Detection of virulent Newcastle disease virus using a phage-capturing dot blot assay

Journal of Virological Methods 136 (2006) 224–229

Detection of virulent Newcastle disease virus using a phage-capturing dot blot assay Thong Chuan Lee a,1 , Khatijah Yusoff a,b , Sheila Nathan c , Wen Siang Tan a,b,∗ a

Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia b Institute of Bioscience, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia c Center for Gene Analysis and Technology, Faculty of Science and Technology, University Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia Received 22 February 2006; received in revised form 15 May 2006; accepted 16 May 2006 Available online 23 June 2006

Abstract Newcastle disease virus (NDV) strains can be classified as virulent or avirulent based upon the severity of the disease. Differentiation of the virus into virulent and avirulent is necessary for effective control of the disease. Biopanning experiments were performed using a disulfide constrained phage displayed heptapeptide library against three pathotypes of NDV strains: velogenic (highly virulent), mesogenic (moderately virulent) and lentogenic (avirulent). A phage clone bearing the peptide sequence SWGEYDM capable of distinguishing virulent from avirulent NDV strains was isolated. This phage clone was employed as a diagnostic reagent in a dot blot assay and it successfully detected only virulent NDV strains. © 2006 Elsevier B.V. All rights reserved. Keywords: Filamentous phage; Dot blot; NDV; Pathotypes; Phage display

1. Introduction Newcastle disease virus (NDV) poses a major threat to the avian species worldwide particularly to domestic poultry. NDV can be grouped into three pathotypes based on the severity of disease; the viscerotropic or neurotropic velogenic strains are highly contagious, causing severe intestinal lesions or neurological disease, resulting in high mortality of flocks; the mesogenic strains cause respiratory and nervous signs with moderate mortality; the lentogenic strains cause only mild and imperceptible respiratory infection (Yusoff and Tan, 2001). The velogenic and mesogenic strains are both virulent and have been frequently identified as the causative agent of outbreaks in many countries. On the other hand, the lentogenic strains are avirulent and have been used as live vaccines to control the disease. However, current diagnosis of NDV is unable to differentiate virulent NDV in a vaccinated flock of chicken. The ability to distinguish the virulent NDV strains in flocks is important because international veterinary regulatory bodies require a definitive diagnosis of

∗ 1

Corresponding author. Tel.: +60 3 89466715; fax: +60 3 89430913. E-mail address: [email protected] (W.S. Tan). Present address: Veterinary Research Institute, 31400 Ipoh, Perak, Malaysia.

0166-0934/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2006.05.017

virulent NDV to enable effective prevention of an outbreak by strict control measures and trade embargo restrictions (Aldous and Alexander, 2001). Conventional methods used for pathotyping NDV strains involve the use of inoculated embryonated chicken eggs or chicks from which parameters such as mean death time (MDT), intracerebral pathogenicity index (ICPI) and intravenous pathogenicity index (IVPI) are determined (Alexander, 2001). These methods are laborious and time consuming. Over the past decade, numerous molecular techniques have been employed to identify and delineate NDV pathotypes: polymerase chain reaction (PCR) coupled to restriction enzyme digestion (Stauber et al., 1995); PCR-sequencing (Seal et al., 1995); a triple one-step PCR (Wang et al., 2001), reverse transcription (RT)-nested PCR coupled with ELISA detection (Kho et al., 2000) and real-time PCR (Aldous and Alexander, 2001; Tan et al., 2004). In addition, monoclonal antibodies against the haemagglutinin-neuraminadase (HN) and fusion (F) proteins have been produced via hybridoma technology for pathotyping NDV strains (Alexander and Manvell, 1997). Most recently, we have shown that a filamentous M13 bacteriophage displaying a peptide bearing the TLTTKLY sequence isolated from a phage displayed peptide library against the velogenic NDV strain AF2240 could be used to differentiate

T.C. Lee et al. / Journal of Virological Methods 136 (2006) 224–229

velogenic NDV strains from mesogenic and lentogenic strains via a newly established indirect phage ELISA (Ramanujam et al., 2004). In the present study, we employed a new biopanning strategy for the selection of a phage displayed heptapeptide capable of distinguishing virulent (velogenic and mesogenic) from avirulent (lentogenic) NDV strains. The phage clone was further developed as a capture reagent in a phage dot blot assay to differentiate virulent from avirulent NDV strains.

