Detection and characterization of Newcastle disease virus in clinical samples using real time RT-PCR and melting curve analysis based on matrix and fusion genes amplification

Aim: Newcastle disease is still one of the major threats for poultry industry allover the world. Therefore, attempt was made in this study to use the SYBR Green I real-time PCR with melting curves analysis as for detection and differentiation of NDV strains in suspected infected birds. Materials and Methods: Two sets of primers were used to amplify matrix and fusion genes in samples collected from suspectly infected birds (chickens and pigeons). Melting curve analysis in conjunction with real time PCR was conducted for identifying different pathotypes of the isolated NDVs. Clinical samples were propagated on specific pathogen free ECE and tested for MDT and ICIP. Results: The velogenic NDVs isolated from chickens and pigeons were distinguished with mean T 85.03±0.341 and m 83.78±0.237 respectively for M-gene amplification and for F-gene amplification the mean T were 84.04±0.037 and m


Introduction
protein (M), phosphoprotein (P), and nucleoprotein (NP), in the order 3'-NP-P-M-F-HN-L-5', [4,5].NDVs Newcastle disease is a highly contagious and fatal were classified into three major pathotypes based on viral disease that affects all species of birds.The the clinical signs induced in infected chickens: clinical signs seen in birds affected by this disease vary velogenic (highly virulent), mesogenic (intermediate widely and are dependent on factors like the virus virulent) and (avirulent) lentogenic strains [6,7,8].strain, host species, age, immune status, environmental The detection and differentiation of NDVs are stress and concurrent infection.In chickens, the disease based on virus isolation using embryonated chicken may vary from sudden death with 100% mortality to eggs, followed by an in vivo estimation of pathosubclinical disease.The disease has a worldwide genicity in chickens, such as the intracerebral pathodistribution, and is a major threat to the poultry genicity index (ICPI) in 1-day-old chicks, the industries due to the huge economic loss associated intravenous pathogenicity index (IVPI) in 6-week-old with it specially in turkeys and chickens, [1].
chickens, or the mean death time (MDT) in chicken Newcastle disease (ND), is caused by NDV, an embryos [8].Studies comparing the deduced amino enveloped virus that contains a liner, non segmented, acid sequence at the cleavage site of NDV, varying in single stranded negative sense RNA genome, in the virulence for chickens, showed that virulent viruses genus Avulavirus of the family Paramyxoviridae.avirulent viruses have G/E-K/R-Q-G/E-R-L [8][9][10][11]."role of six", the genome coding for six major proteins: The advent of real-time PCR methods has improved a large RNA polymerase (L), hemagglutininfurther the significant benefits of RT-PCR in comparison neuraminidase protein (HN), fusion protein (F), matrix Owing to these features, real-time PCR is now homogenized organs aliquot using QIAamp Viral one of the most important techniques for the detection RNA mini kits, Qiagen (USA) following the manufacturer's recommended procedure.and monitoring of virus infections [12][13][14].There are Complementary cDNA was synthesized using different formats available for real-time PCR.The 4µl of eluted RNA with transcriptor first strand cDNA intercalating dyes SYBR Green I assay with the ® Synthesis kits , Roche, (USA) following producer melting curve analysis are the most cost effective and recommendation.easier to establish as compared to other real-time The amplification reaction was performed in detection methods.Melting curve generated by using LightCycler 2.0 (Roche, USA).The reaction mixture Light Cycler instrument with SYBR Green I dye has per 20 µl reaction was (for M gene) 10 pmol of primers been applied for rapid detection and differentiation of (1 µl/each), 5µl cDNA, 5µl of Master mix (Light NDV [15].

® PLUS
Cycler Fast Start DNA Master SYBR Green I, In this study we used SYBR green assay with the Roche, USA), up to 20 µl of dd-H O.For F gene melting curve analysis for rapid detection and 2 differentiation of NDV in clinical samples using set of amplification, same procedures were used except that primers designed for amplification of F (Fusion) and the concentration of forward primer was 30 pmol.The (Matrix) M genes.Our results were confirmed by cycling conditions for matrix gene amplification conventional methods of pathotyping and Real Time primers were performed as follows: initial denaturation NDVs a set of primer-probe designed for amplification sand in sterile PBS containing 0.01% antibiotic to of F gene was used as described and evaluated by [10], ® PLUS make 1:10 (w/v) dilution.Oral and cloacal swab using LightCycler Fast Start DNA Master samples were suspended in 1 ml sterile PBS containing HybProbe (Roch, USA). the antibiotics as described by [8].
Biological pathotyping: To confirm our results, virus Ethical approval: The experiments and sample isolation and virulence level of each isolated NDVs collection procedures were consistent with the rules of was measured by MDT of nine days old chicken SPF the Animal Ethics and Monitoring Committee.
embryos and by ICPI in one day old SPF chicks, as described by [8].Primers and probes: Two sets of primer combinations designed for amplification of matrix and fusion genes

