Sensitive, semi-nested RT-PCR amplification of fusion gene sequences for the rapid detection and differentiation of Newcastle disease virus

Research in Veterinary Science 89 (2010) 282–289

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Sensitive, semi-nested RT-PCR amplification of fusion gene sequences for the rapid detection and differentiation of Newcastle disease virus Lei Zhang 1, Zhiming Pan 1, Shizhong Geng, Xiang Chen, Shunlin Hu, Huimo Liu, Yantao Wu, Xinan Jiao *, Xiufan Liu Jiangsu Key Laboratory of Zoonosis, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, PR China

a r t i c l e

i n f o

Article history: Accepted 9 February 2010

Keywords: Newcastle disease virus Fusion protein Semi-nested RT-PCR

a b s t r a c t A rapid, sensitive and specific semi-nested RT-PCR was developed to detect and differentiate virulent and avirulent strains of Newcastle disease virus (NDV). For a total of 67 NDV strains, the results obtained from the semi-nested RT-PCR were consistent with those from nucleotide sequence analysis, plaque forming assays, mean death time (MDT) measurements and intracerebral pathogenicity index (ICPI). Furthermore, 13 class I NDV strains can be characterized by the semi-nested RT-PCR approach, which was feasible by the conventional methods. The detection limit for the semi-nested RT-PCR was two plaque forming units (PFU) both for NDV strain F48E9 in allantoic fluid and for isolate APMV1/ch/ChinaND4031 in oral or cloacal swabs. In conclusion, this semi-nested RT-PCR method offers a new assay for the rapid detection and differentiation of NDVs. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Newcastle disease (ND), a contagious and widespread avian disease affecting most species of birds, is one of the most serious diseases of poultry caused by ND virus (NDV). Because of the severe nature of the disease and the associated consequences, ND is included as a notifiable avian disease which must be reported to Office Internationale des Epizooties (OIE). NDV is the type virus in the Avulavirus genus of the Paramyxoviridae family. NDV possesses a single stranded, non-segmented RNA genome of ca. 15 kb with negative polarity, encoding for six major structural and non-structural proteins. RNA editing of the P protein produces additional proteins V and W (Huang et al., 2003). The fusion protein, present in the viral envelope is synthesized as the F0 precursor, becoming activated after cleavage into the F1 and F2 fragments, to initiate infection (Nagai et al., 1976; Umino et al., 1990). Depending upon virulence, NDV can be grouped into the avirulent, moderately virulent and highly virulent lentogenic, mesogenic and velogenic pathotypes, respectively. These pathotypes differ in the number of basic amino acids at the cleavage site of the fusion protein (Seal et al., 1995; Toyoda et al., 1989). The velogenic and mesogenic strains are both virulent and have been

* Corresponding author. Address: Jiangsu Key Laboratory of Zoonosis, College of Veterinary Medicine, Yangzhou University, 12 East Wenhui Road, Yangzhou, Jiangsu 225009, China. Tel.: +86 514 8797 1803; fax: +86 514 8731 1374. E-mail address: [email protected] (X. Jiao). 1 These authors contributed equally to this work. 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.02.007

frequently identified as the causative agent of outbreaks in many countries. The lentogenic strains are avirulent and have been used as live vaccines to control the disease. However, outbreaks of ND have continuously been reported in poultry vaccinated with the lentogenic LaSota vaccine and flocks of wild birds (Ezema et al., 2008; Kapczynski and King, 2005; Nakamura et al., 2008). Current diagnosis of NDV is unable to differentiate virulent from avirulent NDVs in vaccinated chickens, therefore, OIE requires a definitive diagnosis of virulent NDV to enable effective prevention of an outbreak by strict control measures and trade embargo restrictions (Aldous and Alexander, 2001). Conventional methods for pathotyping NDV strains involve measuring mean death time (MDT), intracerebral pathogenicity index (ICPI) values and the intravenous pathogenicity index (IVPI) in NDV-inoculated embryonated chicken eggs or chicks (OIE, 2008). Over the past decade, numerous molecular techniques have been employed to diagnose ND, such as a twosome or triple one-step RT-PCR (Shan et al., 2003; Wang et al., 2001), RT-nested PCR coupled with ELISA detection (Kho et al., 2000), real-time PCR (Aldous et al., 2001; Tan et al., 2004; Fuller et al., 2009), RAPID-BAP (Wang et al., 2008), the technique of nucleic acid sequence-based amplification (Cui et al., 2007) and a phage-capturing dot blot (Lee et al., 2006). The strains used in these assays were mainly included as class II types. Recently, there have been two methods developed for the detection of both class I and II NDV isolates (Djikeng et al., 2008; Kim et al., 2008). The meaning is significant if we can develop an ordinary RT-PCR which is less equipment-demanding for detecting class II NDV as well as some prevalent class I strains. Further more, the approach