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2.4. Propagation and purification of NDV strains All of the NDV strains were propagated in allantoic fluid of 9–11 days-old embryonated chicken eggs at 37 ◦ C for 3 days. The allantoic fluid was harvested and the presence of virus was confirmed by the haemaglutination test (Alexander, 1988). The allantoic fluid was directly used in the phage dot blot assay. NDV strains AF2240, 2641/91 P2 and V4 Queensland were purified as previously described (Yusoff et al., 1996) and used in the biopanning.

2. Materials and methods 2.5. Biopanning of displayed peptide library against NDV 2.1. Avian viruses The pathotypes of the NDV strains used in this study were determined by MDT test and nucleotide sequence analysis of the F cleavage site. The velogenic strains were AF2240, Ijok, 986/91 P1, 3410/91 P2, 6270/92 P3, 4059/91 P2, 3147/89 P2. The mesogenic strains comprised 2641/91 P2 and 01/C, and the lentogenic strains were S, 00/IKS, 4989/92 P3, 5953/89 P3, Ulster 2C, V4 Queensland, Hitchner B1, La Sota, F, V4 UPM, 5270/89 P3, 8820/92 P3, 1266/89 P3, 5147/91 P2, 4083/92 P3 and 5731/88 P4. Other avian viruses such as chicken anaemia virus (CAV), fowl pox virus (FPV) and avian influenza virus (AIV) were included as negative controls. 2.2. Mean death time (MDT) assay The MDT assay was performed as previously described (Alexander, 1988). Briefly, 0.1 ml of 10-fold dilution series between 10−6 and 10−9 infected allantoic fluid was inoculated into the allantoic cavity of five 9–10 days-old embryonated chicken eggs and then incubated at 37 ◦ C. These steps were repeated once more with five eggs 8 h later and incubated at 37 ◦ C. Each egg was examined twice daily for 7 days and the time of embryo deaths were recorded. NDV strains were classified into the following groups: velogenic (taking under 60 h to kill); mesogenic (taking between 60 and 90 h to kill); lentogenic (taking more than 90 h to kill). 2.3. PCR and nucleotide sequence analysis Viral RNA was extracted from NDV infected-allantoic fluid (500 ␮l) using the Trizol® reagent (Invitrogen, San Diego, CA, USA) according to the manufacturer’s instructions. The RNA-pellet was resuspended in diethylpyrocarbonate (DEPC: 0.1% v/v) treated distilled water (15 ␮l). cDNA generation and reverse transcription-polymerase chain reaction (RT-PCR) of the F cleavage site were performed as previously described (Kho et al., 2000). The F gene cDNA fragment and PCR product were amplified using the avian murine virus reverse transcriptase (AMV-RT; Promega, Madison, WI, USA) and Taq polymerase (Promega, USA), respectively. Sequencing of the PCR products was performed using the CEQ dye terminator cycle sequencing kit (Beckman Coulter, Fullerton, CA, USA) and sequencing products were analyzed on the CEQ8000 automated sequencer (Beckman Coulter, USA).

