AVIAN DISEASES 56:208217, 2012

Pathogenicity and Immunogenicity of Different Recombinant Newcastle Disease Virus Clone 30 Variants After In Ovo Vaccination
ckerlin,B Dirk Ho per,B Mario Ziller, C Thomas C. Mettenleiter,A Kristina Ramp,A Eylin Topfstedt,A Regula Wa B mer-Oberdo rferAD Christian Grund, and Angela Ro
Institute of Molecular Biology Institute of Diagnostic Virology C dufer 10, D-17493 Greifswald-Insel Riems, Germany Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Su
B A

Received 3 August 2011; Accepted and published ahead of print 25 October 2011 SUMMARY. 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 24:17561765, 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. RESUMEN. Patogenicidad e inmunogenicidad de diferentes virus variantes recombinantes de la cepa Clone 30 del virus de la s de la vacunacio n in ovo. enfermedad de Newcastle despue a de edad en los pollos, A pesar de que las cepas vacunales vivas del virus de la enfermedad de Newcastle pueden ser aplicadas al d as antes de eclosionar. Con base en un sistema stas son pato genas para los embriones de pollo cuando se administran in ovo tres d e tica inversa, se modifico el virus recombinante de Newcastle (rNDV) que se establecio de la cepa vacunal lentoge nica Clone de gene ficas dentro de las prote nas de fusio n de mutaciones espec n (F) y hemaglutinina-neuraminidasa (HN), 30 mediante la introduccio n de las variantes de vacuna seleccionadas (Mast et al. que recientemente se han sugerido como las responsables de la atenuacio en el virus rNDV49. Otro virus recombinante (rNDVGu) se genero para corregir las Vaccine 24:17561765, 2006) lo que resulto diferencias de las secuencias entre el virus rNDV y la cepa vacunal de Newcastle Clone 30. Los virus recombinantes rNDV, n con la cepa Clone 30, que se manifesto por la disminucio n de los rNDV49 y rNDVGu han reducido su virulencia en comparacio s de la inoculacio n in ovo, la ndices de patogenicidad intracerebral y el elevado tiempo promedio de mortalidad. Despue lo un pollo del grupo que recibio la vacuna Clone 30 incubabilidad fue similar para todos los grupos infectados. Sin embargo, so odo de observacio as, mientras que la tasa de supervivencia de los pollos nacidos de los grupos que a un per n de 21 d sobrevivio entre el 40% y 80%, siendo el virus rNDVGu el virus ma s pato geno. Adema s, los virus recombinantes recibieron rNDV se ubico o con un virus de Newcastle virulento a los 21 d as despue n contra la infeccio n por desaf s de la eclosio n. Las indujeron proteccio diferencias en la respuesta de los anticuerpos de los virus recombinantes indican que la inmunogenicidad se correlaciona con la virulencia. En resumen, los datos muestran que las mutaciones puntuales pueden reducir la virulencia del virus de la enfermedad de nas F y HN del virus rNDV no dieron lugar a la n de determinados aminoa cidos en las prote Newcastle. Sin embargo, la alteracio n adicional como lo indico su patogenicidad para los pollos despue s de la inoculacio n in ovo. atenuacio Key words: recombinant NDV, in ovo vaccine Abbreviations: CEF 5 chicken embryo fibroblast; EID50 5 mean egg infectious doses; F 5 fusion protein; FITC 5 fluorescein isothiocyanate; GS 5 genome sequencer; HI 5 hemagglutinin inhibition; HN 5 hemagglutinin-neuraminidase; ICPI 5 intracerebral pathogenicity index; IFA 5 indirect immunofluorescence assay; L protein 5 large protein; M protein 5 matrix protein; MDT 5 mean death time; MOI 5 multiplicity of infection; ND 5 Newcastle disease; NDV 5 Newcastle disease virus; NP 5 nucleoprotein; OIE 5 World Organisation for Animal Health (Office International des Epizooties); P protein 5 phosphoprotein; POD 5 peroxidase; QM 5 quail muscle; RT-qPCR 5 reverse transcriptase real time PCR; SDS 5 sodium dodecyl sulfate; SPF 5 specific pathogen free; TCID50 5 mean tissue culture infectious dose

Newcastle disease (ND) is a highly contagious, severe infection in poultry worldwide that has a great impact on the poultry industry. Therefore, it is listed by the World Organisation for Animal Health (OIE) as a notifiable disease (http://www.oie.int/en/animal-health-

Corresponding author. E-mail: angela.roemer-oberdoerfer@fli.bund.de

in-the-word/oie-listed-diseases-2011/). The causative agent, Newcastle disease virus (NDV), also designated avian paramyxovirus serotype 1 (APMV-1), belongs to the genus Avulavirus within the subfamily Paramyxovirinae, family Paramyxoviridae (14,19). The genome of NDV consists of a nonsegmented, single-stranded RNA of negative polarity. Following the rule of six (26), the genome size can vary between 15,186 nucleotides (nt) (class II genotype IIV,

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209

Fig. 1. Schematic presentation of genomes of newly generated recombinant NDV based on pflNDV-1 (29). Sites of the restriction enzymes used for different cloning steps are given. A) pflNDV49; B) pflNDVGu.

