Expression of Certain Cytokine Genes in Avian Cells Infected with Newcastle Disease Virus

Abstract

Newcastle disease virus (NDV) is an inescapable and financially significant microbe, which actually keeps on tormenting the Indian poultry industry. The illness has a wide variety in seriousness going from asymptomatic to 100% mortality. The causal specialist, NDV, is a negative-sense single-stranded RNA virus. Transmission happens by exposure to fecal and different discharges from tainted birds, and through contact with contaminated feed, water, devices, and apparel. In this study expression of cytokine genes in avian cells is identified as a basic proposal for researchers to tackle new castle disease.

1. Introduction

The Indian economy is rural based and between 60% and 70% of Indian population depends on agriculture for their sustenance. The development of the Indian poultry industry over the most recent forty years from lawn side interest to the present coordinated scientific and vibrant industrial state is incredible. Subsequently, India has become one of the biggest manufacturers of eggs on the world and the development rate in the poultry industry is exceptional at 5–8% per annum. Besides the various issues that threaten the developments in poultry industry like feed cost, poor marketing and limited post harvest technology, incidence of infectious diseases like Newcastle disease (ND), avian infection bronchitis (IB), etc. also poses major hurdles. A large portion of the irresistible infection causes weighty mortality as well as leads to significant manufacturing misfortunes. The virus causing ND has been classified as the prototype of Avian Paramyxo virus (APMV). The eighth report of the International Committee on Taxonomy of viruses characterized Newcastle disease virus (NDV) under the order Mononegavirales in the family Paramyxoviridae, sub family Paramyxovirinae and the genus Avilavirus. Of the nine species in this genus (APMV 1 to APMV 9), APMV 1 APMV 1 with an intracerebral pathogenecity index (ICPI) value >0.7 has been reported to cause ND with respiratory distress and diarrhoea, with higher morbidity and death. The contamination has been accounted for to be far and wide, in those 240 types of birds addressing 27 of the 50 sets of birds that were so far impacted by ND.

The genome of APMV 1 has been reported to contain approximately 15,186 nucleotides comprising of genes namely HN, NP, P, M, F and L. The HN protein has been shown to be multifunctional, playing a role in cell attachment and release as well as playing an important role in the infection process, notably in the globular head and stem regions of the HN gene, then the fusion protein (F) has been shown to facilitate the fusion of the viral envelope with the cell membrane, and NP has been shown to be highly immunogenic. Thus, it has been confirmed that the virulence of NDV is multigenic. NDV strains are classified as velogenic (highly virulent), mesogenic (mid virulence), or lentogenic (nonvirulent). Velogenic strains induce severe neurological and respiratory symptoms, spread rapidly, and cause up to 90% fatality. Mesogenic strains cause coughing, affect egg quality and yield, and cause up to 10% mortality. Lentogenic strains cause minor symptoms with trivial mortality.

Clinical indications vary greatly depending on viral strain, bird species and age, concomitant sickness, and pre-existing immunity. There are four broad clinical syndromes recognized.

Viscerotropic velogenic

• Sudden arrival

• Rapidly spreads

• Severe sadness and appetite loss

• Significant decrease in egg production

• Increased respiratory rate

• Extensive bright green diarrhoea

• Oedematous swellings of the head, comb cyanosis, and conjunctivitis

• Prostration, with many birds dying in a matter of days

• Nervous symptoms in those who survive the initial phase

• High mortality (>90% in vulnerable flocks)

Neurotropic velogenic

• Predominantly acute respiratory and neurological symptoms

• Sudden depression

• Appetite loss

• Decrease in egg output

• Chest discomfort and persistent coughing

• Torticollis, wing and leg paralysis, and gasping anxious symptoms

• Adult mortality rates range from 10% to 20%.

• Young chickens may have substantially greater levels.

Mesogenic

• A decrease in egg production and quality (lasting 1–3 weeks);

• Weight loss;

• Gasping nervous symptoms may appear late in the clinical phase of acute respiratory sickness

• Mortality rate is about 10%

Lentogenic

Commonly subclinical may be

• No mental symptoms

• Mild respiratory symptoms

• Temporary appetite loss

• Decline in egg production;

• Negligible mortality unless concomitant disease is present.

There are several gross lesions.

Young chicks and chickens dying suddenly sometimes don’t have any lesions.

The following symptoms are present in the viscerotropic velogenic form:

• Hemorrhages and necrosis in the walls of the small intestinal tract, gizzard, and proventriculus. Other internal organs frequently get little hemorrhages.

• Thickened and clouded air sacs, acute laryngitis, and tracheitis, congestion, and catarrhal exudates are present in the neurotropic velogenic and mesogenic variants.