Purified NDV strains AF2240, 2641 and V4 Queensland (1.5 ␮g) in TBS (50 mM Tris–HCl; 150 mM NaCl; pH 7.4; 100 ␮l) buffer were coated onto three different microtiter plate wells and incubated overnight at 4 ◦ C. The wells were washed three times with TBST (TBS supplemented with 0.1% Tween20) and incubated for 2 h at room temperature with 5 mg/ml bovine serum albumin (BSA: 300 ␮l). After three washes with TBST, approximately 1011 pfu of phage particles (7-mer disulfide-constraint phage display library diluted in TBS-buffer; 100 ␮l; New England Biolabs, Ipswich, MA, USA) were added to each well. After 1 h incubation at room temperature, the wells were washed vigorously with TBST six times. Bound phages were eluted by incubating with elution buffer [0.2 M HCl–glycine (pH 2.2), 1 mg/ml BSA; 120 ␮l] for 5 min at room temperature and immediately neutralized with 1 M Tris–HCl, pH 9.1 (15 ␮l). Log phase Escherichia coli ER2738 (New England Biolabs, USA) cells were infected with the phage eluate and titered using a standard plaque assay as described (Smith, 1985). The amplified phage particles were subjected to biopanning for a further two rounds. Binding of phage clones to NDV coated on microtitre wells was determined by indirect phage ELISA as described below. 2.6. Indirect phage ELISA Binding of phage clones to purified or unpurified NDV coated on microtiter wells was determined by indirect phage ELISA as described by Ramanujam et al. (2004) with some modifications. Briefly, NDV (1.5 ␮g in TBS; 100 ␮l) was coated onto microtiter plate wells and incubated overnight at 4 ◦ C. The wells were washed three times with TBST and blocked with BSA (5 mg/ml; 300 ␮l) by incubating at 4 ◦ C for 2 h. After six washes with TBST, phage suspension (1010 pfu/ml; 100 ␮l) was added and incubated for 1 h at room temperature. The wells were then washed vigorously with TBST six times and anti-M13 monoclonal antibody conjugated to horseradish peroxidase (HRP, Amersham Pharmacia, Sweden; 1:2500 dilution; 200 ␮l) was added. After 1 h incubation at room temperature, the wells were washed vigorously with TBST six times and then added with 2,2-azino-di(3-ethylbenthiazoline) sulfonic acid diammonium salt (ABTS: 0.2 mg/ml) in citrate–phosphate buffer (28.6 mM anhydrous citric acid, 41.2 mM Na2 HPO4 ·7H2 O; 200 ␮l). Absorbance was measured at 405 nm with a spectrophotometer (Model 550; Bio-Rad, USA).

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2.7. Phage dot blot for pathotyping of NDV Approximately 1011 phage particles were spotted onto a nitrocellulose membrane using a dot blot apparatus (Bio-Rad, USA). The nitrocellulose membrane was subsequently blocked with BSA (5 mg/ml; 5 ml) for 2 h at room temperature. Allantoic fluid (5 ml) containing NDV was added onto the membrane and incubated overnight at room temperature. After five vigorous washes with TBS containing 0.5% Tween-20, chicken anti-NDV polyclonal antibody was used to detect captured NDV. Antichicken IgG conjugated to alkaline phosphatase (AP, 1:2000 dilution; KPL, UK) was used as the secondary antibody. The dark violet color of the spots was observed by adding 5-bromo4-chloro-3-indoyl phosphate (BCIP: 0.45 mM) and nitro blue tetrazolium chloride (NBT: 0.046 mM) in alkaline phosphatase buffer (100 mM Tris–HCl, pH 9.5; 100 mM NaCl; 5 mM MgCl2 ) for 10 min. Uninfected allantoic fluid and other avian viruses such as CAV, FPV and AIV were used as negative controls for this assay. Density of the dot on the nitrocellulose membrane was determined by an imaging system (GelDoc 2000, Bio-Rad, Hercules State, USA) using the Gel Doc software (Bio-Rad, USA). 3. Results 3.1. Biopanning Peptides that interact with the three NDV pathotypes were selected by panning a combinatorial library of disulfide constrained random phage displayed peptides against immobilized NDV particles. Each of the three pathotypes was represented by a strain of NDV: AF2240 (velogenic), 2641/91 P2 (mesogenic) and V4 Queensland (lentogenic). Enrichment of NDV binding phage clones from each round of biopanning was assessed by phage ELISA (Fig. 1). The results demonstrate successful enrichment and amplification of phage clones that are able to bind specifically to the respective NDV strains over

Table 1 Peptide selected from the third round of biopanning experiment against the three pathotypes of NDV NDV used in panning experiment

