Isolation, Identification and Molecular Characterization of Highly Pathogenic Newcastle Disease Virus From Field Outbreaks

Asma Ashraf Muhammad Salah-ud-Din Shah Mudasser Habib Mujahid Hussain Shahid Mahboob Khalid Al-Ghanim About the authors

ABSTRACT

Newcastle disease (ND) is a major infectious disease of the poultry caused by a virulent strain of Avian Paramyxovirus - 1, that is a single strand non-segmented negative sense RNA virus. ND virus is major threat to the poultry industry in many countries of the world. The study was aimed to isolate and identify Newcastle disease virus (NDV) by using a haemagglutination inhibition (HI) test and reverse transcription-polymerase chain reaction (RT-PCR) assay. A total 100 samples of infected and dead birds were collected from different poultry farms. The weight of the birds was ranged 1000-1200g. The birds were divided into 3 groups. Haemagglutination assay (HA) was performed to detect the presence of NDV in suspension of infected homogenized tissues and it was found that HA is not the best method to detect the virus when it is in trace amounts. RT-PCR using NDV specific primers analyzed different clinical and postmortem samples. Reverse transcriptase polymerase chain reaction and specific primers was used for determining the presence of viruses. It was found that the virus was present in most of the infected samples except the serum of infected birds. During multiple sequence alignment (MSA) it was found that, our isolates have high homology (98%) with other reported NDV isolates. Phylogenetic analysis revealed that our isolate was closely related with viscerotropic velogenic types of NDV, which are highly pathogenic Newcastle disease virus.

Key words:
Newcastle disease; epidemic; molecular characterization; avian virus; RT-PCR

INTRODUCTION

Newcastle disease (ND) is an important viral disease of poultry and other bird species disregarding of sex and age [1[1] Alexander DJ (2003). Newcastle disease, other avian paramyxoviruses and pneumovirus infections. J Diseas Poultry 11: 63-99.

[2] Haque MH, Hossain MT, Islam MT, Zinnah MA, Khan MSR, Islam MA. (2010). Isolation and Detection of Newcastle disease Virus from field outbreaks in broiler and layer chickens by reverse transcription-polymerase chain reaction. Bangla J Vet Med 8(2): 87-92.

[3] Orsi MA, Doretto JrL, Camillo SCA, Reischak D, Ribeiro SAM, Ramazzoti A, Mendonca AO, Spilki FR, Buzinaro MG, Ferreira HL, Arns CW. (2010). Prevalence of Newcastle disease virus in broiler chickens (Gallus gallus) in Brazil. Brazil J Microbiol 41: 349-357.
-4[4] Iram N, Shah MS, Ismat F, Iqbal M, Rahman M (2013). Heterologous expression, characterization and evaluation of the matrix protein from Newcastle disease virus as a target for antiviral therapies. Applied Microbiology and Biotechnology. 98:1691-1701]. ND is a major cause of huge economic losses in various parts of the world [5[5] Aldous EW, Mynn JK, Banks J, Alexander DJ (2003). A molecular epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene. Avian Pathol 32: 239-256.-6[6] Diel DJ, Susta L, Garcia SC, Killian ML, Brown CC, Miller PJ, Afonso CL, (2012). Complete Genome and Clinicopathological Characterization of a Virulent Newcastle Disease Virus Isolate from South America. J Clini Microbiol 50(2): 378-387]. Newcastle disease virus (NDV) is belong to a group of the avian paramyxovirus - I [7[7] Yu L, Wang Z, Jiang Y, Chang L, Kwang J (2001). Characterization of Newly Emerging Newcastle Disease Virus Isolates from the People's Republic of China and Taiwan. J Clin Microbiol 39(10): 3512-3519.