early isolates), 15,192 nt (class II genotype VVIII, late isolates), or 15,198 nt (class I) (8). The genome encodes six structural proteins, the nucleoprotein (NP), the phosphoprotein (P protein), the matrix (M) protein, the fusion (F) protein, the hemagglutinin-neuraminidase (HN) protein, and the large RNA-dependent RNA polymerase (L). Two additional proteins (V and W), presumably regulators, are generated by RNA editing during P gene transcription (33). The two surface glycoproteins HN and F mediate receptor-specific binding to the target cell and virus entry. They are known determinants of viral virulence (17,23,24,30). Depending on the severity of clinical disease in chickens, three pathotypes (i.e., lentogenic, mesogenic, or velogenic NDV) can be differentiated by their intracerebral pathogenicity index (ICPI). A major pathogenicity determinant is represented by the proteolytic cleavage site of the F protein (9,21,24,25). The F protein of pathogenic (i.e., mesogenic or velogenic) NDV contains at least one pair of basic amino acids at residues 115 and 116 plus a phenylalanine at residue 117 and a basic amino acid (arginine) at residue 113 (http:// www.oie.int/index/php?id5169&L50&htmfile5chapitre_1.10.9. htm). Infection by highly virulent, velogenic NDV leads to severe disease (5) with mortality rates as high as 100%. In contrast, lentogenic NDV is in use for oral or spray live vaccination, and commonly used strains like La Sota, Clone 30, B1, or V4 do not induce clinical disease in adult chickens. Although vaccination may be performed on the first day after hatch, respiratory distress and a slight reduction in weight gain may occur depending on the age of chickens at the date of vaccination (15). To minimize these problems and to optimize early protection of chickens, in ovo vaccination has been discussed (4). In ovo vaccination is already commonly practiced in the

United States to protect against Mareks disease and infectious bursal disease (28). It offers the opportunity of precise automated application, which entails reduction of stress for animals and reduction of costs. However, lentogenic NDV vaccine strains like La Sota or Clone 30 are pathogenic for chicken embryos, and therefore not suitable for in ovo application. To minimize embryo pathogenicity, classical vaccine strains were either modified by an alkylating agent (1) or injected in ovo as inactivated oil emulsion preparations (34). A recombinant fowlpox virus engineered to express NDV surface proteins F and HN as well as chicken interferon I or II was also tested for this purpose (27). Despite promising results, the only commercial in ovo vaccine against NDV currently available is the Poulvac OVOlineTM NDV-vaccine (Fort Dodge Animal Health Limited, Southampton, UK), which does not guarantee acceptable immune response and hatch and protection rates in specific pathogen free (SPF) chicken embryos (10). Therefore, a more reliable in ovo vaccine against NDV is in strong demand. Recently, Mast et al. (18) isolated an NDV La Sota escape mutant after immunoselection with neutralizing antibodies against epitopes in the F and HN proteins. The selected mutant was more attenuated than the parental NDV La Sota and exhibited a promising hatch rate and good protection after challenge. However, since the selected mutant has been characterized genetically only within the F and HN genes, it remained unclear whether fortuitous alterations in other regions may have contributed to the observed attenuation. Here, we describe the generation of two recombinant NDV that carry specific mutations based on recombinant NDV (rNDV), derived from vaccine strain Clone 30 (29). One of them possesses

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Binding positionA

K. Ramp et al.

Table 1. Sequences of primers used for mutagenesis and RT-PCR.

Name Sequence Application

MPFD72YF MPHNN115SF MPHNL229RF MP2368F MP3667F MP4446F MP5478F MP13852F MP14701F MP15051F P10AFLF P14STUR
A

47414773 67376777 70797116 23682409 36673707 44464490 54785519 13,85213,893 14,70114,742 15,05115,095 11,29711,318 12,91912,900

59-ccc gaa tct gcc caa gta caa gga ggc atg tgc-39 59-ctc tct ctt atc aga ttt ccg gag ctg caa aca aca gtg gg-39 59-ctc tgc gtt cca tca aca gag acg aca ccc aaa atc gg-3 59-agt caa ccc agt cgc gga aac agt cag gaa aga ccg cag aac-39 59-gca act aat act gag aga atg gtt ttc tca gta gtg cag gc-39 59-cac cca gat cat cat gac aca aaa aac taa tct gtc ttg att att-39 59-cct tat ccg taa gca caa cca ggg gat ttg cct cgg cac ttg-39 59-cga ccc caa ctc agt ttt tga att cgg ttg ttt ata gga atc-39 59-aca tca ctt aaa cag tgc acg aga cag atc cta gag gtt aca-39 59-gcc aat tgt att ctt gtt gat tta atc ata tta tgt tag aaaaaa-39 59-tgc aac gac cca tac tct ttc-39 59-att gta gct gca atg ttg g-39

Mutagenesis Mutagenesis Mutagenesis Mutagenesis Mutagenesis Mutagenesis Mutagenesis Mutagenesis Mutagenesis Mutagenesis RT-PCR RT-PCR