Heptapeptide sequences

Frequency of sequences (%)

AF2240 (velogenic)

SWGEYDM QTHLTRA

80.0 20.0

2641/91 P2 (mesogenic)

YASTKPH LDWYARL MNHPSER SLTTHST HGTSSLP LSTHQYR HESRQAL QSKDPLH Unrelated sequences

16.6 10.0 6.6 10.0 6.6 6.6 6.6 6.6 30.4

V4 Queensland (lentogenic)

RTGKADV ETHLTRA KETIPRI Unrelated sequences

6.6 6.6 10.0 76.8

A total of 30 clones were sequenced from the third round of selection against each pathotype.

the three rounds of panning, indicating the selection of high affinity binding phage clones. Table 1 shows the heptapeptide sequences isolated from the third round of panning. Approximately 80% of the phage clones selected against the velogenic strain AF2240 carried the SWGEYDM sequence and 20% harbored the QTHLTRA sequence. However, phage clones selected against the mesogenic (2641/91 P2) and lentogenic (V4 Queensland) strains did not demonstrate any highly conserved peptide sequences compared to those of the velogenic strain. Furthermore, none of the selected sequences appeared in more than one pathotype, indicating the presence of differences in the molecular structures exposed on the surface of NDV pathotypes. 3.2. Indirect phage ELISA using purified NDV Phage clones carrying different peptide sequences were characterized by indirect phage ELISA to determine their ability to distinguish sucrose gradient purified NDV strains. Phage clones carrying the SWGEYDM and QTHLTRA peptide sequences show significantly high binding to velogenic and mesogenic strains, but not to the lentogenic strains (Fig. 2). This demonstrates that both these peptides could be used to differentiate purified virulent from avirulent NDV isolates. The phage clone harboring the dominant sequence, SWGEYDM, was further selected to study its potential as a probe in pathotyping assays. 3.3. Indirect phage ELISA using unpurified allantoic fluid

Fig. 1. Enrichment of NDV binding phage after three rounds of selection against three different NDV pathotypes. Microtiter wells were coated with AF2240 (velogenic strain), 2641/91 P2 (mesogenic strain) and V4 Queensland (lentogenic strain). The wells were blocked with BSA and phage samples were added. AntiM13 monoclonal antibody conjugated to HRP was added followed by ABTS substrate (( ) unpanned library, ( ) first round, ( ) second round and ( ) third round of panning).

In order to assess the ability of the SWGEYDM phage to distinguish different NDV pathotypes, unpurified allantoic fluid infected with different pathotypes of NDV isolates were used in the indirect phage ELISA established by Ramanujam et al. (2004). However, the SWGEYDM phage could not distinguish the unpurified virulent from avirulent NDV isolates (Fig. 3). A direct phage ELISA was therefore performed by coating the

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Fig. 2. Differentiation of purified NDV strains via indirect phage ELISA using selected phage clones. ( ) AF2240 (velogenic), ( ) 2641/91 P2 (mesogenic), ( ) V4 Queensland (lentogenic) and ( ) BSA (negative control). Purified NDV strains were immobilized on microtiter wells and unsaturated area was blocked by BSA. Selected phage particles (1010 pfu/ml) were added into the wells to interact with NDV. Bound phage was detected by adding HRP-conjugated antiM13 monoclonal antibody followed by ABTS substrate. Assays were performed in triplicate and the error bar represents the standard deviation of the mean.

phage onto microtiter wells, blocked with BSA and samples containing NDV were added. Bound NDV was detected by antiNDV polyclonal antibody, anti-chicken antibody conjugated to alkaline phosphatase (AP) followed by p-nitrophenyl phosphate substrate (PNPP). However, this method failed to differentiate the NDV strains. A double antibody sandwich method was also carried out by immobilizing anti-M13 antibody onto wells to capture the SWGEYDM phage. Samples were then added

