ABSTRACT 

 

 

 

 

Title of Dissertation: DEVELOPMENT OF IMPROVED 

RECOMBINANT NDV-VECTORED VACCINES 

AGAINST HIGHLY PATHOGENIC AVIAN 

INFLUENZA VIRUS.  

  

  

Ishita Roy Chowdhury, 

Doctor of Philosophy, 2023  

  

Dissertation directed by: Dr. George Belov, Professor 

Department of Veterinary Medicine, 

University of Maryland, College Park 

College of Agriculture and Natural Resources 

 

                

                      Highly pathogenic avian influenza viruses (HPAIV) are highly contagious and 

economically devastating poultry pathogens with a documented transmission to humans causing 

severe human infections with high mortality. Circulation of these viruses is of public health 

concern as they have the pandemic potential to mutate to increase transmissibility among humans. 

The diversity of zoonotic influenza viruses causing human infections is alarming and effective 

vaccination is needed to control these viruses. Influenza viruses particularly with H7 and H5 

subtypes of HA can naturally switch to a highly pathogenic phenotype through different 

mechanisms. Currently available vaccines are not satisfactory as they are mostly inactivated 

vaccines that require labor-intensive administration methods and provide suboptimal protection of 

vaccinated birds. Viral vectors offer crucial advantages over traditional vaccines, including 



  

induction of outstanding antibody and cytotoxic lymphocyte responses which is important for the 

control of viral infections.  

 

Newcastle Disease virus (NDV) is a promising vaccine vector for HPAIV since it is highly 

restricted for replication in the respiratory tract of poultry, it can be easily administered, and it 

induces both local and systemic immune responses.  H7 influenza viruses are classified into two 

major genetic lineages, American and Eurasian. To develop a universal anti-H7 vaccine, we 

generated NDV vectors expressing chimeric HA sequences covering both North American and 

Asian isolates.  In the first project, we designed NDV-vectored vaccines against HPAI H7N8 

infection. The Hemagglutinin (HA) protein of influenza viruses is responsible for virus attachment 

to host cell and is the major target of the humoral immune response. Accordingly, we developed 

vaccines against HPAIV by generating recombinant NDV vectored H7 serotype-specific vaccines 

expressing HA protein. We also evaluated the protective efficacy of these recombinant vaccines 

against highly virulent H7 challenges in both broiler chickens and turkeys and the results were 

promising for broiler chickens, but for turkeys the vaccination design and scheme need to be 

further modified. 

 

In the second part of the study, we designed some recombinant NDV-vectored vaccines with an 

increased level of expression of H5HA antigen. The transcriptional unit of NDV contains a major 

open reading frame flanked by 5’ and 3’ untranslated regions (UTRs) followed by conserved 

transcriptional initiation and termination control sequences. Previous studies have shown that the 

addition of UTRs of P, M, and F genes positively modulated foreign gene expression. Hence, we 

hypothesized that cognate NDV mRNA UTRs would improve the expression of a protective 



  

antigen by an NDV-vectored vaccine. We generated recombinant NDVs where the HA of the 

HPAIV strain H5N1 is flanked by 5’ and 3’UTRs of NDV genes and determined the growth 

characteristics of these recombinant viruses, their stability, the level of HA expression and their 

transcription and translation modulation. Both studies aimed for the advancement of NDV-

vectored vaccines emphasizing the fact of better expression of the protective antigen and improved 

immunogenicity for avian influenza virus considering two important strains of H5 and H7. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



  

 

 

 

 

 

 

 

 

 

by 

 

 

 

ISHITA ROY CHOWDHURY 

 

 

 

Dissertation submitted to the Faculty of the Graduate School of the 

University of Maryland, College Park, in partial fulfillment 

of the requirements for the degree of 

Doctor of Philosophy 

2023 

 

 

 

 

 

 

 

 

Advisory Committee: 

Professor Dr. George A. Belov, Chair 

Professor Dr. Debabrata Biswas 

Professor Dr. Nathaniel Tablante 

Associate Professor Dr. Yanjin Zhang 

Assistant Professor Dr. Sean Riley 

 

 

 

DEVELOPMENT OF IMPROVED RECOMBINANT NDV-VECTORED 

VACCNES AGAINST HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUS 



  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

© Copyright by 

Ishita Roy Chowdhury 

2023 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 

 

ii 

 

Dedication 
 

This dissertation is dedicated to my parents, Mrs. Shubhra Roy Chowdhury and Mr. Milan Roy 

Chowdhury for their love and support. They are the biggest motivation of my life. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 

 

iii 

 

 

Acknowledgements 

 I would like to greatly thank my advisor and mentor, Dr. George A. Belov, for giving me the 

opportunity to work in his lab. Dr. Belov made me more confident not only as an independent 

researcher but also as a human being. He is the person for whom I could be able to think 

independently during each troubleshooting of my Ph.D. experiments. I also thank him for his 

wonderful support letter of recommendations for which I able to receive some prestigious awards 

during my Ph.D. tenure. I also specially thank Dr. Ekaterina Viktorova (who is dearly known as 

Kate). She is my inspiration all time as I have always seen her working relentlessly in lab long 

hours including weekends, handling multiple projects at once. Besides answering all my queries 

patiently, she always used to inspire me whenever I got frustrated with my results and situations. 

Moreover, I like her friendly and patient nature to deal with all the queries of students, despite 

being at a high position. I would like to thank my previous advisor Dr. Siba K. Samal. Though I 

didn’t have the opportunity to work with him for long time, but still his contribution during my 

Ph.D. is also unparallel. I also immensely thank my committee members, Dr. Yanjin Zhang, Dr. 

Debabrata Biswas, Dr. Nathaniel Tablante, and Dr. Sean Riley for giving their precious time to 

serve as my committee members and for reviewing my dissertation and providing with many 

valuable comments and ideas to improve my Ph.D. dissertation.  

 

I don’t want to thank my parents formally as they are my everything and I just love them. I cannot 

think of coming abroad, doing a Ph.D. without their constant support and motivation through all 

these years. I am lucky enough to get them as parents and their immeasurable sacrifices for giving 



 

 

iv 

 

me the freedom to pursue my dreams. They were and will always be the first ones to whom I share 

my happiness, excitement, tension, frustrations, difficulties during these five years journey. They 

believed in me thoroughly which made me successfully completed this big journey in my life.  

 

I also hugely thank all the faculty members, staffs, postdoctoral fellows, graduate students, and 

lab-mates at the Department of Veterinary Medicine and all my friends at the University of 

Maryland, College Park for making the workplace fun yet competitive work-driven environment.  

I owe my special thanks to all those animals used in this research for which its possible and 

meaningful to carry out the projects. 

 



 

 

v 

 

Table of Contents 
 

 

Dedication………………………………………………………………………………ii 
Acknowledgements…………………………………………………………………… iii 
Table of Contents……………………………………………………………………...vii 
List of Tables………………………………………………………………………… viii 

List o Figures…………………………………………………………………………. ix 

List of Abbreviations…………………………………………………………………...x 
Chapter 1: Introduction…………………………………………………………….. …1 

1.1 Avian Influenza Virus ................................................................... ……………..1 
     1.2 Research Objectives……………………………………………………….4 

Chapter 2: Review of Literature…………………………………………………….5 

    2.1 Avian Influenza Virus……………………………………………………..5 

    2.1.1. Classification……………………………………………………………5 
    2.1.2. Morphology……………………………………………………………..5 

2.1.3. Genome……………………………………………………………………….6 
    2.1.4 Viral proteins……………………………………………………………..7 

    2.1.5 Host Range Restriction……………………………………………………7 
    2.1.6 Pathogenicity………………………………………………………………8 

2.2 Avian Immunity………………………………………………………………....10 

2.2.1. An overview of avian system……………………………………………….10 
    2.2.2. Avian Immune responses to AIV…………………………………………10 

2.2.2.1. Innate Immune responses to AIV…………………………………………...10 
2.2.2.2. Adaptive Immune responses……………………………………………..13 

2.2.2.2.1. Humoral Immune Response……………………………………………13 
2.2.2.2.2. Cellular Immune Response……………………………………………..15 

    2.3. AIV vaccines in use…………………………………………………………..16 
2.3.1. Requirements for a good avian influenza vaccine………………………….16 
2.3.2. Types of AIV vaccines……………………………………………………...16 

