Proventriculus, caecal tonsil, spleen, brain were collected from twelve Newcastle disease (ND) suspected layer flocks from Namakkal. The tissue samples were processed and Haemagglutination test (HA) was conducted. The tissue sample were subjected to RT-PCR targeting M gene of RT-PCR (product size-145bp) and those found positive were used for virus isolation. Virus isolation was done in 9-11 day old embryonated chicken eggs and the allantoic fluid of the embryos that showed characteristic lesion NDV like haemorrhage in occipital region and extremities were harvested and was screened by HA, HI and FPCS for confirmation of the virus. Out of eight isolates, three isolates were characterised as mesogenic by Intra cerebral Pathogenecity index and mean death time. Two mesogenic isolates TN/NKL/F1, TN/NKL/S2 were subjected to detail study. Based on the fusion protein cleavage site sequence the virulence nature of the NDV was confirmed. The coding sequence (cds) of FPCS , mesogenic viruses had an amino acid sequence of 112 RRQKRF 117. For HN gene study the allantoic fluid was ultra centrifuged at 32000 rpm for 3h and the viral pellet was used for RNA extraction. The amplified product size of 1892bp was purified and cds was subjected to sequencing which comprised of 577 amino acids. The two mesogenic isolates were found to have 99% homology with Komarov and 93% homology with R2B. The two isolates had Arginine at 362 position whereas Komarov has Glycine at that position.
The current scenario of poultry is one of the fastest growing sectors in india and growth rate is 6-8% in layers, 10-12% in broilers. Newcastle disease causes huge economic losses to the commercial poultry farmers round the world. (Diel et al., 2012).To control the disease in commercial layer farms live lentogenic and mesogenic vaccines are being used (Dey et al., 2012; Munir et al., 2012; Balachandran et al., 2014).Mesogenic live vaccines are producing virulent form of ND due to immuno suppression and other disease like infectious Bursal disease, Marek’s disease, and feed related mycotoxins due to this immunosuppressive conditions live mesogenic vaccines some time revert to virulence and producing disease.(Alexander, 1997; Jaganathan, 2015). The causative agent of the disease is Newcastle disease virus (NDV) also designated as Avian Paramyxovirus serotype1 , which is a negative sense single stranded RNA virus and belongs to the genus of avulavirus ,within the family of paramyxoviridae (Alexander, 2011).
Genome of NDV size is approximately 15.2 kb (15186 nucleotides) (Alexander and Senne, 2008). The RNA genome contains six major genes that encode the structural proteins in the order 3’-NP-P-M-F-HN-L-5’ (Chambers et al., 1986a; Wilde et al., 1986) as well as two non structural proteins may result from differential initiation (McGinnes et al., 1988) or transcriptional editing of the P gene m-RNA (Steward et al., 1993). The surface of NDV particles contains two important functional glycoproteins such as fusion (F) and haemagglutinin neuraminidase (HN) proteins. The F protein mediated both virus-cell and cell–cell fusion (Hernandez et al., 1996). The HN protein of NDV was a multifunctional type 2 homotetrameric glycoprotein; it possessed both the receptor recognition and neuraminidase activities associated with the virus (Lamb and Kolakofsky, 1996). Thus, the HN protein plays an important role in viral infection. It has recently been shown that the cleavability of the F protein alone does not convert an otherwise non-pathogenic strain into a highly virulent pathotype (Panda et al., 2004). The moderately virulent, or mesogenic, viruses usually cause severe respiratory disease, followed by nervous signs, with mortality up to 50% or more (Alexander et al., 1985). ICPI values ranged from 0.0 to 0.7 are lentogenic, 0.7-1.5 is mesogenic more than 1.5 is velogenic (Cattoli et al., 2011). Mean Death Time (MDT) is the mean time in hours for minimum lethal dose to kill the embryos (9-11 day old embryonated eggs) which can be used for classifying the strains. If embryo dies less than 60 hrs is velogenic, embryo survives more than 90hrs is (lentogenic), embryo died 60-90 hrs is mesogenic (Alexander, D. J., 2003; OIE, 2012).