According to GenBank Accession number Y 18898. the pUC-18 plasmid to modify nucleotides encoding amino acids at position 72 of the F protein (D to Y) and at positions 115 (N to S) and 229 (L to R) of the HN protein, resulting in NA1MUT and pUCAROGMUT, respectively. Afterwards, the ApaI/NotI fragment and the NotI/BsiWI fragment of pflNDV-1 were substituted by the corresponding modified fragment of NA1MUT and pUCAROGMUT, respectively, resulting in the new full-length plasmid pflNDV49 (Fig. 1A). For generation of rNDVGu, the full-length plasmid pflNDV-7, which is distinct from pflNDV-1 (29) by deletion of artificial MluI-sites, was used as a backbone for the generation of the new full-length plasmid with changed nucleotides corresponding to the results of the updated sequence (alterations are given in Table 2). Nucleotides were changed by site-directed mutagenesis of plasmid DNA or RT-PCR of freshly isolated RNA from wild-type virus NDV Clone 30, followed by fragment substitution. First, the P, M, and F genes were adapted to the updated NDV Clone 30 sequence. In detail, ApaI-NotI and NotI-SpeI fragments of pflNDV-7, containing nucleotides that had to be changed (fragment I: nucleotides 2368, 3667, 4446; fragment II: nucleotide 5478) were cloned into vector pGEMH-T Easy (Promega, Madison, WI) for a single site-directed mutagenesis (QuikChange II XL mutagenesis kit, Stratagene) with given primers (biomers.net GmbH, Ulm, Germany) (Table 1). The L-gene sequence was modified in two steps. First, fragment III of NDV Clone 30 was amplified by RT-PCR using freshly prepared RNA from allantoic fluid and primers P10AFLF and P14STUR (Table 1) and cloned into pGEM-T Easy. Second, nucleotides 13,852, 14,701, and 15,051 within the L gene were changed by mutagenesis with given primers (Table 1) using the AflII-SacII fragment of pflNDV-7, resulting in a sequence that corresponded to the updated NDV Clone 30 sequence (fragment IV). Fragments III and IV were linked using the ApaI site of pGEM-T Easy and the StuI site, which is available in fragment III as well as in fragment IV. The corresponding fragment of pflNDV-7 was then substituted by fragments III and IV, using AflII and SacII sites. Finally, all fragments with nucleotide alterations were used for substitution of the corresponding regions within the pflNDV-7 genome, resulting in the new plasmid pflNDVGu (Fig. 1B). Cells and eggs. Quail muscle cells (QM9) and primary chicken embryo fibroblasts (CEFs) used for in vitro characterization, as well as T7-BSR cells, which stably express phage T7 RNA polymerase (6) used for transfection and recovery of virus, were provided by the cell culture collection of the Friedrich-Loeffler-Institut (Insel Riems, Germany). Embryonated SPF chicken eggs for virus propagation, serial passaging, and animal experiments were purchased from Lohmann (Cuxhaven, Germany) and incubated at 37 C and 55% humidity. Viruses. NDV Clone 30 vaccine (NobilisTM) and velogenic challenge virus NDV strain Herts 33/56 were obtained from Intervet, Boxmeer, The Netherlands. Recombinant NDV (rNDV) based on vaccine strain Clone 30 has already been described (26). Recovery of recombinant viruses. Transfection and virus recovery were done essentially as described (35). Briefly, pflNDV49 and

alterations in the F and HN proteins as described by Mast et al. (18) (rNDV 49). The second virus (rNDVGu) contains single site mutations in P, M, F, and L genes to correct sequence variations between rNDV (29) and the parental Clone 30 revealed by new generation whole genome sequencing. Both newly generated NDV as well as rNDV and vaccine strain Clone 30 were biologically characterized in vitro and in vivo. By application of these viruses in ovo residual pathogenicity of NDV could be further dissected. The results on pathogenicity and immunogenicity after in ovo application give further insight into the fine balance between virulence and immunogenicity of NDV and elucidate part of their genetic background.
MATERIALS AND METHODS Determination of genome sequence of parental NDV Clone 30. In order to confirm the reference genomic sequence (GenBank accession number Y18898) of NDV Clone 30 by genome analysis using the Genome Sequencer FLX system (GS FLX, Roche, Mannheim, Germany), two libraries were prepared from RNA extracted from purified virions (library 1) and from allantoic fluid (library 2), respectively. To this end, nine RT-PCR products were generated from each preparation, covering the complete genome in overlapping amplicons (primers available on request) by OneStep RT-PCR kit (Qiagen, Hilden, Germany). The GS FLX system libraries were per et al. (16). The libraries prepared using the modified protocol of Ho were titrated and sequenced using the GS FLX instrument with the LR70 chemistry and protocol (library 2) or the SR70 chemistry and protocol (library 1) following the manufacturers instructions. Prior to mapping, the PCR primer sequences were trimmed from the reads to avoid distorting the mapping results. In order to identify differences between the genomes of the cloned viruses and the reference strain, the reads were aligned to the reference sequence (GenBank accession Y18898) using the GS reference mapper software (Version 1.1.02.15, Roche). The GS reference mapper software produces a compilation of all cases in which more than three sequence reads differ consistently from the reference sequence. This output was used to identify all base exchanges in rNDV (29) in comparison to wild-type NDV Clone 30. Construction of NDV recombinants: rNDV49 and rNDVGu. The plasmid pflNDV-1, expressing the full-length antigenome RNA of lentogenic NDV vaccine strain Clone 30 (29), was used for cloning (Fig. 1). Nucleotide modifications were introduced according to a recently described attenuated NDV (18) using the NotI-truncated pflNDV-1 (NA1) or a modified pUC-18 plasmid that contained the 3899-bp NotI-BsiWI-fragment of pflNDV-1 (pUCAROG). QuikChange II XL site-directed mutagenesis kit (Stratagene, Heidelberg, Germany) was used with mutagenesis primers MPFD72YF/R for the truncated pflNDV-1 and MPHNN115SF/R and MPHNL229RF/R (MWG Biotech, Ebersberg, Germany, primers are given in Table 1) for

