Antibody detection-based differential ELISA for NDV-infected or vaccinated chickens versus NDV HN-subunit vaccinated chickens

Antibody detection-based differential ELISA for NDV-infected or vaccinated chickens versus NDV HN-subunit vaccinated chickens

Veterinary Microbiology 66 (1999) 209±222 Antibody detection-based differential ELISA for NDV-infected or vaccinated chickens versus NDV HN-subunit v...

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Veterinary Microbiology 66 (1999) 209±222

Antibody detection-based differential ELISA for NDV-infected or vaccinated chickens versus NDV HN-subunit vaccinated chickens Andrea M. Makkaya, Peter J. Krellb, EÂva Nagya,* a

Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph Ont., Canada N1G 2W1 b Department of Microbiology, University of Guelph, Guelph Ont., Canada N1G 2W1 Received 27 May 1998; accepted 3 February 1999

Abstract With the advent of subunit vaccines for microbial diseases it is becoming increasingly important to be able to differentiate naturally infected animals from those vaccinated with the corresponding subunit vaccine. For avian viruses such as Newcastle disease virus (NDV), a whole virus-based ELISA cannot make such a differential diagnosis since in both cases the antisera would react with the whole virus. The nucleocapsid protein (NP) gene of the NDV Hitchner B1 strain was cloned, sequenced and expressed to develop a differential ELISA. The B1 NP had 95.7 and 96.1% amino acid identities with the NP of the d26 and Ulster 2C strains, respectively. The B1 NP expressed in a baculovirus expression vector (recNP) was the expected size and reacted with NDV-specific antibodies (Ab) in Western blots and by radioimmunoprecipitation. The ELISA using recNP-coated wells, tested on serum samples from flocks pretested with a commercial NDV kit gave results corresponding to those of the kit. Furthermore, use of both the recNP-based ELISA and a whole virus ELISA allowed the differentiation of birds vaccinated with a NDV haemagglutinin± neuraminidase (HN) expressing fowlpox virus from birds infected with NDV. This provides the basis for establishing an ELISA that discriminates between the antibody response to a recombinant fowlpox vaccine (expressing NDV HN protein) and that to live and inactivated NDV. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Chicken±Viruses; Newcastle disease virus; Differential ELISA; Baculovirus expression; NDV nucleocapsid protein

* Corresponding author. Tel.: +1-519-824-4120 x 4783; fax: +1-519-767-0809; e-mail: [email protected] 0378-1135/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 9 9 ) 0 0 0 1 6 - 4