• Proventriculus hemorrhages occasionally, but infrequently elsewhere

2. The disease

An “infection of birds caused by Avian paramyxovirus 1 (APMV 1) with an ICPI value more than 0.7, possessing three arginine (R) or lysine (K) residues between position 113 and 116 of the F gene, and possessing phenylalanine (F) at position 117” is what the Office Internationale des Epizooties (OIE) defines as ND. The OIE has reclassified the illness, which was formerly included as a list A infection and is now one of the notifiable avian diseases. There have also been reports of significant productivity and financial losses due to the sickness. The production and economic losses caused by ND were shown to be more significant and severe than its economic effects. The economic effects of ND on commercial poultry trade were found to more important and severe [1].

3. Historical perspectives

Between 1926 and 1930, reports of ND in hens were made in several nations, including Indonesia [2], Newcastle-Upon-Tyne, England [3] and Ranikhet Village, Chennai, India [4, 5]. ND has been reported in many countries, including the USA [6], Australia [7, 8], Malaysia [9], South Africa, and Mozambique [10]. The sudden emergence of ND in a virulent form during the beginning of twentieth century was attributed to number of reasons, which include a sudden change in host population, role of feral birds acting as natural reservoir, shift of virus from enzootic form to epizootic form or to the result of a major mutation at the genome level [1, 11]. These views were reinforced by the emergence of ND as panzootic and report of ND in caged birds and feral birds [12, 13, 14, 15].

4. Impacts

Two hundred and forty one species of birds, or 27 of the 50 orders of that class, have been recorded to have ND, which predominantly affects chickens. House crows, pigeons, ducks and geese, emus, water fowl. Due to their apparent disease resistance, village chickens have been reported to be affected by the virus just as severely as commercial poultry.

5. Control

According to reports, NDV still poses a concern and continues to produce serious outbreaks even if control measures like good management practices and biosecurity standards are available at the farm level [8]. As a result, routine vaccination is the major goal of control measures. However, it has been noted that vaccination is not straightforward because it only prevents clinical sickness and mortality and does not stop virus multiplication, which makes the virulent become endemic [1].

Vaccination against ND in chickens has been reported to be carried out with live naturally occurring and artificially attenuated non pathogenic forms of the agent, inactivated viruses or their immunogenic determinants, subunit vaccines, live genetically modified vaccines, DNA vaccines, marker vaccines and edible vaccines. However, most of the currently available vaccines were not found to be able to provide desirable immunity even after using multiple doses [1], which has been justified by regular outbreaks of ND in vaccinated flocks.

While inactivated vaccines give primed birds a sustained high tire immune response, live vaccines were reported to stimulate the production of both humoral and cellular immune responses in addition to mucosal immune responses. Paranteral vaccination with inactivated virus often elicits serum neutralizing antibodies, and no local immune response, in contrast to attenuated NDV when used as live vaccines, which have been shown to have the ability to revert to virulent strains with transfer from bird to bird.

6. The virus

6.1 Classification

The International Committee on Taxonomy of viruses (ICTV) has been classified NDV under the order Mononegavirales, family Paramyxoviridae, subfamily paramyxovirinae, and genus Avulavirus. The genus Avalavirus as on date has reported to have only one species, namely the Avian Paramyxovirus (APMV) that comprises of nine serotypes—APMV 1 to 9. Of the nine serotypes, APMV-1 has been identified to be responsible for clinical ND.

6.2 Morphology

The nucleocapsid of the NDV virus is reported to measure 1000 nm in length, 17–18 nm in width, and an envelope covered in spike glycoproteins measuring 8–12 nm in diameter. The NDV virus particles are described as being pleomorphic and varying in size from 150 to 400 nm. According to the “rule of six theory”, which is unique to members of the family Paramyxoviridae, the genome was also found to be typical of Baltimore group v, single strand of negative sense RNA with a molecular weight of 5.2–5.7 × 10⁶ Da and 15,186 nucleotides. Six significant proteins have also been identified to be encoded by the genomic RNA, especially the haemagglutinin neuraminidase protein (HN), phosphoprotein (P), matrix protein (M), F, and big protein (L). According to reports, Mrna editing at the P gene led to the formation of the two additional proteins, V and W.

6.3 Methodology

6.3.1 Sample preparation

6.3.1.1 Procedure

The experiment was conducted with infected (treatment) and mock infected (control).

1. The cells from chicken embryos (CEs) were grown in six-well tissue culture plates (Nunc, Denmark, Cat # 150229) for preparation of samples.