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Fig. 4. Optimization of the amount of phage clone spotted onto the nitrocellulose membrane. (A) NDV infected allantoic fluid (64 HAU of virus) and (B) noninfected allantoic fluid. WSFFSNI serves as a negative control, it is a phage clone that carries WSFFSNI sequence that interacts specifically with hepatitis B core antigen (Ho et al., 2003).

followed by anti-NDV polyclonal antibody, AP-conjugated antichicken antibody and its substrate. This method also failed to distinguish the strains. 3.4. Phage dot blot assay A dot blot assay was therefore developed using the SWGEYDM phage clone as the capture reagent immobilized onto a nitrocellulose membrane. Then, unpurified allantoic fluid infected with NDV was used as the test sample in the newly established pathotyping system. A minimum of 1011 pfu of phage clone had to be immobilized onto the membrane to produce a visible reaction with NDV-infected allantoic fluid (Fig. 4). Following this, nitrocellulose membranes spotted with the phage clone were incubated with allantoic fluid infected with different NDV strains (4 HAU) and the intensity of the dot was quantitated by an imaging system (Table 2). The assay was able to detect all the 10 virulent strains harboring the fusion (F) protein cleavage site motif, 112 R-R-Q-K/R/E-R-F117 . Three additional NDV strains (S, 00/IKS, 4989/92 P3) could also be detected by the dot blot assay, nevertheless, these strains were earlier classified as lentogenic by the MDT assay although sequence analysis indicated the presence of the 112 R-R-Q-K/R-R-F117 motif.

Fig. 3. Differentiation of unpurified NDV in allantoic fluid via indirect phage ELISA. Allantoic fluids containing different NDV strains were coated onto microtiter wells and blocked with BSA. Phage (1010 pfu/ml) bearing the SWGEYDM sequence ( ) and M13KE wild type phage (with no insert) ( ), serve as the negative control, were then added into the wells. Bound phage was detected by adding HRP-conjugated anti-M13 monoclonal antibody followed by ABTS substrate. Assays were performed in triplicate and the error bar represents the standard deviation of the mean.

4. Discussion NDV can be grouped into three pathotypes based on its virulence (velogenic, mesogenic or lentogenic). These are determined using conventional pathotyping methods such as MDT, IVPI and ICPI, which are the “gold” standards in NDV pathotyp-

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Table 2 Differentiation of NDV pathotypes by nucleotide sequencing of the F cleavage site, mean death time and phage dot blot assay NDV strain

AF2240 Ijok 986/91 P3 3410/91 P2 6270/92 P3 4059/91 P2 3147/89 P2 2641/91 P2 01/C S 00/IKS 4989/92 P3 5953/89 P3 Ulster 2C V4 Queensland Hitchner B1 La Sota F V4UPM 5270/89 P3 8820/92 P3 1266/89 P3 5147/91 P2 4083/88 P4 5731/88 P4 Allantoic fluid CAV FPV AIV

F cleavage site (a.a. 112–117)

Virulence (based on F cleavage site)

MDT (h)

RRQKRF RRQERF RRQKRF RRQKRF RRQKRF RRQKRF RRQKRF RRQKRF RRQKRF RRQRRF RRQKRF RRQKRF GRQKRF GKQGRL GRQGRL GRQGRL GRQGRL GRQGRL GKQGRL GRQGRL GRQGRL GRQGRL GRQGRL GRQGRL GRQGRL

Vi Vi Vi Vi Vi Vi Vi Vi Vi Vi Vi Vi A A A A A A A A A A A A A

48.0 50.5 55.2 60.0 60.0 52.8 79.2 78.4 76.8 96.8 96.4 96.0 100.7 100.5 96.4 101.6 100.6 98.2 99.2 103.2 90.6 100.6 98.3 99.7 91.2

Pathotype (based on MDT)

Dot density (pixels)

V V V V V V V M M L L L L L L L L L L L L L L L L

245.5 132.3 53.6 153.2 96.3 50.4 63.6 89.2 86.2 153.6 79.5 83.1 0.4 0.5 0.4 0.6 0.3 0.3 0.2 0.2 0.4 0.5 0.8 0.1 0.5 0.2 0.4 0.3 1.3