2.3.2.1. Inactivated whole virus vaccine…………………………………………..16 
        2.3.2.2. Viral vectored vaccines…………………………………………………...17 

    2.4. Newcastle Disease Virus……………………………………………………...22 
2.4.1. Classification………………………………………………………………...22 
2.4.2. Morphology…………………………………………………………………22 
2.4.3. Genome……………………………………………………………………..23 

    2.4.4. Viral proteins……………………………………………………………….24 

2.4.5. UTR regions……………………………………………………………….29 

2.4.6. Life cycle of NDV………………………………………………………….29 

2.4.7. Molecular Basis of NDV Virulence……………………………………….31 
    2.4.8. Reverse Genetics of NDV………………………………………………….32 

2.4.9. NDV as a vector vaccine……………………………………………………34 
Chapter 3. Evaluation of protectove efficacy of NDV vectored vaccine candidates against  
H7HPAIV in broiler chickens and turkeys…………………………………………….36 

3.1. Introduction………………………………………………………………….36 
    3.2. Materials and Methods………………………………………………………39 



 

 

vi 

 

3.2.1. Generation of chimeric NDV vectored vaccine candidates……………….39 

3.2.2. Invitro experiments………………………………………………………..40 

3.2.3. Invivo experiments........................................................................................ …47 
3.2.3.1.Protective efficacy of heterologous NDV vectored vaccines in broiler 

chickens………………………………………………………………………………47        

 3.2.3.2. Standard Pathogenicity Test………………………………………………...49 
3.3. Results……………………………………………………………………………50 

      3.3.1. Expression of a consensus H7HA protein by NDV vectors………………...50 

      3.3.2. Characterization of in vitro replication of H7 vaccine candidates…………….53 

 3.3.3. Heterologous NDV vectored prime-boost vaccination protected broiler chickens  against 

H7N8 HPAI……………………………………………………………………………….54. 

    3.3.3.1. Vector specific immunity for immunized and unimmunized broiler chickens….55 

      3.3.3.2. H7- specific immunity for immunized chickens……………………………….55 

      3.3.4. Challenge experiment with HPAI H7N8…………………………………………56 

      3.3.5. Vector-specific immunity in turkeys……………………………………………59 

      3.3.6. H7-specific immunity for turkeys……………………………………………….59 

      3.3.7. Challenge experiment with HPAI H7N8 for turkeys……………………………59 

3.4. Conclusion…………………………………………………………………………….60 

Chapter4: The Effect of 5’ and 3’ Non-Translated Regions on the Expression of a Transgene 

from a Newcastle Disease Virus Vector…………………………………………………….63 

        4.1. Abstract……………………………………………………………………………..63 

4.2. Introduction……………………………………………………………………….64 

4.3. Materials and Methods…………………………………………………………………68 

 4.3.1Generation and recovery of five recombinant NDV vectored viruses expressing H5 avian 

influenza hemagglutinin flanked by NDV-UTRs…………………………………………..68. 

         4.3.2. Cells…………………………………………………………………………….70 

         4.3.3. Antibodies………………………………………………………………………71 

         4.3.4. Chemical and reagents………………………………………………………….71 

         4.3.5. Immunofluorescence microscopy……………………………………………….71 

         4.3.6. Western Blot………………………………………………………………………72 

         4.3.7. mRNA isolation…………………………………………………………………...73 

         4.3.8. Quantitative PCR………………………………………………………………….73 

         4.3.9. In Vitro capped mRNA synthesis and translation efficacy evaluation…………….74 

         4.3.10. mRNA transfection and Luciferase assay………………………………………...75 

         4.3.11. Data processing and statistical analysis…………………………………………...77 

4.4. Results……………………………………………………………………………………77 

    4.4.1. Construction and characterization of NDV vectors expressing avian influenza virus HA 

protein………………………………………………………………………………………….77. 

  4.4.2. The HA gene was most expressed in mammalian cells when flanked by UTRs derived from 

M gene………………………………………………………………………………………….80. 

   4.4.3. Evaluation of the effect of UTRs on the amount of HA mRNAs……………………….83 

   4.4.4. Evaluation of the effect of UTRs on the translation of H5 HA-coding RNA…………85 

   4.4.5. Evaluation of the stability of the insert flanked with the M UTRs……………………87. 

 

 

 



 

 

vii 

 

 

4.5. Discussion…………………………………………………………………...89 

Chapter 5: Conclusion and Perspectives……………………………………………....92 
            Bibliography……………………………………………………………….100 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 



 

 

viii 

 

List of Tables 
 

Table 1: Length of NDV 5’ and 3’ UTRS………………………………………………….69 

Table 2: Primers for qPCR………………………………………………………………….74 

Table 3: Primers for luciferase constructs………………………………………………….75 

Table 4: Table showing the design of the HA inserts flanked by the control and NDV-derived 

UTRs……………………………………………………………………………………….80. 

Table 5. Table with the corresponding Cq values of the mRNA samples infected with the 

respective viruses……………………………………………………………………………85. 

 

 

 

 

 

 

 

 

 



 

 

ix 

 

 

 

List of Figures 
 

Figure 1. Schematic diagram of AIV structure……………………………………….7 

 

Figure 2. Host determinants of the pathogenicity of avian influenza virus in poultry...11 

 

Figure 3A. Structure of NDV…………………………………………………………26 

 

Figure 3B. Schematic representation of the structure of the NDV genome…………26 

 

Figure 4. Plasmid-based recovery of recombinant NDV……………………………38 

 

Figure 5. Representation of antigenically distinctive chimeric NDV vaccine vector...43 

 

Figure 6. Generation of chimeric NDV and LaSota vectored vaccine viruses……….46 

Figure 7. Schematic model of animal experiments for broiler chicken…………….48 

Figure 8. Illustrative of ICPI test…………………………………………………….49 

Figure 9A. Expression of H7 HA and N8 NA proteins by NDV vectors were analyzed 

by Western blot………………………………………………………………………52 

 

Figure 9B.Surface expression of the HA protein was evaluated by immunofluorescence 

analysis……………………………………………………………………………….53 

 

Figure 10. Growth kinetics of prime and boost vaccine candidates was determined by 

infecting DF1 cells with each virus ………………………………………………….54. 

 

Figure 11. HI assay done for Vector -specific immunity…………………………….56. 

 

Figure11C. H7- specific immunity measured for both groups of broiler chickens….56 

 

Figure 12. Challenge of vaccinated chickens with HPAIV H7N8…………………58 

 

Figure 13. Immunogenicity of NDV vectored vaccines in turkeys……………….60 

 

Figure 14. Mortality and shedding of challenge virus in turkeys…………………61. 

Figure 15A. Map of NDV genome organization…………………………………68 

 



 

 

x 

 

Figure 15B. Schematic representation of transcription cassette of NDV………….69 

Figure 16. Construction of five recombinant NDV constructs containing foreign gene 

HA………………………………………………………………………………….71 

 

Figure 17. Diagram of luciferase constructs for translation study………………….76 

 

Figure18.Schematic representation of the principle of Luciferase assay……………77 

 

Figure 19A. Construction of the NDV vectors expressing HA protein under the control 

of cognate UTRs……………………………………………………………………..79 

 

Fig 19B. The growth kinetics of the recombinant viruses in DF1 cells……………..80 

 

Fig: 19C. IFA images of the recombinant LaSota virus infected HeLa cell…………80 

 

 

Figure 20. M-derived UTRs increase the expression of the transgene in human cells...83 

 

Figure 21.A representative qPCR amplification experiment showing nine samples of 

mRNAs isolated from infected HeLa cell……………………………………………85. 

 

Figure22.Cognate UTRs do not increase the amount of the transgene 

mRNA……………………………………………………………………………….86. 