[8] Choi KS, Lee EK, Jeon WJ, Kwon JH. (2010). Antigenic and immunogenic investigation of the virulence motif of the Newcastle disease virus fusion protein. J Vet Sci 11(3): 205-211.
-9[9] Qin Z M, Tan LT, Xu HY, Ma BC, Wang YL, Yuan XY, Liu WJ (2008). Pathotypical Characterization and Molecular Epidemiology of Newcastle Disease Virus Isolates from Different Hosts in China from 1996 to 2005. J Clin Microbiol 46(2): 601-611.]. The disease is characterized by an involvement of digestive, respiratory and nervous systems [10[10] Nanthakumar T, Kataria RS, Tiwari AK, Butchaiah G, Kataria JM (2000). Pathotyping of Newcastle Disease Viruses by RT-PCR and Restriction Enzyme Analysis. J Vet Res Commun 24: 275-286.-11[11] Tiwari AK, Katari RS, Nanthakumar T, Dash BB, Desai G (2004). Differential detection of Newcastle disease virus strains by degenerate primers based RT-PCR. J Comp Immunol Microbiol Infect Diseas 27:163-169.]. This disease can vary in nature from mild to severe, depending upon the type of the virus. In non-vaccinated chickens, the morbidity and death rates may be up to 100% each, depending upon the virulent intensity of NDV. In recent years, outbreaks have continuously occurred in Pakistan resulting in huge losses. Infected samples indicated the presence of virus identified by chicken embryonated egg inoculation and haemagglutination assay [2[2] Haque MH, Hossain MT, Islam MT, Zinnah MA, Khan MSR, Islam MA. (2010). Isolation and Detection of Newcastle disease Virus from field outbreaks in broiler and layer chickens by reverse transcription-polymerase chain reaction. Bangla J Vet Med 8(2): 87-92.].

NDV has a wide range of hosts range, inclusive of approximately 241 species [12[12] Madadgar O, Karimi V, Nazaktabar A, Kazemimanesh M, Ghafari MM, Dezfouli SMA, Hojjati P (2013). A study of Newcastle disease virus obtained from exotic caged birds in Tehran between 2009 and 2010. Avian Pathol 42(1): 27-31.] belongs to 27 orders out of which 50 orders of birds [13[13] Carrasco AOT, Rodrigues JNM, Seki MC, Moraes FE, Silva JR, Durigon EL, Pinto AA (2013). Use of reverse transcriptase polymerase chain reaction (RT-PCR) in molecular screening of Newcastle disease virus in poultry and free-living bird populations. Trop Anim Heal Prod 45:569-576.]. In various developing countries, ND is an endemic and thus have a high economic impact. Due to this disease, poultry industry is facing losses of billions of Rupees annually in Pakistan [14[14] Waheed U, Siddique M, Arshad M, Ali M, Saeed A (2013). Preparation of Newcastle Disease Vaccine from VG/GA Strain and its Evaluation in Commercial Broiler Chicks. Pak J Zool 45(2): 339-344.] and millions of dollars worldwide [15[15] Susta, L., Miller, P. J., Afonso, C. L., & Brown, C. C. (2010). Clinicopathological Characterization in Poultry of Three Strains of Newcastle Disease Virus Isolated From Recent Outbreaks. J Vet Pathol 48(2): 349-360.].