Identification of NDV can be done through HA and HI and molecular techniques like RT-PCR (Mohammed et al. (2013).The modern method to distinguish NDV pathogenicity is by RT-PCR followed by DNA sequencing and this method is more accurate (Seal et al., 1995; Marin et al., 1996).
Materials and Methods
Isolation and Identification of Virus
The isolates obtained in the present study were designated as TN/NKL/F1,TN/NKL/F3, TN/NKL/F7,TN/NKL/S2,TN/NKL/P3,TN/NKL/T-84,TN/NKL/S6,TN/NKL/S3.
Processing of Samples
All the tissue sample processed according to the protocol of OIE (2012) and screened for NDV by HA and RT-PCR of M- gene. Those tissue sample that were found positive for NDV by HA and RT-PCR for M-gene were further subjected to virus isolation in embryonated chicken eggs. The positive processed tissues sample supernatant was treated with antibiotic solution. 10µl of antibiotic and antimycotic solution (Penicilin-1000 units, Streptomicin-100 µg, Amphotericin- 0.25 µg/10µl) was added to 1ml of 50% tissue suspension and incubated at 37ºc for 1hour. The antibiotic treated tissue material was filtered through 0.45µ syringe filter and used for virus isolation. The NDV was Isolated as per standard procedures mentioned in the terrestrial manual of OIE (2012).0.1 ml of processed tissue suspension was inoculated via allantoic route of 9-11 day old Specific Antibody Negative Egg (SAN- ECE). The ECE were incubated at 37ºc and candled daily up to 5 days. The embryo was chilled after death at 4ºc. Allantoic fluid was harvested and stored at -80ºc. The standard HA and HI test was carried out to find out the presence of NDV in the harvested allantoic fluid as per the standard procedures mentioned in the Terrestrial Manual of OIE (2012).
Virulence Assessment of NDV Isolates
The virus isolates were characterised for its Pathogenecity by Intra Cerebral Pathogenecity Index (ICPI) in day old male layer chicks and MDT in 9-11 day old embryonated chicken eggs, following standard procedures mentioned in the Terrestrial Manual of OIE (2012).
Concentration of Virus by Ultracentrifugation for HN Gene Amplification
Collected allantoic fluid was subjected to ultracentrifugation at 32000 rpm for 3hours in a Beckman-Coulter ultracentrifuge. The virus pellet was eluted in 500 µl PBS.
Viral RNA Extraction
The viral RNA was extracted using TRIZOL-CHLOROFORM extraction method as per manufacture instructions of TRIZOL (Takara-RNA isoplus). Reverse transcription carried out using (High Capacity cDNA-Reverse Transcription Kit Cat # 4368814, Applied biosystems by Thermo Fisher Scientific) following manufactures instructions.
Amplification of Fusion Protein Cleavage Site (FPCS) by PCR
Degenerate oligonucleotide primers suggested by Seal et al. (1995) was used for detection FPCS in amplification. RNA extracted from fresh allantoic fluid.
Amplification of HN Gene by RT-PCR
HN gene at 1892bp was amplified using the primers used by(Peeters et al., 2001; Ponnusamy et al., 2009) Sense (5’-GTA GGC TAG CAA GAG AGG CCG CCC CTC AAT -3’ and antisense-5’-CGA GCC CGG GCC GGC ATT CGG TTT GAT TCT TG-3’ ). Components of PCR mix for 10µl reactions used such as PrimeSTAR® Max DNA Polymerase – 2x (Takara cat#R045A-Containing 2 mM Mg2+ and 0.4 mM each dNTP) -5 µl, Template-2 µl(cDNA), Primers (1µl of each forward and reverse), Nuclease Free Water (2µl). Amplification was carried out with an initial denaturation at 980c for 5 minutes followed by a sequence of 35 cycles (denaturation-98 c for 10 sec, annealing -58 for 15 sec, extension-72 C for 60 seconds) and final extension hold at 72 C for 7 min finally stored at -20 C. PCR products were electrophorosed using 1% gel visualized under Gel Doc (Bio Rad).The PCR product were purified using (EZ-10 spin column DNA Gel extraction kit, Biobasic).
