INTRODUCTION:

Poultry industry is important sector in Indonesia as it serves 65% of protein supply and provides job vacancy for 12 million people. This sector is made up of six sub-sectors (feed, breeding, broiler and layer farms, slaughterhouse and processing) with estimated worth more than 34 billion USD. Indonesian primarily consume broiler and layer chickens but duck and quails form the whole poultry product consumption rate too. In Indonesia, rearing activities are differentiated into three types such as extensive traditional system, semi-intensive system, and intensive system^1,2. They are classified according to the amount of the company capital. All these farming systems have similar problem relate to outbreak of viral disease. Common viral disease emerge in poultry (both broiler and layer) need to be concerned are caused by Toravirus, Parvovirus, Entero-like virus, Adenovirus, Calici virus, Avian Influenza virus, Newcastle Disease virus, Avian Reovirus, Avian Enchepalomyelitis, and Marek’s disease virus³. Moreover, World Organization for Animal Health (OIE) has published some viral disease circulate in poultry such as Avian infectious bronchitis², Duck virus hepatitis, Gumboro disease and Turkey rhinotracheitis⁴. These viral diseases cause annual lost due to the decrease of production generates stunting and mortality^3,5,6,7. This condition become the problem for companies to expand the business abroad, because it does not meet standard for biosecurity for poultry production.

Newcastle Disease Virus (NDV) or Avian Paramyxovirus serotype-1 (APMV 1) is the causative agent for Newcastle Disease⁸. It is a contagious disease causes annual outbreak in poultry industry which has six structural proteins and a single stranded negative sense RNA genome^9,10. NDV’s structural protein consists of Nucleoprotein (N), Phospoprotein (P), Matrix protein (M), Fusion protein (F), Haemagglutinin-Neuraminidase protein (HN), and Polymerase protein (L)¹¹. Among these proteins, Fusion protein has the important role in molecular-genotyping of NDV as the virulence virus confirmed by the existence of multiple-basic amino acid sequence located on cleavage site of Fusion protein¹². Fusion protein is type I integral protein located on the surface of the virion which leads to specific antibody synthesis (immunogenic)^12,13. Since Avian Paramyxoviruses differ in many serotypes, particularly, APMV 1 has antigenic variation which commonly isolated from pigeon so-called Pigeon Paramyxovirus serotype 1 (PPMV1)¹⁴.

NDV firstly reported in Indonesia on 1927 by Kraneveld¹⁴. Since then, it becomes major problem in poultry industry even though annual vaccination as prevention has been conducted¹⁵. Many researches had been conducted to analyze the possibility of seed vaccine candidate isolated from the field NDVs^10,16. Up till now, conventional seed vaccines were isolated from chicken (i.e.; La Sota, Komarov, Hitchner). In Indonesia, research related to NDV obtained from pigeons has never been conducted before, because NDV outbreak, all of them, occurred in poultry farm. This research is a pilot project to analyze the possibility of NDV obtained from pigeons as sub-unit vaccine candidate using molecular in-silico methods.

MATERIAL AND METHODS:

Ethical approve:

This research has been approved by the ethics commission of experimental animals of Faculty of Veterinary Medicine, Airlangga University.

Sample isolation and identification:

Samples were collected from domestic pigeons (Columba livia domestica) suspected to be infected with low virulent NDV and showed several symptoms such as respiratory disturbance, limping, loss of appetite and subclinical enteric disturbance/diarrhea sold in live bird market in Surabaya. The samples were euthanized through cervical dislocation method. Organs from each sample, such as brain, caeca tonsil, proventriculus, intestine, lien, lungs, and liver were obtained. Each of organs were homogenized then inoculated into 9-11 old days Specific Antigen Free (SAN) embryonated eggs. They were incubated in 37°C cabinet for 5 days. Observation was conducted once in 24 hours post infection (hpi) and the mortality of embryonated eggs were recorded. Death embryo were stored in -97°C. After incubation period, all living embryonated eggs were stored in -97°C then on the following day, all inoculated embryonated eggs were processed into Hemagglutination assay (HA assay). Before HA assay was conducted, the allantoic fluid of each embryonated eggs was obtained.