ND is a serious threat to the poultry industry [16[16] Narayanan MS, Parthiban M, Sathiya P, Kumanan K (2010). Molecular detection of Newcastle disease virus using Flinders Molecular detection of Newcastle disease virus using Flinders Technology Associates-PCR Technology Associates-PCR. J Veterin Arhiv 80(1): 51-60.]. The rate of mortality and morbidity of poultry in unvaccinated flock [2[2] Haque MH, Hossain MT, Islam MT, Zinnah MA, Khan MSR, Islam MA. (2010). Isolation and Detection of Newcastle disease Virus from field outbreaks in broiler and layer chickens by reverse transcription-polymerase chain reaction. Bangla J Vet Med 8(2): 87-92.] varies from 90-100%, depending upon the strain of ND virus [10[10] Nanthakumar T, Kataria RS, Tiwari AK, Butchaiah G, Kataria JM (2000). Pathotyping of Newcastle Disease Viruses by RT-PCR and Restriction Enzyme Analysis. J Vet Res Commun 24: 275-286.]. The outbreaks of ND are regularly reported from all continents of the world [17[17] Munir M, Shabbir MZ, Yaqub T, Shabbir MAB, Mukhtar N, Khan MR, Berga M. (2012). Complete Genome Sequence of a Velogenic Neurotropic Avian Paramyxovirus 1 Isolated from Peacocks (Pavo cristatus) in a Wildlife Park in Pakistan. J Virol 86(23): 13113-13114.]. "An intermittent form of ND present in Pakistan throughout the year, only a limited number of cases are reported annually. A severe outbreak of ND occurred during 2012 at Jallo Wildlife Park in Lahore, Pakistan, caused by APMV- 1 serotype. Within a week, it took the lives of approximately 190 peacocks with a 100% mortality rate and 50% loss of the susceptible birds. Isolation of virus and serological diagnostics, such as HI Test, ELISA and molecular diagnostic tests like real time PCR confirmed the presence of velogenic Newcastle disease virus" [18[18] virus isolated from outbreaks in Egypt during 2006. Virol J 8(237): 01-04. ]. The study was aimed to isolate and identify Newcastle disease virus (NDV) by using a haemagglutination inhibition (HI) test and reverse transcription-polymerase chain reaction (RT-PCR) assay from the dead birds from the poultry farms of Punjab province of Pakistan.

MATERIALS AND METHODS

Collection of infected samples:

The samples were collected during 2013 from the poultry farms of different areas of Punjab province, Pakistan. A total 100 samples of infected and dead birds were collected from different poultry farms. The weight of the birds was ranged 1000-1200g. The birds were divided into 3 groups. Post-mortems were conducted and infected tissues, i.e. intestine, proventriculus, spleen, lungs and trachea were collected from the dead chickens. After collection, the samples were transported on ice packs and stored at -20 o C to -25 o C for further processing.

Virus isolation in embryonated eggs:

"Virus isolation was carried out as described by [19-20]. Eggs from healthy and ND-seronegative chickens were used which were conventionally raised. Tissue was homogenized as a 10% (w/v) suspension in PBS containing antibiotics (streptomycin, penicillin). After centrifugation, 0.1 ml supernatant was inoculated into the allantoic cavity of 9- to 11-day-old embryonated chicken eggs. Allantoic fluid from dead embryos or in live embryos after 72 hours of incubation was collected and NDV was detected by HA test".

Haemagglutination assay (HA):

The titer of the virus in allantoic fluid or tissue homogenate was determined by hemagglutination assay as described by [20-22].

Reverse transcription-polymerase chain reaction:

Allantoic fluid was used for RNA extraction by following the protocol of Favor Prep. TM Viral Nucleic Acid Extraction mini kit. RNA extracted by using 150µl of allontoic fluid, 570µl of VNE Buffer, 570µl of ethanol, 500µl Wash Buffer 1 and 750µl of wash buffer. Each RNA sample was dissolved in 40 µl sterile RNAse-free water and stored at -70°C.

The complementary DNA was synthesized using 2µl of the total RNA. 1 µl of random primer hexamers, 9 µl of nuclease free water, 4.0 µl of 5X Reaction Buffer, "1.0 µl of Ribo-Lock RNase Inhibitor (20u/ µl), 2.0 µl of 10 mM dNTP Mixture, 1.0 µl of Revert Aid M-MuL V Reverse Transcriptase (200u/ µl) was added and the mixture centrifuged and then was incubated for 60 min at 42ºC". The reaction terminated after heating it at 70ºC for 5 minutes, then after briefly spin the tube before cDNA was used for PCR amplification.

RT-PCR was performed as described by [25[24] Yi J, Liu C (2011). Detecting Newcastle disease virus in combination of RT-PCR with red blood cell absorption. Virol J 8: 202-208.]. NDV-F /NDV-R primers were selected to amplify a 202 bp fragment of the F gene including the cleavage site. "Primer sequences are shown in Table-1. PCR was carried out in a 50 μl reaction containing 5.0 μl 10X buffer, 2.0 μl 25 mM MgCl2, 2.0 μl 10 mM dNTP, 0.2 μl Taq, 0.8 μl NDV-F primer (100 pmol), 0.8 μl NDV-R primer (100 pmol), 1.5 μl cDNA, and 37.7 μl DEPC was added to each tube. The amplification profile started with one cycle at 94°C for 2 min. followed by 35 cycles of 94°C for 15 sec, 48°C for 30 sec and 72°C for 30 sec and final extension of 72°C for 10 min. Sterile RNAse free water or tissue samples from animals slaughtered on day 0, were used as negative controls".