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1. Introduction Newcastle disease virus (NDV) is a widespread and economically important poultry pathogen. Although vaccines have long been available and administered to control Newcastle disease, the virus remains an ongoing threat to commercial flocks. The forms of the disease vary and are dependent upon several factors, but mainly on the strain of the virus (Alexander, 1997). NDV, a member of the family Paramyxoviridae (Rima et al., 1995), is an enveloped virus with helical nucleocapsid symmetry and a single-stranded, negative sense RNA genome, about 15 kb in length. In the 30 ±50 orientation, the genome encodes six major proteins: nucleocapsid (NP), phosphoprotein (P), matrix (M), fusion (F), haemagglutinin± neuraminidase (HN) and RNA-dependent RNA polymerase (L) (Chambers et al., 1986; Wilde et al., 1986). The HN and F glycoproteins are important for virus infectivity and virulence, and either of these two proteins can induce protective immunity (Meulemans et al., 1986; Nagy et al., 1991). NDV-specific antibodies (Ab) have traditionally been monitored by the haemagglutination inhibition test (Alexander, 1997). ELISA procedures based on whole virus as coating antigen have been described (Miers et al., 1983; Snyder et al., 1983; Wilson et al., 1984; Rivetz et al., 1985), and, more recently, commercial kits (e.g. IDEXX Laboratories, Westbrook, ME) are widely being used in diagnostic laboratories. Errington et al. (1995) recently reported on an NDV NP-based ELISA using NP expressed in a baculovirus system and compared their NP ELISA-based scores with HI and the commercial IDEXX ELISA scores. They did not observe any false positive readings for samples scored positive by HI. However, they did report that among sera expected to be negative were some that scored positive by IDEXX ELISA and negative by their NP-based system. Numerous, live and inactivated virus vaccines exist against NDV. There has recently been growing interest in using subunit vaccines against poultry diseases including ND (Meulemans et al., 1988; Nishino et al., 1991; Morgan et al., 1993; Nagy et al., 1993). Consequently, there is a need to be able to differentiate between birds naturally infected with NDV and vaccinated with such a recombinant subunit in the surveillance of NDV. In this study, we sought to develop an ELISA test for Ab detection that would discriminate between the antibody response to a subunit vaccine (HN expressed by a recombinant fowlpox virus) and the response to live and inactivated NDV. We cloned the NP gene of the Hitchner B1 NDV strain and expressed the NP in a baculovirus vector for use as a coating antigen in the ELISA. Serum samples were analysed from chickens vaccinated with a recombinant fowlpox virus expressing the NDV±HN and subsequently infected with NDV. 2. Materials and methods 2.1. Viruses and cells NDV strains Hitchner B1 and LaSota were used (Nagy et al., 1990). Virus was propagated in embryonated eggs and in chicken embryo lung (CELu) cells by standard procedures.

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Fig. 1. Construction of NP plasmid vector and baculovirus transfer vector.

Autographa californica nucleopolyhedrovirus (AcMNPV) and Spodoptera frugiperda (Sf) 9, Sf21 and Trichoplusia ni High Five cells were from Invitrogen. Baculovirus and insect cell techniques were performed as described by Summers and Smith (1987) and O'Reilly et al. (1992).

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2.2. Generation of recombinant baculovirus The strategy for recombinant plasmid construction is outlined in Fig. 1. All molecular biology techniques were based on Sambrook et al. (1989). Complementary DNA was made (cDNA Synthesis System; Gibco-BRL, Burlington, ON) from genomic NDV RNA using a primer (50 -GCCTTCTGCCAAAATGTC-30 ; the initiation codon is in bold) derived from the NP genes of d26 (Ishida et al., 1986) and Ulster 2C (GenBank accession #Z300084). Blunt-ended cDNA was ligated into the SmaI site of pGEM-7Zf(‡) to generate pNP103, with a 2.5 kb insert containing the NDV NP gene. A PCR product containing just the NP gene was generated using the same primer used for cDNA synthesis, and primer B (50 -TGTTGTTGGTCAGTACCC-30 ) with the complement of the termination codon (in bold) and based on the NP gene of the Ulster 2C strain. The PCR product was blunt-end ligated into the SmaI site of pGEM-7Zf(‡) to generate pNP103.22. The 1.5 kb EcoRI/Csp45I fragment from pNP103.22 was blunt-ended with Klenow enzyme and NheI linkers (New England Biolabs, Beverly, MA) were added for cloning into the NheI site of the baculovirus pETL vector (Richardson et al., 1992) to generate pEN159 which contained the NP gene under the control of the polyhedrin (polh) promoter. Recombinant baculovirus was generated as described by Summers and Smith (1987) and purified by three rounds of plaque purification to generate one recombinant referred to as rBNP. 2.3. Sequencing The NP gene in pNP103 was sequenced at the Guelph Molecular Supercentre (Laboratory Services Division, University of Guelph) and sequence data were analysed with `Align' (version 1.02, #1989 Scientific and Education Software). Sequences were deposited into GenBank, accession number AF060483. 2.4. Polyacrylamide gel electrophoresis and Western blot analysis Sf9 cells infected with rBNP at a multiplicity of infection (m.o.i.) of 10 were analysed by sodium dodecyl sulfate 10% polyacrylamide gel electrophoresis (SDS-PAGE; Laemmli, 1970), and Western blotting. Infected cells, harvested at different times postinfection (p.i.), were lysed in an electrophoretic sample buffer (ESB; 62.5 mM Tris [pH 6.8], 10% glycerol, 2% SDS, 5% b-mercaptoethanol, 0.002% bromophenol blue). Western blotting onto nitrocellulose membrane with a 0.45 mm pore size (Schleicher and Schuell, Keene, NH) was as described by Towbin et al. (1979). Blots were blocked in 5% skim milk powder (SMP; Difco, Detroit, MI) in PBS for 3 h at room temperature. NDVspecific rabbit polyclonal serum at a dilution of 1/2000 in 2% SMP in PBS±Tween (0.05% Tween-20 in PBS) was added to the blots and incubated at room temperature for 2 h. Blots were then incubated with goat anti-rabbit alkaline phosphatase-conjugated Ab (Bio-Rad, Mississauga, ON) at a dilution of 1/1000 in 2% SMP in PBS and developed in a 1 : 1 : 200 (by volume) solution of BCIP (Bio-Rad; 15 mg/ml stock in N,Ndimethylformamide), NBT (Bio-Rad; 30 mg/ml stock in 70% N,N-dimethylformamide) in 0.1 M Tris (pH 9.5), 0.5 mM MgCl2.