 

Figure 23. Cognate NDV UTRs strongly increase the mRNA translation………….88. 

 

Figure 24. The M UTRs do not significantly affect the transgene stability……….89. 

 

 

 

 

 

 

 

 

 

 

 



 

 

xi 

 

List of Abbreviations 

AI: Avian Influenza 

AIV: Avian Influenza Virus 

ANOVA: Analysis of Variance 

APMV-1: Avian paramyxovirus 1 

APP: Acute Phase Proteins 

ATCC: American Type Culture Collection 

BM-DC: Bone marrow derived dendritic cells. 

CEF: Chicken Embryo Fibroblast 

cLL: Chicken lung lectin 

CMI: Cell mediated Immunity. 

CTL: Cytotoxic T Lymphocytes 

DC: Dendritic cells 

DF1: Avian Fibroblast cell 

DIVA: Differentiation between vaccinated and infected animals 

DMEM: Dulbecco Modification of Eagle’s medium 

FAO: Food and Agriculture Organization of United Nations 

FBS: Fetal Bovine Serum 

FPV: Fowl pox Virus 

GE: Gene End 

GS: Gene Start 

HA: Hemagglutinin 

HeLa: Human cervical carcinoma 



 

 

xii 

 

Hep2: Human epidermoid carcinoma 

HI: Hemagglutinin Inhibition 

HN: Hemagglutinin-Neuraminidase 

HPAIV: High Pathogenic Avian Influenza Virus 

HRP: Horseradish peroxidase 

HVT: Herpes Virus of turkeys 

IACUC: Institutional Animal Care and Use Committee 

IAV: Type A Influenza Virus 

IBC: Institutional Biosecurity Committee 

IBV: Infectious Bronchitis Virus 

ICPI: Intracerebral pathogenicity index 

IDPR: Intrinsically disordered protein regions 

IFA: Immunofluorescence assay 

IFN: Interferon 

IGS: Intergenic sequences 

IL: Interleukin 

ILTV: Infectious laryngotracheitis virus 

IRGs: Immunity-related GTPases 

L: Large Polymerase protein 

LPAIV: Low Pathogenic Avian Influenza Virus 

M1: Matrix 1 

M2: Matrix 2 

MDA: Maternally derived antibodies 



 

 

xiii 

 

MDV-1: Marek disease virus serotype 1 

MHC: Major Histocompatibility Complex 

MSA: Multiple Sequence Alignment 

MVA: Modified Vaccinia strain Ankara 

NA: Neuraminidase 

NDV: Newcastle Disease Virus 

NEP: Nuclear Export Protein 

NP: Nucleoprotein 

NS1: Nonstructural protein 1 

NS2: Nonstructural protein 2 

OIE: The Office International des Epizooties  

ORF: Open Reading Frame 

PA: Polymerase acid 

PB1: Polymerase Basic 1  

PB2: Polymerase Basic 2  

PMD: P multimerization domain 

qPCR: Quantitative polymerase chain reaction 

RNP: Ribonucleoprotein 

SPF: Specific-pathogen free 

ssRNA: Single-stranded RNA 

STAT1: Signal transducer and activator of transcription 1 

UTRs: Untranslated regions 

VEE: Venezuelan equine encephalitis virus 



 

 

xiv 

 

VLP: Virus-like particle 

vRNA: viral RNAWHO: World Health Organization 

WOAH: World Organization for Animal Health 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  

 

 

 

 

 



 

 

1 

 

Development of improved recombinant NDV-vectored vaccines 

against highly pathogenic avian influenza virus (HPAIV) 

 

Chapter 1 : Introduction 

1.1 Avian Influenza virus : 

Avian influenza virus (AIV) is a member of the genus Influenzavirus of the family 

Orthomyxoviridae. The genome of AIV is comprised of eight segments of negative-

sense single-stranded RNA encoding for 10 proteins: Polymerase basic 1 (PB1), 

polymerase basic 2 (PB2), polymerase acid (PA), hemagglutinin (HA), nucleoprotein 

(NP), neuraminidase (NA), matrix 1 (M1), matrix 2(M2), nonstructural 1 (NS1) and 

nonstructural 2 (NS2)[1]. Based on two main viral surface glycoproteins, AIV is 

divided into 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes. HA protein 

allows virus attachment to the host cell and is a major target of humoral immune 

response [2]. NA protein helps in release and spread of progeny virions by removing 

sialic acids from glycoproteins. Anti-NA antibodies play a major role in reducing 

clinical signs of infection and viral shedding [3]. Wild aquatic birds including ducks, 

gulls and shorebirds are the natural reservoirs of this virus [1].  

HA is synthesized as an inactive precursor HA0 which needs to be cleaved into HA1 

and HA2 subunits that form the mature protein. Low pathogenic avian influenza (LPAI) 

viruses contain an HA cleavage site which can only be cleaved by proteases available 

in the intestinal and respiratory tracts of birds [4]. In contrast, highly pathogenic avian 

influenza (HPAI) viruses contain multiple basic amino acids at HA cleavage site, 

resulting in cleavability of HA by ubiquitous intracellular proteases. HPAI viruses 



 

 

2 

 

cause systemic infection and high mortality in chickens and other terrestrial poultry. 

Two subtypes (H5 and H7) of LPAIVs can naturally switch to a highly pathogenic 

phenotype through different mechanisms like acquisition of basic amino acids in the 

cleavage region of HA by insertions or substitutions and/or recombination with other 

gene segments of viral genome [5,6]. 

 Worldwide, the number of highly pathogenic H7 virus outbreaks has increased as 

compared to last century: H7N1 in poultry farms (Italy), H7N3 in commercial poultry 

(Australia and Canada), H7N4 in commercial chickens (Australia), H7N7 in 

commercial layer hens and poultry (Netherlands, Belgium, Germany, Spain), H7N8 in 

turkeys (Indiana) are a few examples [7,8]. The H7N8 virus was eradicated from Indiana 

after quarantine and depopulation of commercial birds from 10 commercial turkey 

farms has been executed. Since 2013, the emergence of novel H7N9 viruses have been 

a threat to public health as evidenced by severe human infections with high mortality 

in China [9]. In 2017, concurrent outbreaks of H7N9 HPAI and LPAI occurred at 

poultry farms in Tennessee. Subsequently, H7N9 LPAI were detected in commercial 

and backyard flocks in Alabama, Kentucky, and Georgia [10]. 

The number of HPAIV outbreaks has increased in the past 20 years and the H5 and H7 

lineages of viruses have become endemic in poultry [10]. The first H5 avian influenza 

outbreak in this century was caused by H5N1 virus in Hong Kong in 2002 [ 11]. 

Between January 2005 and November 2022, H5 HPAIVs have caused 8534 outbreaks 

and the loss of 389 million poultry around the world (World Organization for Animal 

Health, World Animal Health Information System, in short OIE-WAHIS) [12]. These 

viruses caused three waves of outbreaks in multiple countries in Asia, Africa, Europe, 



 

 

3 

 

and North America.  The first wave was mainly reported in Asian, some African and 

European countries from 2005–2010 and was caused by H5N1 viruses causing more 

than 55 million poultry deaths. The second wave occurred from 2011 to 2019 and was 

caused by multiple subtypes of H5 viruses and approximately 139.9 million poultry 

died. The outbreaks in this period were reported in Asia, Europe, Africa, and North 

America. A reassortant H5N8 virus in which the HA gene segment was from an H5N1 

(HPAI A/Goose/Guangdong/1/1996) lineage virus and other gene segments were from 

multiple other AIVs was circulating in eastern China and is categorized as HPAI H5 

virus clade 2.3.4.4. This clade of viruses has the propensity to form novel subtypes 

which are capable of rapid, global spread [13]. During 2014-2015, H5N8 and related 

reassortant viruses caused outbreaks in wide geographic regions (Asia, Europe, and 

North America). In the U.S., H5N8 HPAIV was first detected in wild waterfowl in the 

Pacific Northwest in 2014 [14]. Other reassortment events of the H5N8 virus with 

LPAIVs led to the divergence of H5 viruses into distinct subtypes, including H5N1, 

H5N2, and H5N8 [15, 16]. The third wave started in 2020 and was mainly caused by 

H5N8 and H5N1 viruses, and approximately 193.9 million poultry died by the end of 

November 2022. The outbreaks in this period were mainly reported in Europe and 

North America, although some were also reported in Asian and African countries. 