SWGEYDM ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.4 0.6 0.0 2.1 0.9 0.4 0.4 0.5 0.3 0.8 0.6 0.6 0.1 0.1 0.1 0.0 0.1 0.1 0.0 0.0 0.0 0.1 0.0 0.0 0.1 0.1 0.1 0.1 0.4

WSFFSNI 5.6 2.3 0.4 0.5 0.4 0.5 0.5 0.5 0.2 0.6 0.3 0.4 0.4 0.5 0.3 0.4 0.2 0.2 0.4 0.3 0.4 0.4 0.4 0.3 0.2 0.2 0.2 0.5 0.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.5 0.6 0.1 0.1 0.1 0.1 0.1 0.0 0.1 0.1 0.1 0.2 0.2 0.1 0.0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.0 0.1 0.2

M13KE wild type 5.3 3.0 0.3 0.6 0.3 0.2 0.3 0.6 0.3 0.7 0.2 0.2 0.5 0.6 0.2 0.3 0.1 0.1 0.3 0.3 0.1 0.3 0.4 0.3 0.3 0.2 0.2 0.2 0.1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.6 0.4 0.1 0.2 0.0 0.0 0.1 0.1 0.0 0.2 0.1 0.1 0.2 0.0 0.0 0.1 0.0 0.1 0.1 0.0 0.0 0.1 0.0 0.1 0.1 0.1 0.1 0.0 0.0

Negative control 5.0 2.4 0.3 0.3 0.1 0.2 0.3 0.6 0.6 0.6 0.3 0.5 0.3 5.6 0.3 5.6 0.2 0.2 0.1 0.2 0.3 0.1 0.6 0.3 0.3 0.2 0.1 0.2 0.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2 0.5 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.1 0.5 0.1 0.5 0.1 0.0 0.0 0.0 0.1 0.0 0.0 0.1 0.0 0.0 0.1 0.1 0.1

Density of the dot on the nitrocellulose membrane was determined by an imaging system (GelDoc 2000, Bio-Rad, USA) using the Gel Doc software (Bio-Rad, USA). Density estimation was performed in triplicate ± represents standard deviation of the mean. Vi: virulent; A: avirulent; V: velogenic; M: mesogenic; L: lentogenic. WSFFSNI serves as a negative control, it is a phage clone that carries WSFFSNI sequence that interacts specifically with hepatitis B core antigen (Ho et al., 2003).

ing. However, such methods are slow, laborious, lack sensitivity and they are often influenced by the immune status of the chickens. Furthermore, virulent NDV infecting other species of birds often did not demonstrate potential pathogenicity in chickens that have been used as the pathotyping tool (Collins et al., 1994). On the other hand, nucleotide sequencing of the F gene for NDV pathotyping is more sensitive and rapid compared to the conventional pathotyping methods. However, the accuracy of nucleotide sequencing is often affected by NDV genome variability, mixed infection and unspecific binding of probes (Aldous and Alexander, 2001). Both of the conventional pathotyping methods and nucleotide sequence analysis of the F cleavage site have been prescribed by Office International des Epizooties (2004) to determine pathogenecity of NDV strains. In this study, we developed and assessed an alternative pathotyping tool by using a phage displayed peptide to differentiate NDV pathotypes. The virulence of 25 NDV strains, representing various pathotypes, were initially assessed based on the MDT in embryonated chicken eggs and nucleotide sequencing of the F cleavage site. The results from MDT and F cleavage sites nucleotide sequencing demonstrated a good correlation for 22/25 samples. Three NDV strains (S, 00/IKS and 4989/92 P3) were shown to be virulent by nucleotide sequencing but avirulent by MDT. The NDV