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2.5. Radioimmunoprecipitation Sf9 cells infected with rBNP were incubated at 48 h p.i. in methionine-free Grace's medium with [35 S] methionine at 100 mCi/ml (ICN; specific activity 1175 Ci/mmol) for 4 h at 288C. Cells were lysed in RIPA buffer (150 mM NaCl, 10 mM Tris [pH 7.2], 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS). NDV-infected chicken CELu were similarly labelled in methionine-free EMEM at 8 h p.i. for 2 h at 378C. Immunoprecipitation with NDV-specific rabbit serum and analysis was as described by Nagy et al. (1990). 2.6. Antigen preparation and conditions for ELISA The rBNP-infected Sf21 cell lysates were used as antigens for ELISA and the appropriate conditions were determined by a series of checkerboard plates as described by Carpenter (1992). Cells infected at an m.o.i. of 5 and harvested at 40 h p.i. were washed twice, resuspended in PBS, and sonicated over a 1.5 min period on ice. Concentrations of soluble protein were determined with a Bio-Rad protein assay kit. ELISA plates (Becton Dickinson Laboratories, Lincoln Park, NJ) were coated at 2.5 mg total protein/well in 0.5 M carbonate±bicarbonate buffer (pH 9.6), and blocked with 5% BSA (Fraktion V, Boehringer Mannheim) for 1 h at 378C. Plates were incubated at 378C for 1.5 h with primary Ab diluted 1/500 in PBS±Tween. Plates were next incubated at 378C for 1.5 h with goat anti-chicken IgG alkaline phosphatase conjugate (Kirkegaard and Perry, Gaithersburg, MD) diluted 1/1000 in PBS±Tween. Plates were developed with Nitrophenyl±Phosphate (Sigma, p-NPP tablets; Sigma, Oakville, ON) and OD405 was read in a Bio-Tek microplate autoreader. Samples considered positive for NDV were from the Animal Health Laboratory (University of Guelph) as a collection of serum samples from individual chickens from flocks which tested positive for NDV Ab. For flock testing, random samples of five birds per flock were tested with the commercial IDEXX kit. The individual samples were divided into 48 pooled serum samples (five individuals per pooled sample) for testing with our NP ELISA. Samples considered negative for NDV were from 58 birds reared in isolation and confirmed to be negative by IDEXX ELISA. Specificity and sensitivity of the ELISA were calculated according to Smith (1995). Sensitivity was the number of true positive samples divided by the sum of the true positive and false negative samples, and the specificity was the number of true negative samples divided by the sum of the true negative and false positive samples. The true positive and negative samples were those scored as positive or negative, respectively, by both IDEXX and NP ELISA. False positives were those that scored negative by IDEXX but positive by NP ELISA while false negatives were those that scored positive by IDEXX but negative by NP ELISA. An OD405 cut-off value for the NP ELISA to differentiate positive from negative samples was set at two standard deviations above the average OD405 from IDEXX-negative samples. Values above this cutoff were scored as positive.