Among the total of  389 million aberrant influenza-associated poultry deaths, H5N1 

viruses were responsible for 204 million, H5N8 viruses for 111 million, and the other 

74 million poultry losses were caused by other H5 viruses [12].  

Depopulation of infected flocks is a common practice to control the spread of AIV 

including HPAIV. However, for the past two decades, vaccine usage has become 



 

 

4 

 

important not only to control AIV infection in poultry, but also to prevent transmission 

of these viruses from birds to humans [17]. Still, currently available vaccines against 

AIV are not satisfactory as they are mostly inactivated virus vaccines which require 

labor-intensive administration methods and provide suboptimal protection of 

vaccinated birds [18,19, 20]. Furthermore, inactivated virus vaccines can be effective only 

when the birds are immunologically mature (>3-week-old ages) [21]. The use of 

attenuated live influenza vaccines in poultry is not recommended due to  a potential 

risk of reassortment or mutations [22]. Thus, effective vaccines and efficient vaccination 

methods are required for better protection of poultry. 

Non-pathogenic strains of Newcastle Disease virus (NDV) are promising vectors for 

the vaccine development against HPAIV since they are highly restricted for replication 

in the respiratory tract of poultry, they can be easily administered, and they induce a 

strong mucosal immune response which is important for the protection against 

respiratory pathogens [23]. In this study, we aimed to design improved NDV-vectored 

vaccines against HPAIV infection. The hypothesis behind this aim is that the HA 

protein of influenza virus is responsible for virus attachment to host cells and is the 

major target for the development of neutralizing antibodies. Hence our aim is to 

develop recombinant NDV vectors expressing HPAIV HA and assessed their 

protective efficacy in chickens and turkeys as well as evaluated a novel strategy of 

increasing transgene expression from the NDV vectors. 

 

 

 



 

 

5 

 

1.2. Research Objectives: 

1.2.1. To evaluate the protective efficacy of NDV vectored vaccine candidates against 

H7 HPAIV in broiler chickens and turkeys. 

 

1.2.2. To determine the effect of 5’ and 3’ non-translated regions on the expression of 

a transgene from a Newcastle Disease Virus vector. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 

 

6 

 

Chapter 2: Review of Literature 

2.1. Avian Influenza Virus (AIV): 

2.1.1. Classification: 

AIV belongs to the family Orthomyxoviridae, genus Alphainfluenzavirus. The AI 

virions have a lipid bilayer envelope derived from host cell membrane and contain a 

segmented, negative-sense, single-stranded RNA genome associated with the viral 

RNA-dependent RNA polymerase [24]. The Orthomyxoviridae family is divided into 

nine different genera: Alphainfluenzavirus, Betainfluenzavirus, Gammainfluenzavirus, 

Deltainfluenzavirus, Isavirus, Thogotovirus, Sardinovirus, Mykissvirus and 

Quaranjavirus, as well as unclassified Orthomyxoviruses  

[25]. Type A influenza viruses are the most widespread of the group, and they infect 

many different avian and mammalian species [1]. They are further classified into 18HA 

and 11NA subtypes based on viral surface proteins hemagglutinin (HA) and 

neuraminidase (NA). Among these, HA subtypes 1-16 and NA subtypes 1-9 are found 

in wild waterfowl and shorebirds and  HA subtypes 17-18 and NA subtypes 10-11 have 

been isolated from bats [26]. 

 

2.1.2. Morphology: 

AIVs are enveloped, pleomorphic particles of around 80-120nm in diameter with 

spherical or filamentous shape [27]. The virus envelope has tightly packed projections 

containing two types of glycoproteins: rod-shaped trimers of HA and mushroom-

shaped tetramers of NA of length 10-14nm that are present in the ratio of four HA to 

one NA [1]. The matrix (M1) protein lies under the lipid envelope. The core of the virus 

https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Tree&id=197912&lvl=3&lin=f&keep=1&srchmode=1&unlock
https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Tree&id=197913&lvl=3&lin=f&keep=1&srchmode=1&unlock
https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Tree&id=1511083&lvl=3&lin=f&keep=1&srchmode=1&unlock
https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Tree&id=2948895&lvl=3&lin=f&keep=1&srchmode=1&unlock


 

 

7 

 

particle is comprised of ribonucleoprotein (RNP)complex consisting of viral RNA 

segments, polymerase basic proteins (PB1, PB2), polymerase acidic (PA) and 

nucleoprotein (NP). Although detectable antibody responses are observed against some 

internal proteins, neutralizing antibodies produced against the two surface proteins HA 

and NA are the major determinants for protective immune response [28]. 

 

 

Figure 1: Schematic diagram of AIV structure: Schematic of the eight viral RNA 

(vRNA) gene segments that comprise influenza A genome. The 5′ and 3′ untranslated 

regions (UTRs) which contain the viral promoters are represented with lines on both 

sides of each gene in left picture. The viral membrane proteins HA, NA, and M2 are 

shown, along with the eight viral ribonucleoproteins (vRNPs), and matrix protein M1 

that supports the viral envelope. A single vRNA gene segment is shown wrapped 

around multiple nucleoprotein (NP) copies with the conserved promoter regions in the 

5′ and 3′ UTRs forming a helical hairpin, which is bound by a single heterotrimeric 

viral RNA-dependent RNA polymerase (PB1, PB2, and PA) (Dou et al, Frontiers in 

Immunology, 2018). 



 

 

8 

 

2.1.3. Genome: 

Influenza A virus genome consists of eight negative-sense, single-stranded viral RNA 

(vRNA) segments. The term negative-sense RNA implies that the RNA genome cannot 

be translated into protein directly and that it must first be transcribed to positive-sense 

mRNA before it can be translated into protein products. The segmented nature of the 

genome allows for the exchange of entire genes between different viral strains. 

Antigenic shift allows the segmented genome of the virus to accommodate HA and NA 

segment from a different influenza subtype. This kind of segment reassortment occurs 

in cells infected with different human and animal viruses thus producing a new 

resulting virus which encodes completely novel antigenic proteins to which the human 

or animal population has no preexisting immunity [29]. Each vRNA segment possesses 

noncoding regions (of varying lengths) at both 3′ and 5′ ends. The noncoding regions 

also include mRNA polyadenylation signal and part of the packaging signals for virus 

assembly[29]. 

 

2.1.4. Viral proteins: 

The structural proteins in the mature virion are divided into surface proteins: 

hemagglutinin (HA), neuraminidase (NA), and membrane ion channel (M2) proteins 

and the internal proteins, nucleoprotein (NP), matrix protein (M1); the polymerase 

complex composed of polymerase basic protein 1 (PB1), polymerase basic protein 2 

(PB2), and polymerase acidic protein (PA)1. Two additional proteins produced by 

influenza viruses are nonstructural protein 1 (NS1) and nonstructural protein 2 (NS2), 

which is also known as the nuclear export protein (NEP) [1]. One protein that is not 



 

 

9 

 

found in all type A influenza viruses is the PB1-F2 protein, an 87–amino acid protein 

translated from a different reading frame from PB1 protein [30]. This PB1-F2 is highly 

conserved among avian influenza viruses and removal of this protein in a HPAIV H5N1 

delayed onset of clinical signs and systemic spread of virus in ducks[31]. This PB1-F2 

protein shown to be involved in apoptosis and play an active role in pathogenesis of a 

reassortant virus within animal host [32]. 

 

2.1.5. Host Range Restriction: 

The attachment of influenza virus is controlled by the linkage type of galactose residues 

on host cell surface [33]. All AIV (with exception of some viruses of H5N1 and H9N2 

subtype) have HAs attaching to alpha 2,3 linked galactose sugars of sialic acid residues 

with high affinity [34]. In contrast, human influenza viruses have HAs which 

preferentially bind to sialic acids where the sugars are attached by alpha 2,6-linkage. 