Results
were used in real time RT-PCR.A velogenic specific After establishing the optimum condition of Real primer probe designed to amplify a wide range of Time RT-PCR, the lentogenic LaSota and HB1 Velogenic NDVs was used to detect the vNDVS in this (Vaccines) and Velogenic NDV strains (FJ939313 and study (Table -1).-2).vaccines LaSota and HB1 (lentogenic strains) were Our results were confirmed using virus isolation, amplified with melting temperature (T ) 87.10ºC and MDT, ICIP and amplification of Velogenic cleavage m site using fluorogenic probe.Results showed that 86.93ºC respectively using the M-gene primer and melting temperature (T ) was 86.55 ºC and 86.62 ºC samples collected from 15 suspected NDV flock were m positive for SYBR Green I real-time PCR and virus respectively using the F-gene primer (Fig. 1).On the isolation respectively.All isolates that fall in the T of other hand the NDV strain FJ939313 that isolated from m chickens and NDV/Pg isolated from pigeon were velogenic category showed chicken embryo MDT test amplified with melting temperature (T ) was 85.28 ºC results ranged between 48 and 55 hours.and ICPI m and 83.94 ºC.values between 1.56 and 1.85, respectively (Table 2).

Samples collected from 21 poultry flock
On the other hand all isolates that fall in the T of m subjected to The SYBR Green I real-time PCR using lentogenic category showed chicken embryo MDT test both F-gene and M-gene primers.Results showed that results ranged between >90 hours.and ICPI values all samples collected from 16 poultry flocks were ranged from 0.8 and 0.85, respectively (Table -2).All positive with the SYBR Green I real-time PCR and the velogenic isolates were positive for fluorogenic probe other 5 flocks failed to show any positive results with detection and amplification.both gene primers.

Discussion
Melting curve analysis revealed that all positive samples were divided into 3 groups based on the The objective of this study was to assess a distinct melting peaks (Table -2).All the chicken diagnostic method for the rapid detection and velogenic isolates were grouped together with T m differentiation of NDV strains using SYBR Green I Primers-Probe sequences were used as previously described, and validated [16].sequence.Melting curve analysis can distinguish collaboration between all authors.products of the same length but different GC/AT ratio 112 117 usually have the motif R/K-R-Q-K/R-R-F and [2,3].The RNA genome of NDV is ~15 kb follow the 112 117 to conventional gel-based PCR assays, real-time PCR RNA extraction and real time RT-PCR: The RNA was offers increased sensitivity and specificity in a rapid extracted from 140 µl of swab fluid or 140 µl of ® format.
Figure-1.(A) Melting-curve analysis of a 101 bp fragment conaining the F cleavage site amplified by F-gene primer.(B) Melting curve analysis of a 121 bp fragment containing the F cleavage site amplified by F-gene primer a. Melting temperature of chicken velogenic strains (Fj939313).b. melting temperature of pigeon velogenic strain (NDV/Pg) c. melting temperature of lentogenic strain (LaSota and HB1).
) were detected and differentiated using both ranged from 86.98 C to 87.01 C (86.99±0.021C) and the F-gene primer amplifying the region around the 86.55 to 86.64 (86.50±0.063)for both M-gene and Fcleavage site and the M-gene primers.The NDV gene amplification respectively (Table

aNV=
Not available, -= Negative, T = Melting temperature, ICIP = Intra cerebral pathogenicity index, MDT = Mean Death Time, Commercial m vaccine produced by Veterinary Vaccine Production Center, Riyadh, Saudia Arabia.* Local NDV isolated from pigeon and characterized by Prof.Dr Saad Sharwi, Professor of Virology, Faculty of Veterinary Medicine, Banha university, Egypt

Table - 1
. Primers and probes used in the study