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2.7. Vaccination experiment Three-week-old Leghorn chickens (Arkell Research Station, University of Guelph) were separated into three groups of five birds each. Group 1 and Group 2 chickens were vaccinated with a recombinant fowlpox virus expressing the HN of NDV with the vaccinia virus promoter P7.5 (rFPV7.5) or P11 (rFPV11), respectively, at Weeks 0 and 2. Group 3 chickens were mock-vaccinated. At Week 4 all birds were infected with 0.1 ml of NDV (LaSota strain) via the intranasal route. Weekly blood samples were collected beginning with a pre-immune bleed at Week 0, and ending with post-infection samples at Week 5. Serum samples were tested using recNP ELISA and whole NDV ELISA. Plates for whole NDV ELISA were coated with a hundred-fold dilution of NDV-infected allantoic fluid (initial HA 1 : 256) at 100 ml/well. 3. Results 3.1. Sequence analysis of the NP gene The open reading frame of the NP gene of the Hitchner B1 NDV strain was 1,467 nucleotides (nt) long and coded for a 489 aa protein with a predicted molecular mass (Mr) of 53.03 kDa. The number of amino acids for the B1 strain NP gene was identical to that from d26 and Ulster 2C. The nucleotide homology of the NP gene was 90.03 and 89.45% with that of d26 (Ishida et al., 1986) and Ulster 2C (GenBank accession #Z30084), respectively. Amino acid identities were 95.71 and 96.11%, respectively. The NP gene sequences of the d26 and Ulster 2C strains showed nucleotide homology and amino acid identity of 96.4 and 98.6%, respectively. 3.2. Expression of the recombinant NP protein in insect cells Sf9 cells infected with rBNP were labelled with [35 S] methionine and cell lysates were immunoprecipitated (Fig. 2). A very prominent band was detected at 53 kDa (Lane 3) for rBNP and the position of this band was identical to that of the NP band in NDV-infected CELu cells (Lane 2). An NDV-specific band was also detected at 43 kDa for rBNP but a corresponding band was not detected for NDV-infected CELu cells and may represent degradation of the 53 kDa NP. Similarly, a 39 kDa protein band was detected in NDVinfected CELu cells but not in the rBNP lanes. This represents the NDV matrix protein that would co-precipitate with the anti-NDV Ab. No proteins of the uninfected CELu (Lane 1) or Sf9 (Lane 5) cells reacted with the NDV-specific Ab. A single band at approximately 30 kDa which corresponds to the position of the polyhedrin protein was detected in the AcMNPV-infected Sf9 cell lysates treated with either anti-NDV or preimmune sera (Lanes 4 and 7, respectively). Polyhedrin is present as a protein complex which does not solubilize in cell lysis buffer and pellets with immunoprecipitated proteins (O'Reilly et al., 1992). In a time course of expression of the recNP in Sf9 cells infected with rBNP at an m.o.i. of 10, a Coomassie blue stained band corresponding in size to recNP (53 kDa) was seen

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Fig. 2. Radioimmunoprecipitation of rBNP-infected [35 S] methionine-labelled Sf9 cell lysates. Lysates in Lanes 1 to 5 were precipitated with NDV-specific Ab, in Lanes 6 and 7 were precipitated with pre-immune serum. Lane 1: uninfected CELu cells; Lane 2: NDV infected CELu cells; Lanes 3 and 6: rBNP infected cells; Lanes 4 and 7: AcMNPV-infected cells; and Lane 5: uninfected Sf9 cells. M: radiolabelled molecular weight marker.