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Table 2.

Sequence differences between NDV Clone 30 (GenBank Accession number Y18898) and their recombinants.
Y18898 rNDVGu rNDV rNDV 49 Region (gene) Resulting alteration

Nucleotide no.

76 79 2368 3667 4446 4757 4759 5478 6754 6755 6756 7096 7097 7098 11,657 12,567 13,852 14,701 15,039 15,041 15,042 15,051

T A T G C G T A A A T C T G G C A G T T T T

T A A A T G T G A A T C T G A T G A T T T A

A C T G C G T A A A T C T G G C A G A G C T

A C T G C T C A T C C A G A G C A G A G C T

ncr NP ncr NP P M ncr M F F F HN HN HN HN HN HN L L L L ncr L ncr L ncr L ncr L gene

MluI site MluI site L to Q Silent D to Y D to Y K to R N to S N to S N to S L to R L to R L to R G to R T to I Silent Silent MluI site MluI site MluI site

rNDVGu were cotransfected with the helper plasmids pCiteNP, pCiteP, and pCiteL expressing the NP, P, and L proteins of NDV Clone 30 into T7-BSR cells using Lipofectamin 2000 (Invitrogen, Karlsruhe, Germany) at a DNA:lipofectamin ratio of 1:1.5 (mg:ml) following the manufacturers instruction. Cells were split 1:2 24 hr after transfection and further incubated for 48 hr. Three days after transfection, supernatant was harvested and inoculated into the allantoic cavity of 9- to 11-day-old embryonated SPF chicken eggs. After 4 to 5 days of incubation, presence of the virus in allantoic fluid was confirmed by hemagglutination test with chicken erythrocytes and, after infection of QM9 cells with harvested allantoic fluid, by indirect immunofluorescence using a polyclonal rabbit anti-NDV serum. Indirect immunofluorescence assay (IFA). Infected cells were fixed with acetone/methanol (1:1). Detection of NDV was done by IFA using monoclonal antibody HN-10 (36) directed against NDV HN protein and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG F(ab)2 (Dako, Glostrup, Denmark) or NDV hyperimmune serum and FITC-conjugated goat anti-rabbit (Sifin, Berlin, Germany). Virus passaging and verification of changed sequences. Recombinant viruses were passaged 10 times in embryonated SPF chicken eggs. To this end, 9- to 11-day-old SPF chicken eggs were inoculated with an adequate dilution of allantoic fluid of the previous passage. Three to 4 days after injection into the allantoic cavity and incubation at 37 C and 55% humidity, allantoic fluid was harvested and examined for the presence of virus by hemagglutination test according to the standard procedure described in the Commission of the European Community Council Directive. Virus harvested after the second egg passage was used for characterization. Viral titers were assessed as tissue culture infectious doses (TCID50/ml) on QM9 cells. To verify presence of the mutations, viral RNA was prepared from allantoic fluid of the second and 10th egg Table 3.
Virus

MDT and ICPI of investigated viruses.

MDT (hr) ICPIA

rNDV rNDV49 rNDVGu NDV Clone 30

127 116 105 97

0.01a,b 0.09c,d 0.13b,d 0.29a,c

A Significance was tested applying Kruskal Wallis test (P 5 4.52E-05) and pairwise Fisher test. Letters indicate significant differences between groups (P , 0.05).