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can differentiate virulent and non-virulent NDV strains and identify virulent NDV strains from chickens vaccinated with live commercial vaccines. The objective of this RT-PCR method was to accelerate detection and differentiation of NDV strains and apply this method to rapid screening and disease monitoring. 2. Material and methods

forward degenerate primers and distinct reverse primer, were designed from the F gene encoding for the cleavage site to differentiate virulent (velogenic and mesogenic) and avirulent (lentogenic) NDV. The first round of amplification using primer pair F1 + F2 generated a 301 bp fragment with all NDV strains. Using primer pair F1 + F3, a 206 bp fragment was detected only in virulent NDV in the second round PCR. The 301 bp products were sequenced by TaKaRa Biotechnology Inc (Dalian, China).

2.1. Viruses 2.5. Construction of recombinant plasmids A total of 15 reference strains NDV were used in this study and kindly provided by the Animal Infectious Diseases Laboratory of the Ministry of Agriculture, Yangzhou University, Jiangsu, China (Table 1). The total number of field isolates, from different areas of China, was 52, and they were isolated during the 2006 ND outbreaks. Other avian viruses such as infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), fowl pox virus (FPV) and avian influenza virus (AIV) were included as negative controls.

The 301 bp fragment amplified from the F gene of strain F48E9 was cloned into pGEM-T Easy vector (Promega, USA). The resulting construct was designated pGEM-T-F. The plasmid pGEM-T-F was then purified using a Qiagen Plasmid Mega Kit (Qiagen, USA) and confirmed by sequencing analysis (TaKaRa, China). The positive plasmid was diluted 200-fold as a positive standard in the assays. 2.6. Semi-nested RT-PCR

2.2. Propagation of NDV strains All NDV strains were propagated in 9-day-old embryonated chicken eggs (Shandong Institute of Poultry Science, Jinan, China), and the presence of viruses was determined by the hemagglutination inhibition (HI) test (Alexander, 1988) using the NDV standard antiserum purchased from Harbin Veterinary Research Institute, Harbin, China. Virus-containing allantoic fluid was aliquoted and stored at 70 °C until use.

2.6.1. cDNA synthesis and first amplification reaction Reverse transcription (RT) and first amplification of the seminested PCR was performed in one-tube. In the first round of the RT-PCR, the reactions were performed in a 2700 GeneAmp PCR system thermal cycler (Applied Biosystems, USA) in a 25 lL reaction volume containing 2 lL of 5  M-MLV buffer (Promega, USA), 1.5 lL 10  PCR buffer (Promega, USA), 0.24 mM each dNTP (Promega, USA), 5 pmol each primer F1 and F2, 1.5 lL of acetylated bovine serum albumin (BSA, 1000 lg/mL; Promega, USA), 0.5 lL of

2.3. Viral RNA extraction Viral RNA was extracted from clarified allantoic fluids or swab specimens using Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions. The viral RNA was resuspended in diethylpyrocarbonate (DEPC) treated distilled water and stored at 70 °C until further use.

Table 2 Primers designed to differentiate virulent and avirulent NDVs. Code

Sense

Sequence

Locationa

F1 F2 F3

+ – –

TAYACCTCRTCYCAGACW(T) bGG GGAGGATGTTGGCAGCATTc AYRGCGCCTATAAASCGTYT

nt 203–222 nt 503–485 nt 408–389

a

2.4. Primers

Numbering according to Toyoda et al. (1989). The amplification would be promoted when the nucleotide W was replaced by T in the second round of the semi-nested RT-PCR. And the primer A is a derivative of the oligonucleotide primer FOP1 described by Kho et al. (2000). Codes for mixed bases position: R = A/G, Y = C/T, W = A/T, S = C/G. c From Kant et al. (1997). b

Primers were designed following multiple sequence alignments of NDV F genes using the DNASTAR software (DNASTAR Inc., USA). Two sets of primers (F1 + F2 and F1 + F3; Table 2), with common