by 36 h p.i. (arrow) and increased in intensity up to 48 h p.i., and remained constant thereafter (Fig. 3, panel A). By Western blotting an NDV-specific protein band corresponding to the unique 53 kDa band in the stained gel, was first detected at 24 h p.i. (arrow) and increased in intensity over time (Fig. 3, panel B). Some bands of lower molecular weight appeared at 36 h p.i. and increased in amount with time. The NP of NDV grown in embryonated eggs, migrated at 55 kDa. NP expression was compared in Sf9, Sf21 and High Five cell lines, at m.o.i. of 0, 1, 5 and 10 and harvested at 24, 36, 48 and at 60 h p.i. By Coomassie blue staining, recNP expression in Sf9 and Sf21 cells was similar at all time points and m.o.i., but expression in High Five cells was extremely low. Based on densitometric analysis of the gels, Sf9 and Sf21 cells infected with an m.o.i. of 5 and collected at 48 h p.i. contained approximately 0.60 and 0.53%, respectively, of recNP relative to the total amount of protein in the lane. RecNP expression was greater in Sf21 cells than in Sf9 cells by Western blot analysis (data not shown). Since optimal production of recNP occurred in

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Fig. 3. Time course analysis of recNP expression by SDS-PAGE and Western blotting. A: Coomassie blue stained gel, and B: Western blot analysis with NDV-specific Ab. The numbers above the lanes indicate rBNPinfected cells harvested at 12±72 h p.i. AcMNPV-infected (wt) and uninfected (un) cell lysates, and purified NDV (NDV) were also run. M: molecular weight markers, in kDa. Arrows show recNP band.

Sf21 cells infected at an m.o.i. of 5 and harvested between 36 and 48 h p.i. these conditions were used for production of recNP for subsequent ELISA studies. 3.3. Development of recombinant NP-based ELISA Appropriate conditions of the recNP-based ELISA were determined through a series of checkerboard ELISAs testing antigen concentrations, primary and secondary Ab dilutions, and use of different blocking agents. The final conditions, as described in Section 2, were then used to screen a number of known positive and negative samples. The specificity and sensitivity of the ELISA were evaluated from a panel of 48 IDEXXpositive and 58 IDEXX-negative serum samples (Fig. 4). Of the samples tested, the recNP ELISA gave a total of 48 true positive, 55 true negative, three false positive, and no false negative readings (i.e. 48 of the 48 positive sera were scored as positive and 55 of the 58 negative sera were scored as negative using the recNP ELISA). The OD405 values for the three false positives were just above the cut-off OD405 value. The specificity and sensitivity of the recNP ELISA were 94.8 and 100%, respectively. Raising the OD405 cutoff value to three standard deviations above the average for the negative values (0.251) would result in 48 true positive samples and 58 true negative samples for 100% specificity and 100% sensitivity.

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Fig. 4. Graphs of absorbance values obtained in testing known positive and negative serum samples in recNP ELISA to determine specificity and sensitivity values. Cut-off values were set at two standard deviations above the average absorbance of negative standard sera (OD405 0.230).

3.4. Vaccination experiment The ability of the recNP ELISA to differentiate between NDV-infected birds, and those vaccinated with an HN subunit vaccine was tested. Chickens were vaccinated with two recombinant fowlpox viruses expressing HN and differing only in the nature of the promoters used, and at Week 4, all chickens were inoculated with the LaSota NDV strain. Blood samples were collected at 1 week intervals and tested using both the recNP ELISA and a whole NDV ELISA. OD405 values for each group were averaged and results for the pre-immune, post-FPV vaccination (Week 4) and post-NDV infection (Week 5) bleeds are presented in Fig. 5. Recombinant FPV-vaccinated birds, which were protected from virulent NDV challenge (unpublished), were positive by whole NDV ELISA at Week 4,