passage using Trizol reagents (Invitrogen). One-Step-RT PCR (Qiagen) with primers which enclosed the changed sequences was carried out and PCR products were directly sequenced with specific primers to confirm the alterations. Sequences of primers are available on request. Western Blot analyses. CEF cells were infected at a multiplicity of infection (MOI) of 5, and cell lysates were harvested 48 hr postinoculation. Viral proteins were separated under denaturing conditions in a 11% sodium dodecyl sulfate (SDS) polyacrylamide gel, transferred to a nitrocellulose membrane, and incubated with rabbit anti-NDV-F serum, which had been prepared by inoculation of rabbits with a vaccinia recombinant expressing NDV F essentially as already described for the generation of a rabbit anti-NDV-HN serum (32). Subsequently, the membrane was incubated with peroxidase (POD)labeled anti-rabbit antibodies (Dianova, Hamburg, Germany) after thorough washing. Binding of POD-labeled antibodies was detected by chemiluminescence with SuperSignal West Pico Chemiluminescence Substrate (Pierce, Rockford, IL) on X-ray films (Hyperfilm MP, Amersham Bioscience, Birmingham, UK). Thereafter, the nitrocellulose was placed in stripping buffer (25 mM glycine-HCl pH 2.0, 1% SDS), washed, and incubated with the rabbit anti-NDV-HN serum and PODlabeled anti-rabbit antibodies followed by detection. After another stripping step, proteins were detected after incubation with a rabbit antiNDV serum by chemiluminescence as described above. Kinetics of replication. QM9 and CEF cells cultured in 24-well plates were infected with NDV Clone 30, rNDV, rNDVGu, and rNDV49 at a MOI of 0.1 (QM9) or 0.01 (CEF) and incubated at 37 C and 3% CO2 atmosphere. The inoculum was removed 40 min after infection. Cells were washed twice and overlaid with fresh medium. QM9 cells were additionally treated with citrate buffer, pH 3.0, for 2 min to inactivate nonpenetrated virus before the fresh medium overlay. This treatment was not tolerated by CEFs. Cell supernatants were harvested and frozen at the indicated time points in two independent experiments. After thawing of the frozen samples, virus titers were determined on QM 9 cells by indirect immunofluorescence using rabbit-anti NDV serum and FITC-conjugated anti-rabbit IgG (Sifin, Berlin, Germany). Determination of mean death time (MDT). MDT was evaluated according to the protocol given by Alexander (3). Briefly, 100 ml of 10-fold dilution between 1023 and 10210 of infectious allantoic fluid in sterile saline was inoculated into the allantoic cavity of each of five 10-day-old embryonated SPF chicken eggs and incubated at 37 C. The remaining virus dilutions were retained at 4 C, and 100 ml of each

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Table 4. Hatch rate of inoculated chicken eggs.
Inoculated

K. Ramp et al.

Hatched

Hatchability (%)A

Effective hatchability (%)

rNDV rNDV /mock rNDV49 rNDV49 /mock rNDVGu rNDVGu /mock NDV Clone 30 NDV Clone 30 /mock

30 30 30 30 30 30 30 30

22 26 16 25 18 25 16 16

73.3 86.7 53.31 83.31 60.02 83.32 53.3 53.3

84.6 64.0 72.0 100.0

A Significance was tested applying pairwise Fisher test and numbers indicate significant differences between groups (P , 0.05). Hatchability refers to hatched chicks in relation to inoculated eggs, while effective hatchability compares vaccinated animals to mock-inoculated sentinel birds.

dilution was used for inoculation of another five eggs 8 hr later. Each egg was examined twice daily for 7 days and any embryo death was recorded. The minimum lethal dose was determined as the highest virus dilution that causes death of all inoculated embryos. The MDT is the mean time in hours to kill each embryo at the dose at which all embryos were killed. Characterization of recombinant virus in vivo. The MDT and the ICPI were determined as described (3,22) to assess the pathogenicity of recombinant viruses in embryonated SPF chicken eggs or SPF chickens, respectively. In ovo vaccination. All animal experiments were conducted with white leghorn chickens hatched from SPF eggs (Lohmann Tierzucht, Cuxhaven, Germany) in accordance with German animal rights legislation (M-V/TSD/7221.3-1.1-087/10). Groups of 30 SPF eggs each were incubated at 38.5 C and 60% humidity until day 18 and subsequently at 37.5 C and 85% humidity until hatching. At day 18 of incubation embryonated eggs were inoculated into the allantoic cavity with 104 EID50 of rNDV, rNDV49, rNDVGu, or wild-type NDV Clone 30, respectively. At the same time, groups of 30 eggs each were inoculated with 0.2 ml isotonic NaCl solution (mock) and were hatched together with the respective vaccine group in the same incubator at the same time, serving as contact controls (sentinel). Subsequently, hatchability was calculated as the percentage of hatched chickens from in ovo vaccinated groups. Control eggs were incubated without inoculation and hatched at a separate location. The presence of vaccine virus was tested by taking oropharyngeal swab samples at day 1 from 10 randomly selected animals of each group and analyzed by a NDV NP gene-specific real-time RT-PCR (RT-qPCR). Subsequently, 15 animals of each vaccine group were housed together with 15 chicks of the respective mock-inoculated sentinel group in positive pressure HEPAfiltered isolation unit (IM1500, Montair Andersen, The Netherlands)