The purified product of HN and FPCS gene of mesogenic isolates were sequenced through sequence scanner software 2 v2.0, ABI, USA (M/s Eurofins genomics Pvt Ltd, Bangalore, India). Internal primers (F-5AGTGTGAGTGCAACTCCCTTA-3), (R-5GACTCCAGTAACACACGAGTT-3) were designed to amplify the nucleotide sequence of HN gene by Sangers Sequence Method and substituted. Sequence analysis of HN gene and FPCS were carried out using the Bio-Edit software (North Carolina University, USA). The parameters used for sequence analysis were multiple alignment (clustal W), sequence identity plotter and sequence identity matrix at both nucleotide and amino acids level.
Out of eight isolates, 7 isolates killed the embryos. Dead embryos on examination revealed the characteristic lesions specific for NDV like haemorrhages in occipital region, muscles and extremities as compared to the control which revealed no lesions. The embryo lesions are depicted in Fig.1. Presence of virus was confirmed by HA, RT – PCR of M-gene in tissue sample and HA, HI, RT-PCR of FPCS in case of allantoic fluid harvested from ECE. RT-PCR for M gene (product size-145bp) in tissue sample depicted Fig.2. RT-PCR for FPCS (product size -254bp) in allantoic fluid is depicted in Fig.3. Based on ICPI value and MDT out of 8 isolates, 3 isolates (TN/NKL/S2, TN/NKL/T-84, TN/NKL/F1) were characterised as mesogenic strains. The mean death time of the isolates was assessed and the results are presented in Table 1. The highest virus dilution that killed all the embryos was 1 in 10-6 and 1 in 10-7. Two mesogenic isolates TN/NKL/F1, TN/NKL/S2 were subjected to detailed study.
Fig. 1: Lesions of virus infected embryos
|Fig. 2: RT-PCR of M – gene (145bp)
M-100bp DNA ladder
T2-TN/NKL/F1 Brain and spleen
T3-TN/NKL/S3 Caecal tonsil
T4- TN/NKL/S6 Spleen
|Fig. 3: RT–PCR of FPCS (254bp)
Lane M-100bp-DNA ladder
Lane 3- R2B (P.C)
Table 1: MDT and ICPI value the different isolates
N.C – not carried out
Sequence Analysis of Fusion Protein Cleavage Site (FPCS)
The product sizes for FPCS were approximately 254 bp in length. The two mesogenic isolates. (TN/NKL/F1, TN/NKL/S2) were found to have FPCS of 112RRQKRF117 confirming these two isolates are virulent.
Sequence Analysis of HN – Gene
The RT-PCR amplicons of the HN gene of two mesogenic isolates (TN/NKL/S2 and TN/NKL/F1) are depicted in Fig.4. The product sizes for HN were approximately 1892. Internal primer (693 bp) was designed to amplify the missing nucleotide sequence of HN gene by Sanger sequence method and substituted. Amplifies product is depicted in Fig. 5. The result of sequence analysis of HN gene summarised below for two isolates. The two virus isolates were found to have amino acid sequence 360 KPRRFGG366 and Komarov vaccine strain have amino acid sequence of 360 KPGRFGG 366 . Two virus isolates have Arginine(R) in 362th position. While Komarov strain having Glycine (G) instead of Arginine.
|Lane M-1 Kb DNA ladder
Lane-1 – TN/NKL/S2
Lane-2 – Vaccine-R2B (P.C)
Lane-3 – TN/NKL/F1
Fig. 4: RT- PCR of HN gene (1892bp)
|Lane M-100 bp DNA ladder
Lane-1 – TN/NKL/S2
Fig.5: Internal Primer for HN gene (693bp)
|Position||Accession No.KT-445901.1 Komarov-HN||Study – HN (Isolates)|
Amino Acid Changes
|Position||Accession No. ALN 97032.1 Komarov-HN||Study-HN|
Newcastle disease is a commonly reported disease in vaccinated commercial flocks in Tamil Nadu (Mohan et al., 2005; Balachandran et al., 2014). Although proper vaccination protects the birds from clinical disease, it does not prevent virus replication and shedding, which results in a source of infection (Chukwudi et al., 2012). This results in outbreaks that are continuously occurring. To overcome this problem poultry producers are using different combinations of live and killed vaccines in a flock. . The most commonly used mesogenic vaccines are Komarov and R2B. Both these vaccines are capable of causing disease in immuno suppressed birds (Alexander, 1997; Jaganathan, et al., 2015) and there will also be circulation of vaccine strains in the poultry population. A Study of comparison between the field and vaccine virus sequence revealed unique genomic and amino acid differences in important antigenic regions of F and HN genes which can be targeted for site directed mutagenesis to evaluate the impact of these substitutions on virus pathogenecity reported by Jakhesara et al. (2014).