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Fig. 5. Histograms of average OD405 values in ELISAs of serum samples from chickens vaccinated with recombinant fowlpox virus expressing the haemagglutinin±neuraminidase of NDV and subsequently infected with NDV. Panel A shows the results of the whole NDV ELISA, and panel B shows the results of the same serum samples in the recNP ELISA. Average values per group are shown for Week 0 (pre-immune), Week 4 (postvaccination) and Week 5 (post-infection).

but were negative by recNP ELISA. These recombinant FPV-vaccinated birds would produce antibodies only to HN of NDV and consequently react with only this NDV polypeptide in the wells. The resulting OD405 values would consequently be lower than that for NDV-inoculated birds whose antibodies would react against all NDV polypeptides. Even though the OD405 values for the vaccinated birds were close to the cut-off values they were clearly above those of the pre-immune sera. Mock-vaccinated birds remained negative throughout Weeks 1±4 in both ELISAs. Serum samples taken at Week 5, 1 week after live NDV inoculation, were positive in both tests for all birds. 4. Discussion The NP of NDV was expressed in a baculovirus expression vector system and was used as a coating antigen for a diagnostic and differential ELISA.

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The NP gene of the Hitchner B1 strain was sequenced and compared to those of strains d26 and Ulster 2C (Ishida et al., 1986; GenBank accession #Z30084), the only complete NP sequences of any NDV strains reported to date. The predicted molecular mass of the B1 NP according to sequence information was 53,029 Da which is in excellent agreement with the size of the virion NP and with the expressed NP determined by PAGE. The B1 NP amino acid sequence showed a high degree of identity to those of Ulster 2C and d26 (95.71 and 96.11%, respectively). Since there is a very high degree of amino acid sequence conservation, the recNP ELISA based on the B1 strain should be effective against all NDV strains. The recombinant NP expressed in insect cells was detected as a single, Coomassie blue-stained band at approximately 53 kDa in size. The same protein was shown to be NDV-specific by Western blotting and radioimmunoprecipitation with rabbit polyclonal serum raised against purified NDV (Figs. 2 and 3, panel B). The recombinant NP of 53 kDa migrated at a size comparable to that of NP (55 kDa) from virus grown in embryonated eggs (Smith and Hightower, 1981; Ishida et al., 1986; Samson, 1988). The slight discrepancy in size between the recNP (53 kDa) and the virion NP (53 or 55 kDa) in Fig. 3 might reflect slightly different forms of the NP proteins from NDV grown under different conditions as has previously been described (Smith and Hightower, 1981). For radioimmunoprecipitation (Fig. 2), CELu cells infected with NDV were used to compare with the rBNP-infected Sf9 cells. Both these samples showed NP bands of the same mobility, estimated at 53 kDa. In addition to the major recNP band several faster migrating bands were seen in Western blots and radioimmunoprecipitation of rBNP-infected cell lysates. In time course experiments, these bands appeared only after the major recNP band was seen, and they accumulated over time. These lower molecular weight proteins may be due to proteolytic degradation of the recombinant NP, since the NP of NDV is susceptible to such degradation (Mountcastle et al., 1970). This is also common for other paramyxoviruses including simian virus 5 (Mountcastle et al., 1974), measles virus (Rozenblatt et al., 1979) and canine distemper virus (Hall et al., 1980). Lower molecular weight species were also obtained by Kamata et al. (1993) when the rinderpest virus nucleocapsid protein was expressed using baculovirus. The 53 and 43 kDa bands present in the radioimmunoprecipitated rBNP-infected cell sample could represent the undigested and digested forms of NP, respectively, as described by Mountcastle et al. (1974). Initial ELISA experiments showed no cross-reactivity of chicken sera with uninfected and AcMNPV-infected Sf21 cell lysates. ELISA plates were therefore coated with cell lysates from rBNP-infected cells. Baculovirus-expressed nucleocapsid proteins have been used as antigens in ELISAs for measles virus (Hummel et al., 1992), rinderpest virus (Kamata et al., 1993), vesicular stomatitis virus (Ahmad et al., 1993) and NDV (Errington et al., 1995). In comparison to commercially available ELISAs, there was a better correlation of the recombinant NP ELISAs with neutralising antibody levels than with commercial ELISAs (Hummel et al., 1992; Errington et al., 1995). The recNP-based ELISA was developed and optimised, and positive and negative sera were tested. Since pooled samples are commonly used to determine overall flock immunity, use of pooled samples for positive sera in determining the specificity and sensitivity of this test was considered appropriate. Specificity and sensitivity of the test