within S3 facilities. Animals were inspected daily for clinical signs and mortality and at day 21 post hatch 10 randomly selected chickens from each group were challenged intramuscularly with 106 EID50 of the velogenic NDV strain Herts 33/56 according to the recommendation by the European Pharmacopoeia (11). NP gene-specific RT-qPCR. RNA from swab samples was extracted with the QiAmp viral RNA mini kit (Qiagen) according to manufacturers instructions and subsequently tested with TaqMan one-step RT-qPCR assay targeting the NDV NP gene, using the SuperScript III One-step RTPCR kit with Platinum Taq DNA polymerase (Invitrogen) on a MX3000P Real-Time PCR System (Stratagene). In all tests, negative RNA preparation controls and negative as well as positive RT-PCR controls were included. Reactions were run in a volume of 25 ml, containing 20 pmol of primers (NDVnp1066F: 59-GACAGYGACCAGATGAGCTT-39; NDVnp1231R: 59-CCTGAGCCTGAGCRTACTC-39) and 4 pmol of a dual-labeled 59-nuclease (TaqMan) probe featuring FAM as fluorophore at the 59 terminus and BHQ1 as quencher at the 39 terminus (59-[FAM]-ACATCATTYTGGMGACT-[BHQ1]-39) containing locked nucleic acids (bold letters). Reaction conditions were 30 min at 50 C for reverse transcription, followed by 15 min at 95 C and 42 cycles of 30 sec at 95 C for denaturation, 30 sec at 55 C for primer annealing, and 30 sec at 72 C for elongation. Fluorescence values were measured at the end of the annealing step of each cycle. Statistics. Kruskal-Wallis test was used for the comparison of MDT and ICPI (Table 3). Pairwise Fisher test was applied for MDT, ICPI (Table 3), for hatchability (Table 4), rate of infection, and survival rate (Table 5). For comparing hemagglutinin inhibition (HI) titers and weights, appropriate Wilcoxon tests were applied. The analysis was performed using R version 2.8.1 (http://www.R-project.org) with package exactRankTests. Box plots were drawn using Sigma Plot, Version 11 (Systat Software Inc., San Jose, CA).

Table 5. Rate of infection, survival rate, and protection rate of hatched chicks.
Rate of infectionA Survival rate,B day 21 (%) Protection after challenge infectionB

rNDV rNDV/mock rNDV49 rNDV49/mock rNDVGu rNDVGu/mock NDVClone 30 NDVClone 30/mock Control
A B

9/10Aa 0/10A 10/10Ba 0/10B 10/10Ca 1/10C 10/10Da 4/10Da 0/10E

12/15a 15/15b 7/13CAcd 12/14CA 6/15d 14/15 1/15Bacd 9/15Bb 15/15d

(80.0) (100.0) (53.8) (85.7) (40.0) (93.3) (6.7) (60.0) (100.0)

10/10a 10/10a 8/8a 10/10 6/6 10/10a 1/1 ndD 9/9a 0/10a

Infection was tested by RT-qPCR, Ct , 35 were considered positive. Significance was tested applying pairwise Fisher test: lowercase letters indicate significant differences between vaccinated groups and between vaccinated groups and control group; uppercase letters indicate significant differences between vaccinated and mock-vaccinated sentinel groups (P , 0.05). C Chickens with straddling legs were euthanatized, reducing the number of birds per group. D nd 5 statistical analysis are not applicable because of the small numbers of animals.

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Fig. 2. Western blot analyses. CEFs were infected with the indicated viruses at a MOI of 5. Lysates of infected cells were harvested 48 h postinoculation and analyzed by Western blotting using three different antibodies (a-NDV F, a-NDV HN, and a-NDV) as described in Material and Methods. RESULTS

Determination of parental NDV Clone 30 sequence. Two DNA libraries, either from RNA of purified virions (library 1) or from RNA directly recovered from allantoic fluid harvested from infected SPF chicken eggs (library 2), were prepared for deep sequencing of NDV Clone 30. The sequencing yielded a total of 1,108,923 nt (11,609 reads) for library 1 and 339,580 nt (2245 reads) for library 2. Mapping the reads along the reference sequence obtained by Sanger sequencing identified 14 and 11 nucleotide differences