HA assay was employed to detect the presence of Hemagglutinin protein in the samples. Positive control used was NDV vaccine containing La Sota. Then positive HA assay samples were processed into Hemagglutination Inhibition assay (HI assay) using Newcastle Disease specific serum (titer of 2⁶). Samples showed positive in both assays were processed into molecular identification.

Molecular identification:

The allantoic fluid of positive samples were extracted according to RNeasy Mini Kit (QIAGEN, Cat No./ID: 74104). Obtained RNA then employed to Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) amplified partial gene of Fusion protein (768 bp) (Table 1). The RT-PCR was set up as Lid on 95°C; pre-denaturation 94°C for 10 minutes; denaturation 94°C for a minute; annealing 56°C for a minute; extension 72°C for 2 minutes; prolong extension 72°C for 10 minutes. The cycle was repeated for 40 times. In the end of amplification, the temperature was remained as 4°C. The amplicon was visualized on 1% agarose gel electrophoresis. The electrophoresis machine was set as 100V for 60 minutes and used marker 100 bp cDNA ladder (Invitrogen). Samples indicated the present of band were processed into sequencing according to QIAquick^® PCR Purification Kit (Qiagen, 28104). After purification, the amplicon was labelled according to BigDye^® Terminator Sequencing RR-100. After labelling the amplicon, the sequence of the nucleotide was read by ABI 310 xL GENETIC ANALYZER (Applied Biosystem). The obtained sequence was analyzed using Biological Sequence Alignment Editor (BioEdit) ver. 7.2.5.

Table 1. Sequence of primer amplify partial gene of fusion protein of Newcastle disease virus.

Sequence 5’-3’		Product (bp)
Forward	TTC CTG CTT GAG CGG CAA TA	768
Reverse	AAG CGG TAG AAC GGA GGT TG	768

B cell epitopes prediction:

The sequence were processed into B cell epitope prediction according to Kolaskar-Tongaonkar antigenicity and Emini surface accessibility^17,18,19. Selected epitopes were processed into further immunogenicity analyses using Vaxijen¹⁶. The immunogenic peptides sequences were processed into Allertop and ToxinPred to pick the one which does not cause allergic and intoxication toward the poultry^20,21.

RESULT:

Sample collection and isolation:

Four pigeons showed subclinical signs of NDV infection was collected from Surabaya. They were euthanized using cervical dislocation method then employed to virus isolation. To confirm the present of NDV, the samples were processed to HA assay. It was conducted to detect the present of Hemagglutinin protein²². Positive HA assay was processed to HI assay to confirm that isolated samples was NDV (Table 2). Two out of four samples were serologically confirmed as NDV. After that, molecular identification was conducted to affirmed that isolated samples were NDV.

Molecular identification:

Before processing the samples into RT-PCR, their RNA was extracted then processed to RT-PCR. The amplicons were visualized through agarose gel electrophoresis (Figure 1). The sequences of the samples were analyzed using Biological Sequence Alignment Editor (BioEdit) ver. 7.2.5.

B cell epitope prediction:

Amino acids from each samples were analyzed using Kolaskar-Tongaonkar antigenicity and Emini surface accessibility to obtain the B cell epitopes^18,19. The epitopes were processed into further immunogenicity analysis using Vaxijen, then Allertop and ToxinPred to pick the peptides sequence which does not cause allergy and intoxication reaction respectively^16,20,21.

DISCUSSION:

Newcastle disease virus infects many birds such as chickens, cockatoo, water fowl species, and pigeons^23,24,25. Among these host systems, the record showed that NDV causes panzootic in poultry (Miller et al., 2015) and pigeons^7,14,26. According to the HA and HI assays, two samples were confirmed as Newcastle Disease Virus; Pigeon/Surabaya/2019/01 and Pigeon/Surabaya/2019/03. The possibility of cross reaction is existed among NDV from different genotypes, which means HI assays will generate positive result even though the specific antiserum origin belongs to different genotype compared to isolated NDV. This founding revealed the possibility to design vaccine from vary genotype to protect poultry from other NDV outbreak. The positive samples were processed into RNA extraction then processed into Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR). According to visualization, they were molecularly confirmed to be NDV (Figure 1). Further analyses were conducted to reveal peptides which has potency to become subunit vaccine candidate, because it is very important to preserve vaccine which is not only capable to trigger specific immune response, but also safe, and not causing allergy and poisoning. According to the analyses, both samples had possibility to become subunit vaccine candidate. Peptides were possible to cause no harmful for the poultry are Swvyihllstf, Ctnvclseiqllhsfa, and Vrpcmvivrl obtained from Pigeon/Surabaya/2019/01 (Table 3). Whilst Pigeon/Surabaya/2019/03 generated two peptides which possible to become subunit vaccine candidate; NLTGRKRRTVG and SDREYSQAIAR (Table 4).