This preferential binding of HAs to alpha 2,3- or alpha 2,6 linkage results in adaptive 

mutations that are essential for switching from bird to human tropism [35]. Host range 

restriction of influenza virus also depends on receptor distribution in the hosts.  Two 

research groups reported a crucial finding explaining why H5N1 is lethal to humans 

but is inefficient in human-to-human transmission [36]. They found that humans have 

alpha 2,6 galactose receptors throughout the respiratory tract from the nose to lungs 

and have alpha 2,3 galactose receptors in and around alveoli deep in the lungs. Hence, 

H5N1 preferentially infects cells in the lower respiratory tract and induces heavy 

damage to the lungs with little involvement of the upper respiratory tract[36]. 

 



 

 

10 

 

2.1.6. Pathogenicity: 

The cleavability of HA protein is one of the major determinants for tissue tropism of 

AI viruses [37]. Post-translational proteolytic activation of precursor HA (HA0) into 

HA1 and HA2 subunits by host proteases is essential for infectivity and spread of the 

virus. Most LPAI viruses have a single basic amino acid (arginine) at the HA cleavage 

site. HAs of LPAI viruses are cleaved externally only after the glycoprotein has been 

transported to the plasma membrane[38]. This restricts the sites of replication to certain 

tissues where appropriate extracellular proteases are present, allowing the virus only to 

cause localized infections in respiratory or intestinal tracts, resulting in less severe 

infections. In contrast, HAs of HPAI viruses are cleaved intracellularly in Golgi 

apparatus by subtilisin-like proteases [39]. Low pathogenic H5 or H7 virus subtypes can 

become highly pathogenic upon acquiring multiple basic amino acids at the HA 

cleavage site which allows for recognition and cleavage by ubiquitous intracellular 

proteases [40]. Since these proteases are ubiquitous, HPAI viruses can replicate in a 

broad range of different host cells and can cause fatal, systemic infections in poultry. 

Some HAs had a glycosylation site which shields the HA cleavage site. Elimination of 

the glycosylation site and changes of non-basic amino acids to basic ones, insertions 

that open the cleavage site and additions of basic amino acids to the cleavage site 

change cleavability and often increase pathogenicity. Thus, virus activation by host 

proteases plays a pivotal role in the spread of infection, tissue tropism and 

pathogenicity.  



 

 

11 

 

 

 

                                                

 

 

 

 

 

 

 

 

Figure 2. Host determinants of the pathogenicity of avian influenza virus in 

poultry: LPAIV are restricted to the respiratory and gastrointestinal tracts where 

extracellular trypsin-like proteases can cleave and activate HA. HPAIV have a broader 

tissue tropism and can replicate in many organs as their HA protein is cleaved by 

intracellular furin-like proteases. (Adaptation from Low Pathogenic Avian Influenza 

Virus, Viruses, 2017). 

 

2.2. Avian Immunity: 

2.2.1. An overview of avian immune system: 

The avian immune system, like that of other vertebrate animals, is divided into specific 

(adaptive) and non-specific (innate) immune mechanisms. The non-specific disease 

innate immune responses target broad groups of pathogens, body temperature, 

LPAIV 

proteases localized in 

upper respiratory system 

and intestine. 

HPAIV 

Ubiquitous 

proteases 

Infected organs 

Uninfected 

organs 



 

 

12 

 

anatomic features, normal microflora, and respiratory tract cilia. Specific immune 

mechanisms (adaptive system) are characterized by specificity, heterogeneity, and 

memory. This system is divided into humoral and cellular immunity. Humoral 

immunity is related to antibodies secreted by B-lymphocytes that develop in a specific 

organ called the Bursa of Fabricius. The cellular immune system is mediated by T- 

lymphocytes which develop in the thymus. The thymus gland in chickens is composed 

of multiple lobes located along the side of the neck and extending into the thoracic 

cavity. Thymus is essential for the maturation of T-lymphocytes. Other cells important 

to the cellular immune response include macrophages, dendritic cells, natural killer 

cells, and effector cells of antibody-dependent cellular cytotoxicity [41]. 

 

2.2.2. Avian immune response to AIV 

2.2.2.1. Innate immune responses to AIV:  

The innate immune system forms the first line of defense against AIVs. Innate immune 

cells such as macrophages, granulocytes, NK (natural killer) cells are recruited to the 

site of infection to control virus replication and prevent further spreading of virus. The 

major differences in susceptibility to AIV infections that are found between chickens 

and waterfowl are caused by these early innate immune responses[42]. Several immune 

genes involved in the innate responses in mammals seem to be missing in avian 

genomes. Chickens lack functional TLR8 and RIG-I genes in their genome which 

affects recognition of intracellular bacteria and RNA viruses, including AIVs and may 

be associated with the high mortality rates observed after infection with HPAIV. 

Research done by Hayashi et al showed that chickenMDA5(chMDA5) preferentially 



 

 

13 

 

senses shorter dsRNA synthetic analogues, poly(I:C), in chicken DF-1 fibroblasts [43]. 

The researchers also found that chMDA5 is involved in chicken interferonβ (chIFNβ) 

induction against avian influenza virus infection. The importance of chMDA5 in innate 

immune response lies in the fact that it indemnifies the function of RIG-I in chicken[43]. 

In contrast, ducks only lack TLR8, and survive infections with HPAIV. A variety of 

proteins are involved in the active responses induced after entry of AIV[42]. Some 

innate inhibitors of viral infections belong to families of proteins that are highly 

evolutionarily conserved and include plasma proteins such as acute-phase proteins 

(APPs), C-reactive protein, serum amyloid A, collagenous lectins (collectins, 

surfactants, and ficolins), pentraxins, alpha-macroglobulin families, and antimicrobial 

peptides (e.g. defensins and cathelicidins) [24]. Chicken surfactant A (SP-A) expression 

is decreased upon application of  IFNα but gene expression is upregulated during AIV 

infection[ 44]. SP-A homologue chicken lung lectin (cLL) is a C-type lectin lacking the 

collagenous domain has moderate activity to AIV invitro [45]. 

  

Macrophages and Dendritic cells (DC): 

Respiratory macrophages and dendritic cells (DC) are essential to detect and respond 

to AIV and for control of the innate and adaptive immune responses. Once the cells are 

in contact with pathogens, they play essential role in nonspecific and specific immunity 

and are important mediators of the inflammatory response. Macrophages function in 

the inflammatory response by producing cytokines and engulfing foreign particles 

through phagocytosis [46]. AIV infection impairs macrophage functions in vivo. 

Research by Kodiahalli et al. showed that pulmonary macrophages isolated from 



 

 

14 

 

turkeys infected with the LPAI strain A/Turkey/Minnesota/534/78 (H6N1) exhibited 

suppressed  phagocytosis and pulmonary microbicidal activity 10 days post infection 

[47].  

Comparison of LPAIV and HPAIV infection responses in chickens indicated that in 

contrast to LPAIV, infection of chicken bone marrow derived dendritic cells (BM-DC) 

with H7N1 or H5N2 HPAIV resulted in increased viral load and a significant increase 

in IL-8 (CXCLi2), IFN-α, and IFN-γ mRNA expression [49]. Infection of chicken DC 

with LPAIV and HPAIV strains H7N1 or H5N2 induced differential up-regulation of 

TLR1, TLR3, and TLR21 mRNA expression, which may be related to the differences 

in cytokine responses induced by these HPAIV and LPAIV strains [49]. 

 

Interferon (IFN): 

In chickens, type I interferons are a multigene family of IFNα and IFNβ genes. Type II 

IFNs are represented by a single gene encoding IFNγ. Type I IFNs are responsible for 

antiviral activities, inhibition of cell proliferation, cell differentiation and migration. 