2. The CE cells from one well from each group were taken after confluent monolayer to serve as the sample for day 0. The remaining cell cultures in both the treatment and control groups were infected with the seventh passaged NDV (D58) virus and MEM, respectively.

3. For the viral adsorption, the plates were incubated at 37°C for 1 hour.

4. The un-adsorbed virus was thoroughly washed off with MEM medium and the cell cultures were maintained in maintenance medium (2 ml/well).

5. The plates were incubated at 37°C and 5% CO₂ (CO₂ Incubator, Model 3131, Thermo Scientific, USA).

6. Throughout a 5-day time course as sample 1–5, the CE cells of one well from each group were collected every day at intervals of 24 hours.

6.3.2 Harvesting of CE cells

Prior to trypsinization using 250 μl of 0.1% trypsin 1:250 (Invitrogen, Canada, Cat # 27250-018) and 0.5 mM EDTA (Life-Technologies, USA, Cat # 15576-028) solution (made in sterile 1× PBS and sterilized by 0.22 m syringe filter) for 2–3 minutes, the cultures were immediately rinsed twice in pre-warmed PBS before being used to harvest the cells. To stop the trypsinization, 750 μl of growth media was added. Centrifugation at 500 g (∼2400 rpm) for 10 minutes at room temperature (25°C) was used to collect the cells. 750 μl of supernatant was discarded and remaining 250 μl along with cells pellet was preserved at −40°C for RNA isolation.

7. Reverse transcriptase polymerase chain reaction (RT-PCR)

7.1 Material for RT-PCR

7.1.1 RT-PCR kit

The iScript^TM cDNA Synthesis kit (BIO-RAD, USA, Cat # 170 – 8891) was used to synthesize cDNA for cellular (CE-fibroblast) genes (β-actin, IFN-α, IFN-y, MHC-I and DDX1) and viral genes (M and F genes).

7.1.2 For RNA extraction required materials

Cellular and viral RNA was extracted from CEF samples using the following chemicals

1. TRIZOL^® LS reagent (Invitrogen, USA, Cat # 10001 96-010)

2. Chloroform (Qualigens, India, Cat # 22465)

3. Isopropanol (Merck, India, Cat # 9634)

4. 75% ethanol (Changshu Yangyuan Chemical, China, Cat # XK-13-201-00185)

5. Protease, nuclease free water (GeNei^TM, Bangalore, Cat # 105437)

7.1.3 Other materials/reagents for PCR

Material/reagent	Supplier with Cat #	Purpose
PCR tubes	Axygen, USA, Cat # MCT-02-C	For cDNA synthesis and PCR
Microfuge tube 1.5 ml	Axygen, USA, Cat # MCT-150-C	For RNA isolation
Ampliqon (Taq DNA Pol 2.0× Master Mix Red)	Biomol, Denmark; Cat # AMP 180301	For amplification of DNA
DNA marker	100 bp (100–3000 bp) Cat #; 1 kb (300–10,000 bp), Cat #; Axygen, USA	In order to research the genes unique DNA migration pattern
Agarose	Genei^TM India, Cat # 105193	For gel electrophoresis of PCR product
Ethidium bromide solution	Sigma-Aldrich, USA, Cat # 46067-50ML-F	Using a final concentration of 10 μg/ml for staining the gels

7.1.4 RNA extraction from cells and viruses

With a few minor adjustments during the RNA pellet washing stage, the TRIZOL^® LS reagent was used to extract the RNA from preserved pelleted CE cells in accordance with the manufacturer’s instructions.

1. The pelleted CE cells was suspended in supernatant and 250 μl of this suspension was taken into DNAse, RNase free microfuge tube.

2. After adding 750 μl of TRIZOL^® LS reagent, the mixture was vortexed to combine the components.

3. For the complete dissociation of nucleoprotein complexes, this mixture was incubated for 5 minutes at room temperature (20°C).

4. Days after the initial incubation, 200 μl of chloroform was added, and the mixture was vortexed for 15 seconds and again incubated at room temperature for 10 minutes.

5. A refrigerated centrifuge machine (Eppendorf model # 5415 R) was used to centrifuge the contents at 12,000 g for 15 minutes at 4°C.

6. After separating the upper aqueous phase, RNA was precipitated by adding an equivalent volume of isopropanol.

7. To pellet the precipitated RNA, this mixture was centrifuged at 12,000 g for 10 minutes at 4°C while being held at 20°C for 20 minutes.

8. Then discarded the supernatant and the pellet was then centrifuged twice with 75% ethanol for 5 minutes at 7500 g before being air-dried for the remaining 5 minutes. The RNA pellet was once again dissolved in 20 μl of DNase RNase free water.