Table 1 Reference strains and isolates of NDV used in this study. Strain

Host

F0 cleavage site (a.a. 112–117)

Virulence (based on F0 cleavage site)

GenBank number

Class/genotype

LaSota Clone30a JS/9/05 Mukteswar/57 HER/33 070422 JX-1/94 ZJ1 JS-2/98 JS/5/05 QH-1/79 F48E9 D/ZJ/1/04 D/ZJ/1/02 D/SD/16/05 IBDV IBV FPV AIV H5 AIV H9

Chicken Chicken Goose Chicken Chicken Pigeon Chicken Goose Chicken Goose Chicken Chicken Duck Duck Duck – – – – –

GRQGRL GRQGRL RRQRRF RRQRRF RRQRRF RRQRRF RRRKRF RRQKRF RRQKRF RRQKRF RRQKRF RRQRRF GKQGRL ERQGRL EQQERL – – – – –

A A Vi Vi Vi Vi Vi Vi Vi Vi Vi Vi A A A – – – – –

AF077761 Y18898 FJ430160 EF201805 M24702 FJ766526 AF458021 AF431744 AF458013 EU044798 AF378250 AF079172 FJ597613 FJ597580 FJ597584 – – – – –

II/II II/II II/III II/III II/IV II/VIb II/VIf II/VII II/VIIe II/VIId II/VIII II/IX II/Ib I/2 I/3b – – – – –

Vi: virulent; A: avirulent.

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dimethyl sulfoxide (DMSO, 1100 lg/mL; Sigma–Aldrich, USA), 40 U M-MLV (Promega, USA), 1.5 U Taq DNA polymerase (Promega, USA), 5 U RNasin (Promega, USA) and 10 lL undiluted RNA or 2 lL diluted plasmid. The reaction was carried out at 42 °C for 60 min and then at 94 °C for 3 min followed by 40 cycles of 94 °C for 30 s, 57 and 72 °C, 45 s each with a final extension at 72 °C for 5 min. 2.6.2. Second amplification reaction In the second round of PCR amplification, the reaction mix consisted of 2.5 lL 10  PCR buffer (Promega, USA), 0.24 mM each dNTP (Promega, USA), 5 pmol each primer F1 and F3, 1.0 U of Taq DNA polymerase (Promega, USA) and 0.5 lL of the first round RT-PCR product or 2 lL of diluted plasmid. The distilled water was added to adjust a final 25 lL reaction volume. The PCR mixture was preheated at 94 °C for 5 min and then cycled 30 times at 94 °C for 30 s, 56 °C and 72 °C, 40 s each with final extension at 72 °C for 5 min. The PCR products were analyzed by electrophoresis on agarose gels (1.5% w/v), stained with ethidium bromide (EB). 2.7. Determination of sensitivity and specificity of semi-nested RT-PCR Chicken fibroblast cells (CEF) were prepared from 9-day-old SPF embryonated chicken eggs. Cell monolayers were grown in 24-well plates. The F48E9 allantoic fluid was 10-fold serially diluted, and 100 lL of each serial dilution was added per well to confluent CEF cells. Four repeated wells were used for each serial dilution. After 60 min adsorption, cells were overlaid with M199 and F10 (containing 2% fetal bovine serum and 0.75% agarose) and incubated at 37 °C for 48 h. The cells were then overlaid with M199 and F10 (containing 5% fetal bovine serum, 1% agarose and 0.015% neutral red). Simultaneously, RNA was extracted from aliquots of 100 lL of each dilution for the development of seminested RT-PCR. The specificity of the PCR was evaluated with all the reference strains and isolates of NDV, and five other infectious avian viruses including avian influenza (H5 and H9 subtypes), infectious bursal disease, infectious bronchitis and fowl pox viruses. 2.8. Blind study and sequencing The 52 field isolates were checked with the semi-nested RT-PCR established above. The 301 bp PCR products were sequenced as described in 2.4 and deposited in GenBank under accession numbers GU289432 to GU289483. 2.9. Phylogenetic analysis To establish the genetic lineage among the NDV strains, phylogenetic analysis was performed using the partial nucleotide sequences at 30 ends of 52 F genes (nt 203–503) obtained from the blind study and eight published F gene subsequences from GenBank. Phylogenetic trees were constructed based upon the comparison of the nucleotide sequences of F gene fragments by aligning with the Clustal W program. Phylogenetic trees were drawn by using the neighbor-joining method of the MEGA program (version 4.0), based on the variable-region (nt 203–503) nucleotide sequences of the F gene with 1000 bootstrap trials to assign confidence to groupings. 2.10. Virulence assessment A total of 52 field isolates were tested with the plaque forming assay. Based on the topology of the phylogenetic tree, the plaque purified representative isolates were selected for MDT and ICPI tests according to OIE procedures (OIE, 2008).