within library 1 and library 2, respectively. Five nucleotides of library 1 and two nucleotides of library 2 had only minor reads and were not considered further. Consequently, nine prominent changes compared to Sanger sequencing remained (Table 2), which resulted in four alterations of the amino acid sequence of the P protein (one), the F protein (one), and the L protein (two). Generation of recombinant NDV49 and rNDVGu. Based on the recombinant full-length plasmid pflNDV-1 of Clone 30 (29), two new plasmids pflNDV49 and pflNDVGu were constructed in a multistep cloning process. In the F protein gene of pflNDV49 nucleotides 4757 and 4759 were changed from G to T and from T to C, respectively, resulting in a modification of amino acid 72 from aspartic acid (D) to tyrosine (Y). Amino acids 115 and 229 in the HN protein were changed from asparagine (N) to serine (S) and from leucine (L) to arginine (R), respectively, by mutation of nucleotides 67546756 from AAT to TCC and of nucleotides 70967098 from CTG to AGA. These mutations were introduced based on results which showed an association of these mutations with attenuation of NDV for in ovo vaccination (18). The plasmid pflNDVGu possesses all alterations according to the newly established NDV Clone 30 sequence, resulting in the appropriate amino acid modification within the P, F, and L proteins (Table 2). The corresponding recombinant virus was rescued after cotransfection of the full-length plasmid with helper plasmids into T7-BSR cells, followed by propagation in embryonated SPF chicken eggs as already described (29). Presence of the modifications in the NDV genome was confirmed by amplification of the respective genome regions and direct sequencing of the PCR product of the second as well as of the 10th virus passage. Expression of NDV-specific proteins was analyzed by indirect immunofluorescence (data not shown) and Western blot analyses (Fig. 2) using monospecific antiNDV F and HN sera, and NDV hyperimmune serum demonstrating, as expected, no differences of protein pattern. Characterization of generated recombinant virus. Replication of rNDV49 and rNDVGu in CEF as well as in QM9 cells was comparable to the replication of parental NDV Clone 30 and rNDV. All viruses replicated in CEFs to final titers of 105106 TCID50/ml; whereas, no virus propagation was detected after infection of QM9 cells (Fig. 3) as expected for lentogenic NDV strains. To analyze whether the introduced mutations modified virulence, MDT and ICPI of NDV Clone 30, rNDV, rNDV49, and rNDVGu were determined. Both MDT and ICPI (Table 3) were typical for a lentogenic NDV, demonstrating that the rNDV49 did not become more virulent after alteration of the genome sequence. In contrast, the ICPI of rNDVGu was significantly higher than that of rNDV and rNDV49 (P , 0.05). Compared to the parental NDV Clone 30, rNDVGu displays a similar ICPI (Table 3). In ovo vaccination. Recombinant viruses rNDV, rNDV49, and rNDVGu as well as vaccine strain NDV Clone 30 were subsequently used for in ovo vaccination at 18 days of embryo age. The hatchability of groups inoculated with recombinant viruses ranged from 53% to 73% while in the mock-inoculated control groups embryo hatchability was 80%. Only the mock-inoculated Clone 30 control group of birds had a lower hatch rate (53%) (Table 4). Virus infection of hatched chickens of inoculated eggs was verified by RT-qPCR. Chickens hatched from mock-inoculated eggs, housed together with chickens from rNDVGu (1 out of 10) and NDV Clone 30 (4 out of 10) infected eggs also showed presence of virus (Table 5). Major differences between groups became evident in a comparison of the 21-day survival rates. Whereas only one of the animals vaccinated with NDV Clone 30 survived for 21 days after

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Fig. 3. Replication kinetics of recombinant NDV (rNDV), rNDV49, rNDVGu, and wild-type NDV Clone 30. A) In CEFs infected with MOI 0.01; B) in quail muscle cells (QM9) infected with MOI 0.1.

hatch (P , 0.05), survival rates of groups vaccinated with rNDVs varied between 80% (rNDV), 53.8% (rNDV49), and 40% (rNDVGu), respectively. By comparison, the groups of mockinfected chicks, serving as sentinels, had survival rates up to 100% (rNDV/mock), but as low as 60% when co-housed with NDV Clone 30 infected eggs (Table 5). Impact on the development of the chickens by in ovo vaccination becomes manifest in a comparison of the weight of the animals before challenge as compared to the control group (Fig. 4). A significantly lower weight compared to the control group was evident for all vaccinated groups and interestingly,

also for the Clone 30/mock-infected as well as rNDV/mockinfected, and rNDV49/mock-infected sentinel groups (Fig. 4). Comparing vaccinated and mock-infected sentinel groups only the weight of rNDVGu/mock show a tendency (P 5 0.01869) to be higher than rNDVGu, but by multiple analyses this difference did not prove to be significant. With respect to antibody response measured by hemagglutination inhibition (7) at day 21 post hatch, all animals from the vaccinated groups tested seropositive, with highest antibody titer in the rNDVGu inoculated animals (8.561.5)log 2 (6 means standard deviation). Additionally, the

Fig. 4. Body mass of in ovo vaccinated and mock-vaccinated sentinel chickens Body weight of chicken was determined before challenge (i.e., 21 days post hatch) and are depicted for in ovo vaccinated animals and the control animals (A) and for mock-vaccinated animals serving as sentinels (B). The number of surviving animals used for the analysis is given above box plots and significant differences (P , 0.00833) are indicated (*), comparing in ovo vaccinated chickens to the control animals (A), or mock-vaccinated sentinels to rNDVGu/mock group (B).

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Fig. 5. Antibody response of in ovo vaccinated chicken before challenge infection with virulent NDV. Serum samples from chickens immunized with respective vaccines (A) and mock-vaccinated sentinel birds (B) collected at the day of challenge (i.e., 21 post hatch) were tested for NDVspecific antibodies by HI. The number of animals that are considered seropositive (HI .2 log2) is given above box plots. Significant differences (P , 0.00833) are indicated (*) for A) between respective vaccinated groups and the control animals or for B) between mock-vaccinated sentinels and the Clone 30/mock group. Letters (ad) indicate significant differences between vaccinated groups (A) or mock-infected groups (B) as well as between vaccinated and mock-infected groups (P , 0.01667) (IIII).

one chicken from the Clone 30 inoculated group that survived the 21-day observation period exhibited a high antibody titer (210). Interestingly, the majority of animals from the mock-infected sentinel group also tested seropositive (Fig. 5); the highest antibody titer was detected in the group Clone 30/mock (27.660.7), followed by rNDVGu/mock (25.960.9), and both had significantly higher antibody titers than rNDV/mock (23.861.2) and rNDV49/mock (23.661.2) groups (26.761.4). However, antibody titers of mockinfected sentinel animals were significantly lower than those of the respective vaccinated groups. Independent of the antibody level, the vaccinated as well as the mock-infected contact groups were protected after challenge with a virulent NDV (Herts33/56); whereas, all nonvaccinated chickens died within 2 days after challenge infection.
DISCUSSION