Chicken IFNα/β bind to heterodimeric cell surface receptors IFNAR1/IFNAR2 and 

differentially regulate interferon- stimulated genes [48]. Chicken IFNα induced a more 

potent antiviral state than IFNβ due to their differential binding to IFNAR1 and 

IFNAR2 and regulation of IFNAR1 and IFNAR2 during embryonic development [49]. 

A study done by Roll et al demonstrated that the induction or suppression of interferon-

regulated genes in chickens is both tissue and time specific [44].  

Research by Haq et al. have shown that small interfering RNA can downregulate 

chicken IFN γ expression in vitro when delivered directly as well as when expressed 



 

 

15 

 

by a recombinant avian adeno-associated virus-based vector [50]. IFNγ is a 

macrophage-activating factor which plays a crucial role in upregulation of MHC class 

I and II expression, maturation and differentiation of various immune cells, and 

activation of T helper 1-type immune responses [24]. 

Only one copy of type III IFN (IFNλ) has been identified in chickens and ducks with 

high homology to human IFNλ3.  Type III IFN plays a major role in the antiviral 

responses at epithelial surfaces in chickens which is well researched in  in vivo, invitro 

and in-ovo experiments against NDV, AIV and infectious bronchitis virus (IBV)[51]. 

  

2.2.2.2. Adaptive Immune response : 

2.2.2.2.1. Humoral Immune response : 

The humoral immune response in poultry against viral infection includes systemic and 

mucosal antibody production. The systemic antibody response in chickens and turkeys, 

like in other avian species starts with the production of IgM measured as early as 5 days 

post-infection and IgY which is detected shortly after. While avian influenza viruses 

produce 10 viral proteins, only antibodies against the surface proteins HA and NA have 

been shown to be protective.  

Shirvani et al. showed that the intact HA protein induced a neutralizing antibody 

response in chickens against an HPAIV H5N1 and provided complete protection 

against a lethal challenge, whereas, HA1 or HA2 subunits neither induced significant 

levels of neutralizing antibody response nor provided protection [52]. Thus, the epitopes 

responsible for the induction of neutralizing antibodies and protective immunity are 

present on both HA subunits in their native conformation, but the epitopes are lost when 



 

 

16 

 

the two subunits are separated. The importance of HA protein for protection is further 

proven by the protection of birds with subunit vaccines that contain only HA protein. 

Antibody titers to HA protein are commonly measured by an indirect antibody test, 

such as the hemagglutinin inhibition (HI) test.  

The NA protein is an enzymatically active protein that is important in cleaving sialic 

acid allowing the virus to be released from cell surface. The NA protein elicits 

neutralizing antibody in chickens and NA specific vaccines can be protective against 

HPAI challenge [53]. 

Avian influenza viruses that infect poultry invade two primary mucosal regions – the 

respiratory tract and the gastrointestinal tract. Avian IgA is found in secretions of gut, 

respiratory and reproductive tracts of birds.  The IgA antibodies in the mucosal surfaces 

play an important role in the recovery of infected birds and in protecting from further 

infections, particularly against LPAIVs which are primarily restricted to mucosal 

replication. Avian IgA possesses a J chain and secretory component. The Joining (J) 

chain is a protein component that links monomers of antibodies IgM and IgA to form 

polymeric antibodies capable of secretion. The ontogeny of chicken lymphocytes 

expressing J chain was investigated in the embryonic bursa of Fabricius, the spleen, 

and the thymus. In adult chickens, the highest percentage of lymphocytes expressing J 

chain was found in the spleen. It helps in secretion of IgA or IgM but not be. associated 

with the expression of surface immunoglobulin[54]. Although IgA does not fix 

complement, the immunoglobulin does have several effector functions including viral 

neutralization, inhibition of bacterial adherence, and acting as an opsonin for mucosal 

phagocytes [55]. 

https://en.wikipedia.org/wiki/Antibodies
https://en.wikipedia.org/wiki/IgM
https://en.wikipedia.org/wiki/IgA


 

 

17 

 

The mucosal immune response also has a role in protection from the HPAI infection 

because the initial exposure to the virus is through a mucosal surface. However, few 

studies directly addressed the mucosal immune response in chickens and turkeys. It 

was shown that chicken can produce IgA immune response after infection with NDV 

and infectious bronchitis virus, with the evidence of IgA providing a protective immune 

response against challenge with virulent strains of these respiratory viruses [28]. 

 

2.2.2.2.2. Cellular Immune responses:  

In avian species, lymphomyeloid tissues develop from Bursa of Fabricius or thymus 

gland which are populated by hematopoietic stem cells. The stem cells then develop 

directly into immunologically competent B (bursa) and T (thymus) cells. Cell-mediated 

immunity (CMI) is a part of adaptive immune response which uses highly specific 

antigen receptors on B- and T-lymphocytes that are generated by random processes of 

gene rearrangement. Two well-defined avian T-cell lymphocytes subpopulations: T 

helper (TH) and T cytotoxic lymphocytes (CTL) cells are characterized by antigenic 

glycoproteins found on their surfaces. The subsets of T-lymphocytes, CD4+ helper 

cells and CD8+ cytotoxic cells constitute the principal cells of CMI response.  T-cells 

displaying CD4+ markers generally function as TH cells whereas those displaying 

CD8+ function as CTL cells. Two subpopulations of TH cells described as TH1 and TH2 

are differentiated based on cytokine profiles they secret following stimulation. TH1 

cells secrete cytokines that support induction of a cell-mediated immune response 

including interferon-γ and interleukin (IL)-12. TH2 cells produce IL-4, 5, and 13 which 

are responsible for aiding in the humoral immune response. In poultry, target cell 



 

 

18 

 

recognition by CD8+ T cells and the ability of CD8+ T cells to kill target cells in the 

context of the major histocompatibility complex (MHC) class I restriction have been 

well studied [56]. MHC plays a dominant role in presentation of antigens by antigen-

presenting cells to CTLs and optimal activation of CTLs depends on identical MHC 

class I antigens. Antigen-presenting cells, like dendritic cells, macrophages, B cells 

process antigen and display epitopes to T cells in MHC class II molecules and provide 

other signals needed to initiate immunity[42].  

 

Chicken MHC: 

Chicken MHC determines resistance and susceptibility to many infectious viral 

diseases. The BF-BL region of chicken MHC is defined as genetic locus with 

polymorphic classical MHC genes responsible for graft rejection and antigen 

presentation to T-lymphocytes. CD8 CTLs primarily recognize class I molecules 

encoded by BF2 gene while CD4 cells recognize class II molecules encoded by BLB2 

gene [57]. Oher genes in BF-BL region include those that encode accessory molecules 

like TAP1 and TAP2 (transporter associated with antigen presentation) which pump 

peptides from cytoplasm into lumen of endoplasmic reticulum for loading class I 

molecules [51].     

 

2.3. AIV Vaccines in use: 

2.3.1. Requirements for a good avian influenza vaccine:  

The ideal vaccine should be potent, safe, stable at room temperature, easily 

administered through oral or mucosal route, sufficient to induce protection after a 



 

 

19 

 

single dose, and cheap. It should also enable differentiation between vaccinated and 

infected animals (DIVA). There are currently two types of vaccines against AIV in 

use: inactivated whole AIV vaccine and live recombinant vaccines [28].  

 

2.3.2. Types of AIV vaccines:  

2.3.2.1. Inactivated whole virus vaccine:   

Conventional AIV vaccines are produced with the whole virus of a specific subtype 

grown in embryonating chicken eggs (allantoic fluid injected with that virus strain). 

The viruses are inactivated by β-propiolactone or formalin, and are adjuvanted with 

mineral oil paraffin [58]. Limitations of this type of vaccine include the requirement 

to administer multiple doses to individual birds against each subtype of AIV; the 

difficulties of distinguishing vaccinated from naturally infected birds by common 

serological tests; incomplete inactivation of vaccine viruses that can exacerbate an 

outbreak; biohazards associated with manufacturing the vaccines as well as the need 

to use large amounts of antigen to elicit an adequate antibody response  significantly 

increasing the production cost[59]. Generally, for the inactivated vaccines, the primary 

vaccination course comprises two or three injections; further “booster” doses may be 

required at intervals to maintain protective immunity.  