9. The concentration of RNA was determined to be 260/280 Å using a spectrophotometer (Bio-tek Instruments, Inc., μ Quant).

7.1.5 cDNA synthesis

Following the manufacturer’s instructions, the iScript^TM cDNA Synthesis kit (Bio-Rad, USA) was used to create cDNA synthesis.

1. The reaction mixture (20 μl) used to synthesize cDNA has the following ingredients

   Component	Volume	Final concentration in the reaction
   5× iScript reaction mix	4 μl	1×
   iScript reverse transcriptase	1 μl	1×
   Nuclease-free water	10 μl	1×
   RNA template (100 fg–1 μg)	5 μl	1×
   Total volume	20 μl	1×

2. The above reaction components were added and mixed properly by vortexing and spinned for few seconds to accumulate all of the components at the tube's bottom.

3. Reverse transcription was carried out in the tubes using a thermocycler (Applied Biosystem, Singapore, Model #2720) at 25°C for 5 minutes, 42°C for 30 minutes, and terminated at 85°C for 5 minutes.

4. Following cDNA synthesis, 10 μl of each sample’s cDNA was pooled, serially diluted, and used as a standard curve in a relative quantitative PCR (qPCR) assay. The 10 μl of the cDNA was diluted ten times and kept at −40°C for a farther use.

7.1.6 Polymerase chain reaction

Polymerase chain reaction (PCR) was performed using Ampliqon (Bio-Basic Inc.) following manufacturer’s instructions for FPCS of F gene and M gene fragment. The primers designed for qPCR were used.

The reaction was set up as follow

Component	Volume	Final concentration in the reaction
Taq Master Mix Red	10 μl	1×
Forward Primer (10 pmol)	1 μl	0.5 pmol
Reverse Primer (10 pmol)	1 μl	0.5 pmol
Template cDNA	2 μl	—
Nuclease free water	6 μl	—
Total volume	20 μl	—

1. For amplification of M gene fragment, PCR was carried out as per cycle sequence provided below.

   

2. For the purpose of amplifying FPCS gene specific cDNA, PCR was performed using the cycle sequence shown below

   

3. The FPCS of the F gene and M gene fragment PCR products were electrophoresed at 150 V for 15 minutes in a 2.0% agarose gel and compared with a 100 bp DNA marker. The gel was then photographed and scanned using a gel doc system (Bio-Rad).

7.1.7 Other materials/reagents for qPCR

Sl. no.	Material/reagent	Supplier with Cat #	Purpose
1	Real time PCR tube (0.2 ml) strips and masterclear cap strips	Eppendorf, North America, Cat # 951022109	For qPCR
2	Micro tips (0.2–10 μl)	Tarsons, Kolkata; Cat # 52100	For dispensing the reagents
3	Real time PCR plates (96 wells/plate)	Applied Biosystem, USA, Cat #	For qPCR

7.1.8 qPCR

Using SYBR^® Green JumpStart^TM Taq Ready Mix^TM (Sigma, USA, Cat # S4438) in accordance with the manufacturer’s instructions, qPCR was performed. SYBR Green jumpstart Taq Ready Mix combines the convenience of an easy-to-use ready-Mix solution with the performance boost of jumpstart Taq antibody for hot start PCR with SYBR Green I. It contains a fluorescent dye and the reagents for performing high-throughput qPCR and is provided in 2× concentration.

1. The composition of reaction mix (10 μl) used for real time PCR is as follow

   Component	Volume	Final concentration in the reaction
   2X JumpStart Taq Ready Mix	5 μl	Taq DNA Polymerase—1.25 units, Tris HCl 10 mM, KCl 50 mM, MgCl2 3.5 mM, dNTP 0.2 mM,stabilizers
   Forward primer (10 μM)	0.4 μl	0.4 μM
   Reverse primer (10 μM)	0.4 μl	0.4 μM
   Template cDNA	___μl	2–20 ng
   Nuclease free water	q.s. to 10 μl
   Total volume	10 μl

2. The above components are combined and added to a 200 μl PCR tube with a thin wall and carefully combined by vortexing before being quickly centrifuged for a few seconds to let allow the contents settle at the bottom of the tube.

3. Real time PCR was performed in a real time thermocycler (Mastercycler^® ep realplex, model # 22331, Eppendorf, Germany) for the amplification and relative quantification of cellular genes. The procedure started with a heat denaturing step at 94°C for 3 minutes, then the sequence cycle, final extension, and melting curve as follows

After each extension step, a single fluorescence `n end-point was measured. To establish the specificity of each amplification, the melting curves for PCR products were examined between 70°C and 95°C.

Conflict of interest

Authors express no conflict of interest.