2.11. Detection of NDV in oropharyngeal and cloacal swab specimens from experimentally infected chicken by the semi-nested RT-PCR Twenty 4-week-old White Leghorn chickens (Shandong Institute of Poultry Science, Jinan, Shandong, China) were divided into two groups (A and B). Group A (10 chickens) was inoculated with 0.1 mL of commercial LaSota vaccine (Yangzhou VacBio Company, China) containing 106 50% egg infective doses (EID50) via the oral and intranasal route, respectively. Two weeks after inoculation, immunized chickens were challenged with 106 EID50 of a VIId virus strain (APMV1/ch/ChinaND4031) administered in a 50 lL volume into the right eye and 50 lL into the choana (Miller et al., 2007). Group B (10 chickens) were kept in another facility and left as controls. Chickens were housed, handled, and immunized following approval by the institutional animal experimental committee. Oropharyngeal and cloacal swabs were collected on day 2 postchallenge, and placed into 1 mL brain heart infusion (BHI) broth (Difco, Becton–Dickinson, USA) with a final concentration of 2000 U/mL penicillin G, 2.5 lg/mL streptomycin, 6 lg/mL gentamycin, 2.5 lg/mL fungizone. After centrifugation at 6000g for 5 min at 4 °C, supernatants were divided into two aliquots, one for RT-PCR, and the other for the plaque forming assay. After the plaque forming assay, PFU-purified NDV was collected using 10 lL filter tips (Axygen Scientific, USA) and suspended in 200 lL M199 and F10 medium without serum. Then, 100 lL medium was inoculated into 9-day-old embryonated chicken eggs for HI testing. 3. Results 3.1. Specificity of the semi-nested RT-PCR The specificity of the primers were determined by performing the semi-nested RT-PCR on RNA extracted from the allantoic fluids of the embryonated eggs infected with 15 NDV reference strains as well as 5 non-NDV infectious avian viruses. The 301 bp fragments were amplified from all 15 NDV strains using the primer pair F1 + F2. Conversely, primer pair F1 + F3 did not amplify the 206 bp fragments from avirulent strains, suggesting that they are specific to virulent strains (Figs. 1 and 2). Amplification was not possible for AIV, IBV, IBDV or FPV by RT-PCR (data not shown). 3.2. The sensitivity of the semi-nested RT-PCR The sensitivity of the established semi-nested RT-PCR was assessed, and the F48E9 virus titers in allantoic fluids were confirmed with the plaque forming assay (Fig. 3A). After gel electrophoresis, a 206 bp band was visible until the 106 dilution of allantoic fluids (Fig. 3B, lane 10), which was equivalent to 2 PFU. The outer primers (F1 + F2) had a detection limit out to the 103 dilution, which was approximately 2000 PFU. Therefore, the semi-nested RT-PCR was about 1000-fold more sensitive than the non-nested RT-PCR. 3.3. Differentiation of virulent and avirulent NDV strains by seminested RT-PCR and sequencing results For the 52 field isolates tested in the blind study (Table 3), the results from the semi-nested RT-PCR were consistent with the sequencing and plaque forming assay results. Of the 21 virulent strains, 18 were of the genotype VIId and contained the sequence motif 112R-R-Q-K-R-F117 at the cleavage sites of the F gene. The remaining three virulent strains were genotype IX and had the motif 112R-R-Q-R-R-F117 at the cleavage sites. The 31 avirulent strains exhibited various sequence motifs at the cleavage sites: 112E-K-QG-R-L117, 112E-R-Q-G-R-L117, 112G-K-Q-G-R-L117, 112G-R-Q-G-R-L117