In ovo vaccination was hampered in the past due to residual virulence of naturally occurring NDV strains. Since the advent of reverse genetics for NDV it is possible to specifically alter the NDV genome and thus develop viruses with tailored properties (25,31) that consist of defined genetic material. Here, we investigated, whether point mutations within the F and HN protein of an in vitro escape mutant of NDV La Sota vaccine strain are responsible for its observed attenuation (18). To this end, recombinant NDVs (rNDV49, rNDVGu) were generated with specific alterations compared to recombinant NDV Clone 30 (29). In vitro replication, ICPI, or MDT of rNDV 49 and rNDVGu were comparable with rNDV (29). Both newly generated rNDVs showed a similar electrophoretic protein profile and replicated well in CEFs, but not in QM9 cells. Replication properties were in agreement with

earlier observations for NDV with a monobasic amino acid sequence at the cleavage site of the F protein (31). The recombinant NDVs (rNDV, rNDV49, and rNDVGu), as well as wild-type NDV Clone 30 were lentogenic with a MDT . 90 hr. However, slight differences in virulence became apparent, resulting in a ranking of viruses NDV Clone 30 . rNDVGu . rNDV , rNDV49 with decreasing virulence. This finding coincides with the ICPI data and the survival rate of chickens after in ovo vaccination. Surprisingly, rNDVGu with restriction of one defined sequence resulted in a decreased virulence compared with vaccine strain Clone 30. Even though NDV Clone 30 was derived from NDV strain La Sota as a biological clone, it is feasible that minor species might still be included that are not reflected by genome sequencing, considering only prominent alterations in comparison to the reference sequence. However, these minor species can possibly influence pathogenicity of the virus. Additional attenuation was observed for rNDV, which differed in four amino acid alterations in which one was in the P protein and two within the L protein. Therefore, two proteins of the ribonucleoprotein (RNP) complex are changed and may be responsible for the observed difference in virulence. This finding matches with results of Dortmans et al. (12) who found that the activity of the RNP complex is directly related to virulence and that point mutation within the P and L protein can result in alteration of virulence (13). The attenuation of rNDV and rNDV 49 was confirmed after in ovo vaccination by a higher survival rate of hatched chickens compared with NDV Clone 30. However, both recombinant viruses are not yet sufficiently attenuated for use as in ovo vaccine. Compared with a previous study, in which a 105 TCID50 per dose was used and seven chickens hatched from a total of 30 embryos (20), hatchability improved considerably using 104 TCID50 per dose with 16 to 22 hatched chickens out of 30 inoculated eggs.

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Nevertheless, hatchability after in ovo inoculation with recombinant NDVs is still reduced compared to mock-infected eggs. Even more dramatic is the impact on the health status of the hatched chickens, with reduced survival rates and impaired growth, with some of the chickens dwarfed. Interestingly, even from mock-infected eggs that served as sentinels, 6 out of 15 chicks of the NDV Clone 30/mock group died from infection transmitted by the virus-inoculated animals; whereas, the sentinel group to recombinant NDVs had survival rates between 85.7% and 100%. Concerning immunogenicity with respect to induced level of serum antibodies, the order of viruses was analogous to the virulence. Twenty-one days post hatch, the only chicken that survived NDV Clone 30 in ovo vaccination had the highest HI titer (210). Moreover, the antibody titers of rNDVGu-inoculated chickens were significantly higher than those that received rNDV or rNDV49. Regarding the induced serum antibody level, a comparable ranking was observed between the groups of mock-inoculated animals that contracted the respective virus by contact after hatching, indicating differences in immunogenicity. Upon challenge, all chickens were protected from highly pathogenic NDVs, independent from the vaccine virus. Furthermore, all chickens from mock-infected eggs that hatched and were raised together with vaccinated chickens were protected from challenge infection, indicating efficient transmission of vaccine virus by chickens hatched from vaccinated eggs. Our results are in good agreement with previous data, showing that NDV vaccine strains are pathogenic to chickens when inoculated in ovo (1,2,20). By site-directed mutagenesis recombinant viruses could be generated that exhibit a reduced pathogenicity after in ovo vaccination with 104 EID50. However, none of the recombinant NDVs described here are sufficiently qualified for an in ovo vaccine. In addition, our experiments demonstrate that, despite similar ICPI values, slight differences in virulence of different lentogenic NDVs can be distinguished after in ovo application. Furthermore, investigation of rNDV49 indicated that the suggested specific alteration in F and HN (18) actually did not further attenuate rNDV. Generally, regardless whether vaccines are applied after hatching or in ovo, our results underscore that a well-defined balance between immunogenicity and virulence is critical for success of vaccination.
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ACKNOWLEDGMENTS We thank Martina Lange, Cornelia Illing, and Katja Hartwig for excellent technical assistance.