Table 1:
Specific primers used for RT-PCR

Agarose Gel Electrophoresis:

PCR products were subjected to agarose gel electrophoresis. For this 10 μl of PCR product along with 2 μl of 6X loading dye were mixed and loaded on 1.5% agarose gel along with 100bp ladder. The gel was run in 1X TAE buffer till the dye reach near other end. At the end, the gel was stained with ethidium bromide and observed under UV light.

Nucleotide sequence analysis:

The PCR product was purified and its nucleotide sequence was determined using both forward and reverse primers. The nucleotide sequence was analyzed using BLAST software and its homology was searched against available nucleotide sequences from GenBank. Phylogenetic analysis was performed using a partial nucleotide sequence of the fusion protein gene and phylogenetic tree was drawn on the basis of observed divergence using software DNAMAN by Lynnon Biosoft, Canada.

RESULTS AND DISCUSSION

Clinical signs and symptoms:

The infected broilers showed clinical symptoms of depression, dizziness, gasping, paralysis of neck, legs or wings and loss of appetite. Swelling of the eyes and discharge from eyes were also observed. Greenish yellow colored diarrhea was very prominent. Similar signs and symptoms were also reported by [1, 11[11] Tiwari AK, Katari RS, Nanthakumar T, Dash BB, Desai G (2004). Differential detection of Newcastle disease virus strains by degenerate primers based RT-PCR. J Comp Immunol Microbiol Infect Diseas 27:163-169.].

Postmortem lesions:

Typical Postmortem lesions are shown in Figure 1. During postmortem examination of dead birds, it was observed that the necrotic lesions were present in the mucosa of intestine, proventriculus and gizzard. Hemorrhagic lesions were very prominent in the mucosa of the proventriculus. "The air sacs were filled with whitish translucent material, but lungs were normal in size. Enlarged spleen and liver were also observed". The similar postmortem lesions in birds [16 [16] Narayanan MS, Parthiban M, Sathiya P, Kumanan K (2010). Molecular detection of Newcastle disease virus using Flinders Molecular detection of Newcastle disease virus using Flinders Technology Associates-PCR Technology Associates-PCR. J Veterin Arhiv 80(1): 51-60.and 23[22] Nadeem Y, Chaudhary TM, Shah M S, Ashraf A (2004). Oil adjuvanted newcastle disease vaccine production using local viral isolates. Proceedings of 24th PakiCong Zool pp: 51- 56.].

Fig 1:
(a) Typical conjunctivitis (b) Postmortem examination showing hemorrhages of intestine (c) Enlarged spleen and hemorrhages in mucosa of proventriculus, along with normal organs for comparison.

Fig 2:
Agarose gel electrophoresis showing RT-PCR results.

Detection of NDV by Haemagglutination Assay:

Haemagglutination assay (HA) was performed to detect the presence of NDV in suspension of infected homogenized tissues. Results are shown in Table 2. HA is not the best method to detect the virus when it is in trace amounts. When the homogenized viral suspension was allowed to multiply in allantoic fluid of embryonated eggs, then HA can detect successfully the virus in allantoic fluid. [5[5] Aldous EW, Mynn JK, Banks J, Alexander DJ (2003). A molecular epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene. Avian Pathol 32: 239-256.] also found that HA is one of the rapid and successful techniques to detect the NDV.