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Fig. 1. Specificity of semi-nested RT-PCR assay. Lane M: DL2000; lanes 1, 3, 5, 7, 9, 13, 15, 17 and 19 contain the first round PCR products; lanes 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 contain second round PCR products. Lanes 1 and 2: LaSota; lanes 3 and 4: Clone30a; lanes 5 and 6: F48E9; lanes 7 and 8: JS/9/05; lanes 9 and 10: Mukteswar/57; lanes 11 and 12: HER/33; lanes 13 and 14: 070422; lanes 15 and 16: JS-2/98; lanes 17 and 18: JX-1/94; lanes 19 and 20: JS/5/05; lane 21: PBS control.

or 112E-Q-Q-E-R-L117. According to GenBank blast results, the strains with the cleavage site 112E-Q-Q-E-R-L117 and 112E-R-Q-GR-L117 are classified into class I. 3.4. Phylogenetic analysis and virulence assessment

Fig. 2. Specificity of semi-nested RT-PCR assay. Lane M: DL2000; lanes 22, 24, 26, 28 and 30 contain the first round PCR products; lanes 23, 25, 27, 29 and 31 contain second round PCR products. Lanes 22 and 23: QH-1/79; lanes 24 and 25: D/ZJ/1/04; lanes 26 and 27: ZJ1; lanes 28 and 29: D/ZJ/1/02; lanes 30 and 31: D/SD/16/05.

The sequences obtained in the blind study from the 301 bp products were compared with other NDV sequence data available in GenBank (Lomniczi et al., 1998; Seal et al., 1995). A phylogenetic tree was constructed (Fig. 4), and the virulence assessment results were listed in Table 4. Based on GenBank blast results, the following representative virulent strains were selected: APMV1/ch/ChinaND241, APMV1/ch/ChinaND4031, APMV1/ch/ ChinaND4222 and APMV1/ch/ChinaND5322. These virulent strains shared 100%, 100%, 100% and 98.7% nucleotide identity with the

Fig. 3. Sensitivity of semi-nested RT-PCR assay. The plaque forming assay showed that a 106 dilution of allantoic fluids was equivalent to 2 PFU. Therefore the concentration of F48E9 was 2  107 PFU/ml (panel A). The sensitivity of the semi-nested PCR assay was determined by 10-fold serial dilution of virus stocks. PCR products were analyzed by electrophoresis on a 1.5% agarose gel stained with ethidium bromide. Lanes 1, 3, 5, 7, 9 and 11: first round PCR products; lanes 2, 4, 6, 8, 10 and 12: second round PCR products. Lane M: DL2000; lanes 1 and 2: 2  104 PFU; lanes 3 and 4: 2  103 PFU; lanes 5 and 6: 2  102 PFU; lanes 7 and 8: 20 PFU; lanes 9 and 10: 2 PFU; lanes 11 and 12: 0.2 PFU (panel B).

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Table 3 Differentiation of virulent and avirulent NDV strains using semi-nested RT-PCR in a blind study.

a

Virus

F0 cleavage site (a.a. 112–117)

The ability of plaque forming

Class/genotype

APMV1/cha/ChinaND1 APMV1/ch/ChinaND2 APMV1/ch/ChinaND3 APMV1/ch/ChinaND5 APMV1/ch/ChinaND6 APMV1/ch/ChinaND7 APMV1/ch/ChinaND8 APMV1/ch/ChinaND9 APMV1/ch/ChinaND10 APMV1/ch/ChinaND11 APMV1/ch/ChinaND12 APMV1/ch/ChinaND13 APMV1/ch/ChinaND14 APMV1/ch/ChinaND15 APMV1/ch/ChinaND16 APMV1/ch/ChinaND17 APMV1/ch/ChinaND18 APMV1/duck/ChinaND19 APMV1/ch/ChinaND20 APMV1/ch/ChinaND21 APMV1/ch/ChinaND23 APMV1/ch/ChinaND241 APMV1/ch/ChinaND25 APMV1/ch/ChinaND26 APMV1/ch/ChinaND27 APMV1/ch/ChinaND28 APMV1/ch/ChinaND29 APMV1/ch/ChinaND30 APMV1/ch/ChinaND31 APMV1/ch/ChinaND32 APMV1/ch/ChinaND33 APMV1/ch/ChinaND34 APMV1/ch/ChinaND35 APMV1/ch/ChinaND36 APMV1/ch/ChinaND37 APMV1/ch/ChinaND38 APMV1/ch/ChinaND39 APMV1/ch/ChinaND4031 APMV1/ch/ChinaND41 APMV1/ch/ChinaND4222 APMV1/ch/ChinaND43 APMV1/ch/ChinaND44 APMV1/ch/ChinaND45 APMV1/ch/ChinaND46 APMV1/ch/ChinaND47 APMV1/ch/ChinaND48 APMV1/ch/ChinaND49 APMV1/ch/ChinaND50 APMV1/ch/ChinaND51 APMV1/ch/ChinaND52 APMV1/ch/ChinaND5322 APMV1/ch/ChinaND54 Standard plasmid