S strain is a known mesogenic strain that has been widely used as a live vaccine. This shows that nucleotide sequencing is more sensitive than MDT in pathotyping of NDV strains. Both virulence assessments were done to provide a comparison to the pathotyping method developed in this study. The use of phage display technology allows for the selection of peptides with specific binding characteristics from a vast library of random peptides. In this study, we used this technology to isolate peptides that specifically bind to the glycoproteins HN and F which are important in virus infectivity and virulence (Yusoff and Tan, 2001). Thus, these proteins may provide a good target to select ligands that are able to distinguish different virulence. We have earlier shown that a disulfide-constrained peptide, TLTTKLY, displayed on filamentous bacteriophage isolated from a direct biopanning experiment was able to interact specifically with velogenic NDV (Ramanujam et al., 2002, 2004). This phage clone was able to differentiate the velogenic from mesogenic and lentogenic NDV strains. In this study, we employed a more elaborate direct biopanning strategy targeted at NDV strains representing three different NDV pathotypes as compared to the single NDV pathotype by Ramanujam et al. (2002, 2004). This would allow us to isolate more phages displaying different peptides that could be used to differentiate NDV pathotypes.

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Ramanujam et al. (2002, 2004) have demonstrated that the phage clone carrying TLTTKLY can differentiate the velogenic from the mesogenic and lentogenic NDV strains using purified samples in an indirect ELISA. However, this phage could not be used as a capturing reagent in a dot blot assay or ELISA for detecting unpurified NDV samples, because the phage bound nonspecifically to host proteins in allantoic fluid. In addition, the drawback of using purified samples in clinical diagnostics is that it is often time consuming and laborious. This is not feasible for screening large flocks of birds in an outbreak where fast and reliable results are required. In order to develop an indirect ELISA that does not require purified samples, the phage clone carrying SWGEYDM was tested against unpurified allantoic fluid. However, the phage clone could not distinguish virulent from avirulent NDV isolates. In order to develop an assay that is easy, sensitive and requires less sample concentration, the phage clone carrying SWGEYDM was used as a capturing reagent in a dot blot assay. We have demonstrated that the phage clone carrying SWGEYDM can be used to differentiate the virulent from avirulent NDV strains in the dot blot assay, but it did not work in an indirect phage ELISA. This is likely due to the change of the conformation of the peptide displayed on the gpIII protein when the phage is coated onto plastic wells and thus affects the avidity of interaction. In comparison to the other pathotyping methods, the phage-capturing dot blot results are comparable to the nucleotide sequencing methods, where it is able to detect all virulent strains carrying the 112 R-R-Q-K/R/E-R-F117 motif at the fusion protein cleavage site. The SWGEYDM peptide contains two acidic residues, E and D at positions 4 and 6, respectively. This suggests that ionic interactions play an important role for the binding of this peptide with positively charged residues on the surface of a virulent strain. Sequence homology analysis of this peptide with the BLASTP program revealed that the first four residues, SWGE, matched residues 444–447 of a virus activating protease (VAP, GeneBank accession no. p25155) known as coagulation factor X precursor. The VAP which is normally found in the chorioallantoic membrane of chicken embryos, has been demonstrated to cleave a single arginine (R) site in the F proteins of Sendai virus, NDV and influenza virus (Ogasawara et al., 1992). We speculate that the SWGEYDM peptide mimics the VAP and most probably interacts with positively charged residues in the F proteins. This could be the reason why the peptide SWGEYDM is able to bind to virulent strains but not to avirulent strains. However, more experiments have to be carried out to study the three-dimensional structure of the peptide and its targets either by X-ray crystallography or nuclear magnetic resonance (NMR). The phage clone carrying SWGEYDM does not interact with any avirulent strains, thus, the newly developed phage dot blot assay may prove to be useful in detecting virulent NDV in the case of mixed infection. This cannot be performed by RT-PCR, where the primer may bind nonspecifically with the nucleic acid of avirulent strains (Aldous and Alexander, 2001). The newly developed phage-capturing dot blot is able to detect NDV in unpurified samples at concentrations as low as 4 HAU with 1011 pfu of phage carrying the peptide SWGEYDM. In addi-