 

2.3.2.2. Viral vectored vaccines:  

Viral vectored vaccines express protective antigen or antigens of one or more 

pathogens from the genome of a non-pathogenic virus (vector). Such vaccines can be 

rapidly developed against newly emerging pathogens by recombinant DNA methods. 

https://www.sciencedirect.com/topics/immunology-and-microbiology/antibody-response


 

 

20 

 

The advantage of this type of vaccine is the ability of viruses to infect host cells so they 

can efficiently express various antigens required for protective immunity. Some vectors 

are capable of limited replication while others are designed to be replication defective 

and are restricted to single cycle of infection [60]. 

The main advantages of viral vectors are as follows: (a) high efficiency of antigen 

expression; (b) specific delivery of genes to target cells; and (c) induction of robust 

immune responses and increased cellular immunity because the antigen is expressed 

upon (limited) vector replication. Besides, viral vector-based vaccines have the 

advantage to be used as DIVA vaccine as these vaccines can induce an antibody 

response that is different from the antibody response produced by wild type strain of 

the virus. Various types of viral vectors have been developed and evaluated in animal 

studies and clinical trials. 

 

Types of vectored vaccines against AIV based on viruses other than NDV: 

1. Avipoxvirus -based vaccine [Fowl Pox virus (FPV) vectors]:  

Fowl pox virus (FPV) recombinant vectors belong to Avipoxvirus genus that can infect 

chickens, turkeys, and many other species of birds. FPV have large double -stranded 

DNA genomes which accommodates expression cassettes with foreign genes encoding 

immunogenic viral proteins [61]. They are first generation poultry recombinant vector 

vaccines used for the expression of antigens of various avian pathogens like NDV, 

infectious laryngotracheitis virus (ILTV), AIV and Mycoplasma gallisepticum [62, 63]. 

 

 



 

 

21 

 

Advantages:  

1. Anti-FPV (vector) maternally derived antibodies (MDAs) do not interfere with 

recombinant FPV vaccine protection [62, 64]. 

2. This vaccine can be used to differentiate vaccinated birds from infected ones as the 

commonly used serological tests of avian influenza detects antibodies to NP protein, 

so these tests can easily differentiate vaccinated birds from challenged birds having 

antibodies to the HA and not the NP [65].  

3. Another major advantage that FPV vector can be used for vaccination of day-old chicks 

and they can achieve protection after a single vaccination[66]. 

Disadvantages: 

1. The available FPV vaccines are only available for H5 subtype, with no commercially 

available vaccines for other subtypes. A study by Mazet et al. showed that  the 

immunogenicity and efficacy of FPV vaccines in young chicks with maternally derived 

antibodies depends on the vaccination scheme (prime-boost) and the type of vaccine 

used in their parent flocks since the level of protection is very low against an antigenic 

variant of a highly pathogenic  avian influenza H5 isolate when vaccinated with FPV 

vectored with avian influenza gene insert[66]. 

2. Another study by Swayne et al. (2000) showed that efficacy of a recombinant FPV-

vectored vaccine was severely impaired when given as a secondary vaccine after a 

primary FPV immunization or infection. This lack of consistent protection against 

HPAI limit the use of FPV-based vaccines to chickens without previous fowl pox 

vaccinations. In addition, prior exposure to field fowl poxvirus could be expected to 

limit protection induced by this vaccine [67]. 



 

 

22 

 

2. Herpes Virus of turkeys (HVT) Vectors:  

Another extensively studied and worldwide used second generation poultry 

recombinant vaccines are herpesvirus of turkeys (HVT)-vectored vaccines. HVT has a 

double stranded DNA genome of approximately 160kbp. These vaccines are first 

introduced for the control of virulent Marek’s disease, a highly contagious tumorigenic 

disease caused by Marek disease virus serotype 1 (MDV-1). Several different 

laboratories have used HVT to express AIV proteins for the protection against HPAIV 

[68,69]. 

Advantages: 

1. HVT vaccines can be administered subcutaneously to day-old chicks or by in-ovo 

injection to 18-days old embryos [70]. 

2. Recombinant HVT (rHVT) vaccines have certain advantages over conventional 

modified live poultry vaccines as they do not produce postvaccination reactions, are 

phenotypically stable, do not revert to virulence, and rarely transmit horizontally [ 71]. 

3. Since HVT possess large DNA genome, it has been evaluated for use as viral vector 

carrying protective antigen genes of multiple poultry pathogens [72,73]. 

4. HVT can be administered by mass vaccination as they can be safely injected in ovo. 

5.  These vaccine vectors induce lifelong immunity [74]. 

6. HVT-vectored vaccines can overcome maternally derived antibodies (MDA) because 

the virus spreads primarily cell to cell, which is important as most chicks have maternal 

antibody against HVT. The HVT-vectored vaccines provided sufficient protection 

against NDV and MDV challenges in commercial chickens with maternal antibodies 

against NDV-F and MDV1 antigens [75]. 



 

 

23 

 

Limitation: The main disadvantage of this vaccine is that liquid nitrogen is required to 

maintain the viability of infected cells as HVT vectored vaccines are produced as cell-

associated viruses  [76]. 

 

3. RNA replicon (alphavirus) vaccines:  

Alphaviruses are membrane enveloped positive-sense, single-stranded RNA viruses 

which include Semlinki Forest Virus (SFV), Sindbis Virus and Venezuelan equine 

encephalitis virus (VEE). A recently licensed vaccine for AIV in the US is the RNA 

replicon system which uses an alphavirus backbone to express HA protein of avian 

influenza. The essential characteristic of this vaccine is that the vector does not contain 

all genes necessary for viral packaging, so that it cannot spread beyond the originally 

infected cell. Hence the vaccine has characteristics of a live vaccine which induces 

humoral, and cell mediated immunity, but it has the safety of an inactivated vaccine 

[77]. The RNA replicon system was first developed and commercialized in the US for 

swine influenza where the flexibility of the production system allows targeting the 

vaccine to the circulating field strain [78].  

Advantages:  

1. The self-amplifying positive strand RNA genome makes alphavirus vectors attractive 

for short-term transient heterologous gene expression [77].  

2. Alphavirus vectors take advantage of the extremely efficient RNA replication, resulting 

in production of around 200,000 RNA copies from each RNA molecule. 

3. Alphavirus vectors have the flexibility of being used as recombinant viral particles, 

RNA replicons (naked RNA), or layered DNA/RNA vectors for delivery. The naked 



 

 

24 

 

alphavirus RNA vectors are transcribed in vitro RNA from an expression vector coding 

for viral nonstructural replicase genes and the foreign gene of interest inserted 

downstream of the strong alphavirus subgenomic RNA promoter. The production of 

recombinant particles requires co-transfection of the in vitro transcribed vector RNA f 

and a helper vector supplying the viral structural genes into mammalian cell lines. The 

generated particles are capable of one round of infection in a broad range of host cells. 

The layered DNA vector system includes a DNA delivery vector that expresses the 

alphavirus vector RNA under a cytomegalovirus promoter. Furthermore, engineering 

of alphavirus vectors with an additional sub genomic RNA promoter allows the 

generation of replication-competent particles, which can provide improved delivery 

and extended gene expression.  

4. The insert capacity of alphavirus vectors is up to 8 kb, but also delivery of multiple 

constructs with different inserts is feasible. 

5. They have broad host range including neuron cells [79]. 

Limitations:  

1. The main drawback of alphavirus replicons is their low immunogenicity sufficient for 

only partial protection. Schultz-Cherry et al. determined that when Venezuelan equine 

encephalitis virus replicon particles containing the gene expressing HA of H5N1 were 

used to vaccinate day-old chicks subcutaneously or for in-ovo injection of embryos, 

chicks were only partially protected, while birds of 2 weeks age were completely 

protected after a single dose, suggesting that alphavirus replicon immunogenicity and 

efficacy depend on bird’s age and vaccination protocol [80]. 