RRQKRF RRQKRF RRQKRF GKQGRL GKQGRL GKQGRL EQQERL GKQGRL GKQGRL ERQGRL EKQGRL EQQERL GRQGRL GRQGRL GKQGRL GKQGRL GKQGRL ERQGRL EQQERL EQQERL GKQGRL RRQRRF RRQRRF RRQRRF EQQERL GKQGRL EQQERL EKQGRL EQQERL GKQGRL GKQGRL EQQERL GKQGRL EQQERL EQQERL EKQGRL RRQKRF RRQKRF RRQKRF RRQKRF RRQKRF RRQKRF RRQKRF RRQKRF RRQKRF RRQKRF RRQKRF RRQKRF RRQKRF EQQERL RRQKRF RRQKRF –

+ + + – – – – – – – – – – – – – – – – – – + + + – – – – – – – – – – – – + + + + + + + + + + + + + – + + –

II/VIId II/VIId II/VIId II/Ib II/Ib II/Ib I/3b II/Ib II/Ib I/2 II/Ib I/3b II/II II/Ib II/Ib II/Ib II/Ib I/2 I/3b I/3b II/Ib II/IX II/IX II/IX I/3b II/Ib I/3b II/Ib I/3b II/Ib II/Ib I/3b II/Ib I/3b I/3b II/Ib II/VIId II/VIId II/VIId II/VIId II/VIId II/VIId II/VIId II/VIId II/VIId II/VIId II/VIId II/VIId II/VIId I/3b II/VIId II/VIId –

Semi-nested RT-PCR First round

Second round

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + – – – – – – – – – – – – – – – – – – + + + – – – – – – – – – – – – + + + + + + + + + + + + + – + + +

Accession number

GU289432 GU289433 GU289434 GU289435 GU289436 GU289437 GU289438 GU289439 GU289440 GU289441 GU289442 GU289443 GU289444 GU289445 GU289446 GU289447 GU289448 GU289449 GU289450 GU289451 GU289452 GU289453 GU289454 GU289455 GU289456 GU289457 GU289458 GU289459 GU289460 GU289461 GU289462 GU289463 GU289464 GU289465 GU289466 GU289467 GU289468 GU289469 GU289470 GU289471 GU289472 GU289473 GU289474 GU289475 GU289476 GU289477 GU289478 GU289479 GU289480 GU289481 GU289482 GU289483 –

ch stands for chicken.

strains DQ858356, EF579731, EF128054 and EU258640, respectively. The non-virulent strains APMV1/ch/ChinaND5, APMV1/ch/ ChinaND8, APMV1/ch/ChinaND11 and APMV1/ch/ChinaND14 exhibited a high degree of similarity with the strains AY965077, EF564825, DQ097893 and FM200802 (98.3%, 96.0%, 97.3% and 100%, respectively).

amplification was observed in the negative controls. As the plaque forming assay showed, the virus quantities varied greatly (Fig. 5a and b with respect to lane 5, lane 6; c was a negative plaque control). All eggs except the controls were no longer viable 48 h after inoculation of the medium containing the viruses purified from the plaque assay. The HI assay was also used to confirm the presence of NDVs (HI = 7). Negative plaque controls have no HI titer.

3.5. Detection of NDV from clinical specimens 4. Discussion All chickens immunized with the commercial LaSota vaccine were experimentally infected with 106 EID50/0.1 mL of virulent NDV. To detect the NDV shedding, oropharyngeal and cloacal swabs were obtained from all chickens on day 2 after challenge. All swabs were positive using the semi-nested RT-PCR, while no

Avirulent NDV strains are widely distributed in nature and have been extensively used as live vaccines. Due to the high similarity in antigenicity between virulent and avirulent NDV strains, there is a possibility that avirulent strains may interfere with the diagnosis

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Fig. 4. Phylogenetic tree of NDV strains based on partial nucleotide sequences from F genes (203–503). Divisions and genotypes are indicated. The tree was constructed by the neighbor-joining methods of MEGA 4.0, with 1000 bootstrap trials to assign confidence to groupings.