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were 94.8 and 100%, respectively, and no false negative readings were observed (Fig. 4). Since IDEXX itself might not be 100% accurate this might lead to some lack of correlation with the recNP ELISA. The few false positive samples were very close to the cut-off values determined, and thus may be an artifact of a cutoff set too low. The cut-off value used in the recNP ELISA was two standard deviations above the average OD405 of the negative standards. If the cutoff was three standard deviations above the average negative value, the three false positives would be true negatives. Since the sensitivity and specificity of this recNP ELISA are high we feel that it is a reliable test for determining the presence of NDV-specific antibodies. Errington et al. (1995) earlier described a baculovirus-expressed NP-based ELISA for NDV Ab. They did not report any false positive NP ELISA readings (for samples positive by HI) although they reported some false negatives relative to some IDEXX-positive samples which they felt may have been due to PMV-2 or PMV-3 interference. In our recNP-based ELISA we did not observe any false negative samples, even using the IDEXX ELISA kit which is more sensitive than HI. One of the differences in the protocols of the two NP-based ELISAs was the cut-off values set; in our study we did not use an S/P ratio as described by Errington et al. (1995). An S/P ratio is the ratio of the unknown value to that of a weak positive control, after the subtraction of the negative control value from both. It is possible that incorporating weak positive standards and an S/P ratio cutoff into the recNP ELISA described herein could eliminate the false positives and thus increase the specificity of our test to 100%. Thus our test is potentially more reliable than that of Errington et al. (1995) for the detection of NDV Ab. Furthermore, we showed that the recNP described can be used to differentiate between birds exposed to NDV in either a whole NDV vaccine or by natural infection, from those vaccinated with a subunit vaccine not containing the NP. Presently, there is a great deal of interest in the development of recombinant and protein subunit vaccines. For NDV, protective antibodies are induced against either the HN or F proteins. The recNP ELISA described here could be used to differentiate between birds naturally infected with NDV from those immunised with HN or F protein subunit vaccines since antibody against the NP would be present only in birds exposed to whole virus. To evaluate this, sera from chickens which were vaccinated with recombinant fowlpox viruses expressing the HN only of NDV, were tested. Chickens immunised with either recombinant FPV construct were weakly positive using whole NDV ELISA after two vaccinations, whereas using recNP ELISA they were negative (Fig. 5). Unvaccinated chickens were negative in both ELISAs. All chickens, after receiving an intranasal inoculation of live NDV, became strong positives according to both ELISAs. The OD405 values of the whole NDV ELISA after vaccination with the FPV-HN were considerably lower than after exposure to NDV, but were still positive for all birds within a group and significantly higher than that in pre-immune sera. These indicate that a positive result in a whole NDV ELISA can be due to antibodies to HN alone and that the recNP ELISA can be used to differentiate between subunit-vaccinated birds and those exposed to whole virus. Most importantly, a positive result with a recNP-based ELISA would indicate immunisation with a whole NDV vaccine or/and infection with NDV and excludes birds immunised with only a subunit vaccine.