Table 2:
Comparison of results of Haemagglutination assay (HA) and RT-PCR

Confirmation of NDV by RT-PCR:

RT-PCR using NDV specific primers analyzed different clinical and postmortem samples. Results of comparison of both techniques are shown in Table 2. It was found that the virus was present in most of the infected samples except the serum of infected birds. It might be because virus presence in the blood is for short periods during infection. RT-PCR is very sensitive technique to detect the presence of NDV in different tissue samples, even if the virus was present in minute quantity [2[2] Haque MH, Hossain MT, Islam MT, Zinnah MA, Khan MSR, Islam MA. (2010). Isolation and Detection of Newcastle disease Virus from field outbreaks in broiler and layer chickens by reverse transcription-polymerase chain reaction. Bangla J Vet Med 8(2): 87-92., 24[23] Ashraf A, Shah MS (2014). Newcastle Disease: Present status and future challenges for developing countries. Afr J Microbiol Res 8(5): 411-416.]. Moreover, [25[24] Yi J, Liu C (2011). Detecting Newcastle disease virus in combination of RT-PCR with red blood cell absorption. Virol J 8: 202-208.] " reported one-step RT-PCR test coupled with restriction enzyme assay (REA) as fast and specific method for the detection and typing of APMV-1 from field samples".

Nucleotide sequence analysis

During multiple sequence alignment (MSA) it was found that, our isolates have high homology (98%) with other reported NDV isolates. Phylogenetic tree and multiple sequence alignment are shown in Figure 3 and Figure 4. Phylogenetic analysis revealed that our isolate was closely related with viscerotropic velogenic types of NDV, which are highly pathogenic Newcastle disease virus. It was found that our isolate (ND-NIAB-Pak) was grouped in a different cluster, which was differentiated from other reported NDV isolates. Moreover, it was revealed that our isolate is closely related with isolates of NARC-Pak, Israel, Kudus and Sragen, while it was distantly related with isolates of Iran, Guangdong, Japan, India and USA. [5, 26-27] did epidemiological investigations of NDV by phylogenetic analysis using a partial nucleotide sequence of a fusion protein gene. Our findings are in line with their results.

Fig 3:
Phylogenetic tree showing relationships among reported isolates of NDV and new Pakistani isolate (ND - NIAB) based on partial nucleotide sequence of Fusion gene.

Fig 4:
Multiple sequence alignment of partial fusion gene (203bp) of different reported NDV isolates along with NDV isolate of Pakistan (ND-NIAB-Pak).

CONCLUSION

It was concluded the virus was present in most of the infected samples except the serum of infected birds.

The multiple sequence alignment (MSA) exhibited, that these isolates have, high homology (98%) with other reported NDV isolates.

Phylogenetic analysis revealed that our isolate is closely related with viscerotropic velogenic types of NDV, which are highly pathogenic Newcastle disease virus.

ACKNOWLEDGMENTS

The authors would like to express their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the Research Group Project No. RG-1435-012.

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  • Erratum
    In Article "Isolation, Identification and Molecular Characterization of Highly Pathogenic Newcastle Disease Virus From Field Outbreaks", with the number of DOI: http://dx.doi.org/10.1590/1678-4324-2016160301, published in journal Brazilian Archives of Biology and Technology, vol. 59, the 01 page.
    that read:
    "Asma Ashraf1; Mohammad Slah U Din2; Muhammad Habib1; Mujahid Hussain2; Shahid Mahboob1,3*; Khalid Al-Ghanim3;
    1Government College University Faisalabad, Faisalabad, Pakistan; 2Nuclear Institute of Agriculture and Biology Faisalabad, Pakistan; 3King Saud University, Department of Zoology, College of Science, P. O. Box 2455, Riyadh, Saudi Arabia. "
    Read:
    "Asma Ashraf1; Muhammad Salah-ud-Din Shah2; Mudasser Habib2; Mujahid Hussain2; Shahid Mahboob2,3*; Khalid Al-Ghanim3;
    1Government College University Faisalabad, Faisalabad, Pakistan; 2Nuclear Institute of Agriculture and Biology Faisalabad, Pakistan; 3King Saud University, Department of Zoology, College of Science, P. O. Box 2455, Riyadh, Saudi Arabia."

Publication Dates

  • Publication in this collection
    2016

History

  • Received
    15 Jan 2016
  • Accepted
    25 Apr 2016
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