire genome of IBDV isolates.

Interactions in vivo between the proteins of infectious bursal disease virus: capsid protein VP3 interacts with the RNA-dependent RNA polymerase, VP1.

Little is known about the intermolecular interactions between the viral proteins of infectious bursal disease virus (IBDV). By using the yeast two-hybrid system, which allows the detection of protein-protein interactions in vivo, all possible interactions were tested by fusing the viral proteins to the LexA DNA-binding domain and the B42 transactivation domain. A heterologous interaction between VP1 and VP3, and homologous interactions of pVP2, VP3, VP5 and possibly VP1, were found by co-expression of the fusion proteins in Saccharomyces cerevisiae. The presence of the VP1-VP3 complex in IBDV-infected cells was confirmed by co-immunoprecipitation studies. Kinetic analyses showed that the complex of VP1 and VP3 is formed in the cytoplasm and eventually is released into the cell-culture medium, indicating that VP1-VP3 complexes are present in mature virions. In IBDV-infected cells, VP1 was present in two forms of 90 and 95 kDa. Whereas VP3 initially interacted with both the 90 and 95 kDa proteins, later it interacted exclusively with the 95 kDa protein both in infected cells and in the culture supernatant. These results suggest that the VP1-VP3 complex is involved in replication and packaging of the IBDV genome.

Efficient rescue of infectious bursal disease virus from cloned cDNA: evidence for involvement of the 3'-terminal sequence in genome replication.

To study the mechanism of replication of infectious bursal disease virus (IBDV), and to determine factors on the IBDV RNA which are involved in viral replication, we used cloned full-length cDNA of both the A- and B-segments to generate infectious IBDV. Infectious IBDV was rescued from plasmids that contained full-length IBDV cDNA behind a T7 promoter, by transfecting these plasmids into cells which were infected with a recombinant Fowlpox virus that expressed T7 RNA polymerase. By using the cDNA transfection system we evaluated the effect of the length of the 3' terminus of the A-segment plus strand of IBDV. Although wild-type IBDV predominantly contains four cytosines at the 3' terminus, no difference in virus yield was found when virus was rescued from cDNAs containing three to six adjacent cytosines. When the 3' terminus was shorter than three cytosines the efficiency to generate infectious IBDV from cDNA was reduced, but IBDV could still be recovered reproducibly. The rescued viruses from cDNAs containing 3'-terminal deletions appeared to have a restored 3'-terminal sequence. The missing nucleotides are probably restored by using complementary bases of a stem-loop structure as template.