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Table 4 MDT and ICPI of representative strains.

a b

Isolates

MDT (h)

ICPI

Pathogenicity

APMV1/ch/ChinaND4031 APMV1/ch/ChinaND4222 APMV1/ch/ChinaND5322 APMV1/ch/ChinaND241 APMV1/ch/ChinaND5 APMV1/ch/ChinaND8 APMV1/ch/ChinaND11 APMV1/ch/ChinaND14

52.8 50.4 45.6 36.0 –a – – –

1.95 1.89 1.88 2.00 NT b NT NT NT

Virulent Virulent Virulent Virulent Avirulent Avirulent Avirulent Avirulent

‘‘–” means the challenged egg was live within 7 days. Not tested.

of ND. Furthermore, previously designed primers may no longer be appropriate given the evolution of NDV in the past decade (Miller et al., 2010). In Kant’s assay, the universal NDV detection primer pair A + B is effective in amplification virulent strains but failed to detect virus in 1/3 flocks from which non-virulent virus was isolated (Kant et al., 1997). The nucleotide variation around the cleavage site of the F gene has been exploited for the pathotypic characterization of NDV isolates and is considered the primary molecular determinant for NDV virulence (Aldous and Alexander, 2001; Glickman et al., 1988). Isolates with at least three basic amino acids at the cleavage site followed by a phenylalanine are designated virulent, whereas there is only one in the avirulent NDV strains (OIE, 2009). So it is difficult to design perfect matched or mismatched primers for NDV differentiation in the cleavage site of the avirulent strains due to the high nucleotides variation. In the present study, the nested RT-PCR was employed to solve this problem where first round of PCR amplification was developed to confirm the NDV infection with degenerate primer pair F1 + F2 both 85% complementary to F-genomic sequences, and the second round of PCR was performed to discriminate virulent from avirulent NDV strains and to improve the sensitivity. By applying this strategy, we successfully characterized NDV pathotypes and cir-

cumvented the difficulty of designing primers specific for F0 cleavage sites in avirulent strains. According to sequence analysis in this study, the virulent strains own a basic acid K/R in the cleavage site at position 115, whereas avirulent strains embody a non-basic acid G/E. It have been shown that the last nucleotide of primer F3 could pair with the first base of triplet codon K/R, but failed to hybridize with the first base of triplet codon G/E in avirulent strains. Further analysis revealed that these field strains in this blind study were responsible for the prevalent outbreaks in China (Liu et al., 2008, 2009). The genotype VIId (18 of 21) was the predominant genotype involved in ND outbreaks worldwide, which constitutes the fourth panzootic of ND. The number of avirulent strains successfully characterized was 31, including 13 prevalent class I strains. Although we tested numerous strains originating from fowls, geese and pigeons, some extraordinary NDV strains have been reported to exist. Werner et al. (1999) found a new motif 112R-R-K-KR-F117 in pigeon variant PMV-1 (PPMV-1) isolates of both high and low virulence to chickens in ICPI tests. Additionally, the PPMV-1 isolates with the virulent motif 112G-R-Q-K-R-F117 exhibited a low virulence towards chickens (Collins et al., 1996; Zanetti et al., 2001). Recently (Tan et al., 2008) reported that another virulent NDV possesses the lentogenic motif 112G-R-Q-G-R-L117 which might be generated in nature from the LaSota live vaccine virus strain under host immune pressure. Theoretically, these strains could hardly be differentiated by molecular methods, but we are yet to isolate them. Besides allantoic fluid samples, clinical swab specimens in which residual vaccine viruses may be present in the swabs and interfere with the differentiation of field strains from vaccine strains were also tested in our study. However, our results suggest that the assay developed in our laboratory was able to detect the corresponding virus from each specimen with the same sensitivity as those from allantoic fluid. In summary, a molecular diagnostic technique based on seminested RT-PCR to detect NDV has been developed. The NDV RTPCR assay system is equivalent sensitivity to viral culture and of-

Fig. 5. Detection in clinical specimens. a, b, c plaque forming results (panel A); Lane M, DL2000; Lane 1–8, cloacal swabs; lane 9–16, oropharyngeal swabs; lane17, standard plasmid; lane18, control allantoic fluid; lane n and n + 8 (n = 1–8) represent the swabs from the same individual (panel B) .

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