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tion, this test is easy to perform and can be adopted in the field as a quick test for screening a large number of samples. Acknowledgements We thank Dr. Abdul Rahman Omar for providing some of the viruses used in this study. This study was supported by the IRPA grant 01-02-04-003-BTK/ER/006 from the Ministry of Science, Technology and Innovation (MOSTI) of Malaysia. T.C. Lee was supported by the National Science Fellowship (NSF) from MOSTI. References Aldous, E.W., Alexander, D.J., 2001. Detection and differentiation of Newcastle disease virus (avian paramyxovirus type 1). Avian Pathol. 30, 117–128. Alexander, D.J., 1988. Newcastle disease diagnosis. In: Alexander, D.J. (Ed.), Newcastle Disease. Kluwer Academic, Norwell, Massachusetts, pp. 147–160. Alexander, D.J., 2001. Gordon memorial lecture. Newcastle disease. Br. Poult. Sci. 42, 5–22. Alexander, D.J., Manvell, R.J., 1997. Antigenic diversity and similarities detected in avian paramyxovirus type 1 (Newcastle disease virus) isolates using monoclonal antibodies. Avian Pathol. 26, 399–418. Collins, M.S., Strong, I., Alexander, D.J., 1994. Evaluation of the molecular basis of pathogenicity of the variant Newcastle disease viruses termed pigeon PMV-1 viruses. Arch. Virol. 134, 403–411. Ho, K.L., Yusoff, K., Seow, H.F., Tan, W.S., 2003. Selection of high affinity ligands to hepatitis B core antigen from a phage-displayed cyclic peptide library. J. Med. Virol. 69, 27–32. Kho, C.L., Mohd-Azmi, M.L., Arshad, S.S., Yusoff, K., 2000. Performance of an RT-nested PCR ELISA for detection of Newcastle disease virus. J. Virol. Meth. 86, 71–83. Office International des Epizooties, 2004. Terrestrial Animal Health Code. Office International Epizooties, Paris, 2.1 pp. Ogasawara, T., Gotoh, B., Suzuki, H., Asaka, J., Shimokata, K., Rott, R., Nagai, Y., 1992. Expression of factor X and its significance for the determination of paramyxovirus tropism in the chick embryo. EMBO J. 11, 467–472. Ramanujam, P., Tan, W.S., Nathan, S., Yusoff, K., 2002. Novel peptides that inhibit the propagation of Newcastle disease virus. Arch. Virol. 147, 981–993. Ramanujam, P., Tan, W.S., Nathan, S., Yusoff, K., 2004. Pathotyping of Newcastle disease virus with a filamentous bacteriophage. Biotechniques 36, 296–300. Seal, B.S., King, D.J., Bennett, J.D., 1995. Characterization of Newcastle disease virus isolates by reverse transcription PCR coupled to direct nucleotide sequencing and development of sequence database for pathotype prediction and molecular epidemiological analysis. J. Clin. Microbiol. 33, 2624–2630. Smith, G.P., 1985. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315–1317. Stauber, N., Brechtbuhl, K., Bruckner, L., Hofmann, M.A., 1995. Detection of Newcastle disease virus in poultry vaccines using the polymerase chain reaction and direct sequencing of amplified cDNA. Vaccine 13, 360–364. Tan, S.W., Omar, A.R., Aini, I., Yusoff, K., Tan, W.S., 2004. Detection of Newcastle disease virus using a SYBR green I real time polymerase chain reaction. Acta Virol. 48, 23–28. Wang, Z., Vreede, Mitchell, J.O., Viljoen, G.J., 2001. Rapid detection and differentiation of Newcastle disease virus isolates by a triple one-step RT-PCR. Onderstepoort J. Vet. Res. 68, 131–134. Yusoff, K., Tan, W.S., 2001. Newcastle disease virus: macromolecules and opportunities. Avian Pathol. 30, 439–455. Yusoff, K., Tan, W.S., Lau, C.H., Ng, B.K., Ibrahim, A.L., 1996. Sequence of the haemagglutinin-neuraminadase gene of the Newcastle disease virus oral vaccine strain V4 (UPM). Avian Pathol. 25, 837–844.