Analysing of the whole HN gene reveals the following facts. This gene has only one long reading frame. This starts at the ATG codon at nucleotides 6412 to 6415 and continues to a termination codon at nucleotides 8142-8145 of the whole genome. This result is in accordance with the findings of Ke, G et al., 2010. HN protein is a type II homotetrameric glycoprotein with a monomer length of 577 amino acids for most NDV strains. The ability to bind a sialic acid containing receptor is one of the functions of the HN protein and plays a key role in the initial steps of the NDV life cycle. Several amino acid residues R-174, Y-526, E-401 and R 416 in HN protein were reported to be involved in the activity of sialic acid binding (Connaris et al., 2002). The salient features of HN protein include the following conserved amino acids –R-174, I-175, D-198, K-236,R-416,R-498,Y-526 and E-547 (Dey et al., 2014). In the present study the HN gene of two mesogenic isolates having 577 amino acids and coding sequence of 1734 nucleotides. However, the HN gene of ND virus reported to have different lengths of coding sequence comprising of 571,577 581,616 amino acids (Sakaguchi et al., 1989). The amino acid sequence of the HN glycoprotein of NDV has 14 cysteine residues, two of which are non conserved , first cysteine (amino acid 6) and the second cysteine (amino acid 123) while the rest are absolutely conserved-amino acid 172, 186,196,238 , 247,251, 344, 455, 461,465,531,542 (Ponnusamy et al., 2009). Proteins with mutations in the non–conserved cysteine residues, formed antigenically mature oligomers which were transported to the cell surface like wild type (McGinnes and Morrison, 1994).
In the present study also all the conserved cysteine residues were unaltered in the HN protein of the two isolates. First cysteine was replaced by serine and the second cysteine was replaced by tryptophan. The amino acid sequence of the two mesogenic isolates were compared with the available mesogenic vaccines sequence and already studied NDV isolates at Gene Bank (KJ636208.1, HQ902590.1). Both the study isolates (TN/NKL/S2,TN/NKL/F1) under study were found to show 99% homology with Komarov and other already studied previous NDV isolates in Namakkal (NCBI-Blast) , 93% with R2B strain. There was a wide variation in the amino acid sequence of HN gene of Komarov and R2B about 29 amino acids. In the present study there were 41 amino acid variations between the isolates and R2B. Only one amino acid variation at position 362, arginine instead of glycine was found in the isolates. This clearly indicates that the isolates are more related to Komarov strain than R2B. Sequence identity matrix for HN gene of isolates is depicted in Fig.6 and Fig.7.
Fig.6: Sequence identity matrix for nucleotides of HN gene
Fig.7: Sequence identity matrix for amino acids of HN gene
However when such homology study was done with F protein by Anand et al. (2008) there was 100% homology between the mesogenic isolates and the mesogenic vaccine strain. This indicates that the F gene is more stable than the HN gene as already reported by Kattenbelt et al. (2006). The reason for these observed difference are unknown, but may be due to pressure by the immune system. Both the mesogenic isolates showed similar value in the ICPI (1.45 and 1.43). Similar result in the sequence study of F protein and HN protein. This may be due to the fact that the isolates were from out breaks from adjacent farms caused by the same ND virus. Since the homology of the virus is 99% with that of Komorov. There is every possibility of the vaccine virus after few point mutations has caused the disease.
Improper implementation of biosecurity measures and use of live mesogenic vaccines in birds without paying attention to the factors contributing to immuno suppression can lead to ND outbreak. Mesogenic vaccines are the only choice for escaping from virulent ND in endemic area. Hence judicial use of mesogenic vaccine following proper vaccination schedule and routine elimination of factors contributing to immuno suppressive conditions in flocks will decrease the incidence. For future study whole genome of field mesogenic isolates will generate more information.