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The feasibility of using baculovirus-expressed nucleocapsid protein as an antigen for an ELISA to differentiate between vaccinated and infected animals was also explored by Ahmad et al. (1993) for vesicular stomatitis virus, and Kamata et al. (1993) for rinderpest virus. As in our study, both groups showed that a baculovirus-expressed recombinant NPbased ELISA was capable of differentiating subunit-vaccinated animals from those infected with whole virus. Acknowledgements This work was funded by the Natural Sciences and Engineering Research Council (NSERC) of Canada. We thank Paul Huber for his photographic and technical expertise and Sean Walter for his technical assistance. We are indebted to Dr. D. Key (Animal Health Laboratory, University of Guelph) for providing the pre-tested chicken serum samples. References Ahmad, S., Bassiri, M., Banerjee, A.K., Yilma, T., 1993. Immunological characterization of the VSV nucleocapsid (N) protein expressed by recombinant baculovirus in Spodoptera exigua larva: Use in differential diagnosis between vaccinated and infected animals. Virology 192, 207±216. Alexander, D.J., 1997. Newcastle disease and other avian Paramyxoviridae infections. In: Calnek, B.W., Barnes, H.J., Beard, C.W., McDougald, L.R., Saif Y.M. (Eds.), Diseases of Poultry, 10th edn., Iowa State University Press, Ames, IA, pp. 541±569. Carpenter, A.B., 1992. Enzyme-linked immunoassays. In: Rose, N.E., de Macario, E.C., Fahey, J.L., Friedman, H., Penn, G.M. (Eds.), Manual of Clinical Laboratory Immunology, 4th edn., American Society for Microbiology. pp. 2±9. Chambers, P., Millar, N.S., Bingham, R.W., Emmerson, P.T., 1986. Molecular cloning of complementary DNA to Newcastle disease virus, and the nucleotide sequence analysis of the junction between the genes encoding the haemagglutinin±neuraminidase and the large protein. J. Gen. Virol. 67, 475±486. Errington, W., Steward, M., Emmerson, P.T., 1995. A diagnostic immunoassay for Newcastle disease virus based on the nucleocapsid protein expressed by a recombinant baculovirus. J. Virol. Meth. 55, 357±365. Hall, W.W., Lamb, R.A., Choppin, P.W., 1980. The polypeptides of canine distemper virus: Synthesis in infected cells and relatedness to the polypeptides of other morbilliviruses. Virology 100, 433±449. Hummel, K.B., Erman, D.D., Heath, J., Bellini, W.J., 1992. Baculovirus expression of the nucleocapsid protein gene of measles virus and utility of the recombinant protein in diagnostic enzyme immunoassays. J. Clin. Microbiol. 30, 2874±2880. Ishida, N., Taira, H., Omata, T., Mizumoto, K., Hattori, S., Iwasaki, K., Kawakita, M., 1986. Sequence of 2,617 nucleotides from the 30 end of Newcastle disease virus genome RNA and the predicted amino acid sequence of viral NP protein. Nucl. Acids. Res. 14, 6551±6564. Kamata, H., Ohkubo, S., Sugiyama, M., Matsuura, Y., Kamata, Y., Tsukiyama-Kohara, K., Imaoka, K., Kai, C., Yoshikawa, Y., Yamanouchi, K., 1993. Expression in baculovirus vector system of the nucleocapsid protein gene of rinderpest virus. J. Virol. Meth. 43, 159±166. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680±685. Meulemans, G., Gonze, M., Carlier, M.C., Petit, P., Burny, A., Long, L., 1986. Protective effects of HN and F glycoprotein-specific monoclonal antibodies on experimental Newcastle disease. Avian Path. 15, 761±768. Meulemans, G., Letellier, C., Gonze, M., Carlier, M.C., Burny, A., 1988. Newcastle disease virus glycoprotein expressed from a recombinant vaccinia virus vector protects chickens against live-virus challenge. Avian Path. 17, 821±827.

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