Table 2. Result of NDV isolation and identification.

Sample	Organ	HA titer	HI titer
Pigeon/Surabaya/2019/01	brain	2⁸	+
	caeca tonsil	-	-
	proventriculus	-	-
	intestine	2⁸	+
	lien	-	-
	lungs	-	-
	liver	-	-
Pigeon/Surabaya/2019/02	brain	-	-
	caeca tonsil	-	-
	proventriculus	-	-
	intestine	-	-
	lien	-	-
	lungs	-	-
	liver	-	-
Pigeon/Surabaya/2019/03	brain	2¹⁰	+
	caeca tonsil	-	-
	proventriculus	2¹⁰	+
	intestine	2¹⁰	+
	lien	-	-
	lungs	-	-
	liver	-	-
Pigeon/Surabaya/2019/04	brain	-	-
	caeca tonsil	-	-
	proventriculus	-	-
	Intestine	-	-
	lien	-	-
	lungs	-	-
	liver	-	-
Control (vaccine)		2⁶	+

Figure 1. Agarose gel electrophoresis confirmed that isolated samples were NDV. M: Marker; A: positive control; B: Pigeon/Surabaya/2019/01; C: Pigeon/Surabaya/2019/03.

Figure 2. Graphic of B cell epitopes prediction using Kolaskar-Tongaonkar antigenicity obtained from Pigeon/Surabaya/2019/01.

Figure 3. Graphic of B cell epitopes prediction using Kolaskar-Tongaonkar antigenicity obtained from Pigeon/Surabaya/2019/03.

Figure 4. Graphic of B cell epitopes prediction using Emini surface accessibility obtained from Pigeon/Surabaya/2019/01.

Table 3. B cell epitopes which could be used as subunit vaccine candidate (marked as green highlight) obtained from Pigeon/Surabaya/2019/01.

S. No.	Peptide	Length	Vaxijen	Allertop	ToxinPred
1	FSRLYPAAFSVF	12	Probable non-antigen
2	SWVYIHLLSTF	11	Probable antigen	Probable non allergen	Non-toxin
3	RSFVVPSA	8	Probable non-antigen
4	WLVHLCLACCCFLPTCPRHCLNCQYQAQL	29	Probable antigen	Probable non allergen	Toxin
5	NLVLRCSLD	9	Probable non-antigen
6	CTNVCLSEIQLLHSFA	16	Probable antigen	Probable non allergen	Non toxin
7	VRPCMVIVRL	10	Probable antigen	Probable non allergen	Non toxin

Table 4. B cell epitopes which could be used as subunit vaccine candidate (marked as green highlight) obtained from Pigeon/Surabaya/2019/03.

S. No.	Peptide	Length	Vaxijen	Allertop	ToxinPred
1	CEAWLPHHAGGSSDRSTRASLANLYP	26	Probable antigen	Probable allergen
2	NLTGRKRRTVG	11	Probable antigen	Probable non-allergen	Non-toxin
3	SDREYSQAIAR	11	Probable antigen	Probable non-allergen	Non toxin
4	V	1	Can not be predicted
5	SACTSGNMLNPEVHRRQEKQLERYRE	26	Probable non-antigen
6	CPRHCLNCQYQAQL	14	Probable antigen	Probable non-allergen	Probable toxin
7	DKLKQIPHASE	11	Probable non-antigen
8	T	1	Can not be predicted

Table 5. B cell epitopes obtained from Pigeon/Surabaya/2019/01 could not pass the screening.

S. No.	Peptide	Length	Vaxijen	Allertop	ToxinPred
1	SSDRST	6	Probable non-antigen
2	TGRKRRT	7	Probable antigen	Probable non-allergen	The peptides were too short
3	SSDREYS	7	Probable antigen	Probable allergen
4	EVHRRQEKQ LERYRERM	17	Probable non-antigen

Table 6. B cell epitopes obtained from Pigeon/Surabaya/2019/03 could not pass the screening.

No.	Peptide	Length	Vaxijen	Allertop	ToxinPred
1	SSDRST	6	Probable non-antigen
2	TGRKRRT	7	Probable antigen	Probable non allergen	The peptides were too short
3	SSDREYS	7	Probable antigen	Probable allergen
4	EVHRRQEKQLERYRERM	17	Probable non-antigen

Figure 5. Graphic of B cell epitopes prediction using Emini surface accessibility obtained from Pigeon/Surabaya/2019/03.

The positive samples were processed into RNA extraction then processed into Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR). According to visualization, they were molecularly confirmed to be NDV (Figure 1). Further analyses were conducted to reveal peptides which has potency to become subunit vaccine candidate, because it is very important to preserve vaccine which is not only capable to trigger specific immune response, but also safe, and not causing allergy and poisoning. According to the analyses, both samples had possibility to become subunit vaccine candidate. Peptides were possible to cause no harmful for the poultry are SWVYIHLLSTF, CTNVCLSEIQLLHSFA, and VRPCMVIVRL obtained from Pigeon/Surabaya/2019/01 (Table 3). Whilst Pigeon/Surabaya/2019/03 generated two peptides which possible to become subunit vaccine candidate; NLTGRKRRTVG and SDREYSQAIAR (Table 4). Newcastle Disease Virus-infecting pigeon has been discovered since 1981 (Pigeon Paramyxovirus serotype 1/PPMV1). It is firstly reported in Italy infecting racing pigeons²⁷. Then it spread to Great Britain, Eurasia, Bangladesh, and China^26,28,29,30. In Indonesia, NDV-infecting pigeon is not the main concern because most NDV infection relate to outbreak in poultry farm. Primarily, the main symptom is associated with neurological disturbance. In some cases the main clinical signs are drowsiness and enteric disturbance²⁶. Newcastle Disease Virus-infecting pigeon commonly belongs to genotype VI of Avian Paramyxovirus 1 (APMV1). These genotypes were suggested to be appeared as the result of multiple passages between chicken and pigeon²⁷.

Vaccination is the prevention method to protect animals or human from viral infection³¹. Part of antigen which role in specific antibody recognition is called epitopes³². It attaches to part of antibody (paratope) which leads to antibody-antigen neutralization³³. B cell epitopes prediction has purpose to predict peptides originate from the antigen which trigger the host-humoral immune response (specific antibody)³². These epitopes can be administrated to the host, hence it is called as subunit vaccine. Subunit vaccine consist of small-purified immunogenic peptides which originate from particular part or product of antigen, such as toxin, fragment or surface molecule. Subunit vaccine works differ according to the type of the particles. Protein antigens usually works on T cell dependent adaptive immunity while polysaccharide antigen works on T cell independent response³⁴. Subunit vaccines can induce specific immune response but less warring than whole-antigen vaccine³⁵. The use of vaccination to prevent Newcastle Disease Virus Infection is very important since the use of antiviral drugs never been administered in poultry to treat this disease. In human medicine, some antiviral drugs can be combined to prevent secondary effect of particular virus³⁶. The arrangement of subunit vaccine, as the way to eradicate NDV, must consider the safety of the poultry. Hence, even though the epitopes are obtained from the particular region of the whole genome, it need to be examined to prevent allergic and toxicity. Further analyses (both in-vivo and in-vitro experiments) toward these peptides must be conducted to know how it works in triggering immunity response in poultry.

ACKNOWLEDGMENTS:

This research was funded by Airlangga University through Credential Grant Program. The authors would express the gratitude toward Faculty of Veterinary Medicine for providing all necessities during the research.

CONFLICT OF INTEREST:

The authors stated that there is no conflict of interest during the research and writing process.

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Received on 04.12.2020 Modified on 12.07.2021

Research J. Pharm. and Tech. 2022; 15(5):2059-2064.

DOI: 10.52711/0974-360X.2022.00340