 

 

25 

 

2. Another drawback of recombinant alphavirus particles is the lack of an efficient 

packaging system limiting the recombinant virus production [77]. 

 

2.4 Newcastle Disease Virus: 

2.4.1. Classification: 

 Newcastle Disease Virus (NDV) is a member of the genus Orthoavulavirus, family 

Paramyxoviridae, subfamily Avulavirinae [24]. The subfamily Avulavirinae comprises 

paramyxoviruses isolated from avian species. Their virions have hemagglutinin and 

neuraminidase activities and they have been classified into 20 serotypes (APMV 

serotypes 1 through 20). All NDV strains are classified as Avian Orthoavulavirus 1, 

formerly Avian paramyxovirus 1 (APMV-1). NDV is an important poultry pathogen, 

and it is most extensively studied virus in the subfamily Avulavirinae [81]. 

 

2.4.2. Morphology: 

NDV particles are 100 to 500 nm in diameter, pleomorphic, but mostly spherical in 

shape. The virion is enveloped with a lipid membrane obtained from host cell plasma 

membrane. The envelope contains two transmembrane glycoproteins—HN and F. 

These proteins are present as homo-oligomers (HN tetramer and F trimer) and form 

densely packed spike-like projections on the outer surface of the envelope. Beneath the 

envelope lies a layer of non-glycosylated matrix (M) protein, tightly associated with 

phospholipid membrane. Inside the viral envelope is the nucleocapsid, which has the 

classical herringbone morphology with the mean diameter of 17 nm. The 

viral nucleocapsid consists of  RNA genome  and replicase complex proteins—the 



 

 

26 

 

nucleocapsid (N), the phosphoprotein (P) and the large polymerase protein (L) [82]. The 

viral genomic RNA in association with N and P proteins can function as a template for 

viral transcription. 

 

 

Fig 3A: Structure of NDV: The six main viral proteins are shown here: nucleo-protein 

(NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-

neuraminidase (HN), and polymerase protein (L). ssRNA (single stranded RNA is 

wrapped by NP, P and L proteins. (Cuoco et al, Neurosurg Focus, 2021). 

3B. Schematic representation of the structure of the NDV genome: The red arrow 

indicates the cleavage site of the F protein. The representative amino acid motifs are 

the virulent- and avirulent-type cleavage sites (Hu et al, Veterinary Research, 2022). 

 

2.4.3. Genome: 

The genome of NDV is a non-segmented, single-stranded RNA of negative polarity. 

The length of NDV genome (15,186 to 15,198 nucleotides) must be in an even multiple 

of six (“Rule of Six”) for efficient replication, which is believed to be determined by 

the interaction of the genome RNA with the N protein [24]. The NDV genome contains 

six transcriptional units separated by short intergenic regions that encode nucleocapsid 

A 



 

 

27 

 

protein (NP), phosphoprotein(P), matrix protein (M), fusion (F) protein, 

hemagglutinin-neuraminidase (HN) and large polymerase (L) protein. Two additional 

proteins, V and W, are expressed from mRNAs which are derived from the P gene via 

RNA editing [83].  The N gene is preceded by a short leader region and L is followed 

by a short trailer region which contain cis-elements important for the RNA replication 

[84]. 

 

2.4.4. Viral proteins: 

NDV genome encodes eight proteins: N, P, V, W, M, F, HN and L. N, P, and L together 

with RNA genome form the nucleocapsid which is considered the minimum infectious 

unit sufficient for initiation of transcription and replication of the viral genome. The 

proteins M, F and HN are associated with the envelope. The M or matrix protein forms 

the inner layer of the envelope and is not an integral membrane protein. F and HN are 

the only two integral membrane proteins present on virion surface, which are the major 

antigens against which the host produces neutralizing antibodies. 

 

Nucleocapsid protein (N): 

The nucleocapsid (N) protein is 489aa long with a molecular weight of 55kDa and is 

the most abundant viral protein found in infected cells. N protein selectively binds to 

genomic and antigenomic RNAs in infected cells to form helical nucleocapsid 

structures. The genomic RNA in association with N, P and L proteins form 

ribonucleoprotein complex (RNP) acting as a template for RNA synthesis. The amino-

terminus of N protein is responsible for its interaction with viral RNA and for the 



 

 

28 

 

formation of herringbone-like structure [85]. It has been observed that the first few 

amino acids in the amino terminus of NDV N protein form a soluble complex with P 

protein. The carboxy terminus is not required for nucleocapsid assembly but 

supposedly plays a regulatory role in its polymerization [86]. It protects viral RNA from 

degradation and impairs recognition of viral RNAs from host innate immune responses 

[87]. 

 

Phosphoprotein (P): 

The P protein of NDV is 395 aa long, heavily phosphorylated at multiple serine and 

threonine residues, has a molecular weight of around 50-55kDa, and is an essential 

component of viral RNA-dependent RNA polymerase[88]. It is expressed from unedited 

P mRNA and functions as a homo-oligomer in paramyxoviruses while its homolog in 

paramyxoviruses is a tetramer [89]. This protein plays a key role in viral replication and 

transcription. It helps in stabilizing the L protein in the P–L complex which then 

functions as viral RNA-dependent RNA polymerase. The P–L complex carries out 

genomic replication, first synthesizing full-length plus strand antigenomic RNA which 

is used as a template to synthesize minus-strand genomic RNA. It has also been 

observed that P protein modulates virulence depending on the cell type and the NDV 

strain [88]. Different domains of P protein carry out different functions of the P–N 

complex while interacting with N protein during virus replication. A study by Karlin et 

al (2003) showed that paramyxovirus P protein consists of an intrinsically disordered 

N terminal domain (PNT) and a C- terminal domain (PCT) [90]. The ordered domains 

of PCT are P multimerization domain (PMD) and C-terminal X domain. PMD and XD 



 

 

29 

 

are separated by intrinsically disordered protein regions (IDPR)).These IDPRs play key 

roles in N-P-L complex formation [91]. The carboxy-terminal 45 amino acids (247–

291) participate in P–P and P–N interaction [92]. 

 

V and W proteins:  

During transcription of the P gene the viral polymerase occasionally inserts one or two 

non-templated G nucleotides at a conserved editing site, generating mRNAs coding for 

the V and W proteins, respectively, which have the same 135 amino acid N-termini as 

the P protein but different C-termini [87, 83]. The V protein of NDV is 239 aa long with 

molecular weight of 36 kDa. It is a multifunctional protein playing important roles in 

both virus replication and virulence. The V protein regulates viral RNA replication by 

interacting with the N protein [93]. It also plays a major role in NDV pathogenesis by 

antagonizing the antiviral effects of IFN in a species-specific manner. It blocks IFNα 

signaling in avian but not in mammalian cells by targeting signal transducer and 

activator of transcription 1 (STAT1) degradation [94]. Furthermore, the V protein plays 

important role in preventing apoptosis in a species-specific manner [95]. 

 

W protein:  

Incorporation of two G residues at the P gene editing site produces W mRNA which is 

of low abundance. W protein of NDV is 221aa long [24]. W proteins from different 

NDV strains localize in either the cytoplasm (e.g., NDV strain SG10) or the nucleus 

(e.g., NDV strain La Sota) [96]. Compared to the P and V proteins, the W protein has 

the same N-terminal domain but a unique C-terminal domain. While the V protein is 



 

 

30 

 

known as a key virulence factor and an important interferon antagonist across the 

family Paramyxoviridae, very little is known about the function of the NDV W protein. 

It was shown showed that variations in NDV W protein subcellular localization (in the 

nucleus and/or mitochondria) impact IFN-β production, consequently affecting NDV 

virulence, replication, and pathogenicity [96]. Research done by Park et al. showed that 

expression of the NDV V protein or the Nipah virus V, W, or C proteins rescues NDV-

GFP replication from the transfection-induced IFN response. The V and W proteins of 

Nipah virus block activation of an IFN-inducible promoter in primate cells. 

Interestingly, the amino-terminal region of the Nipah virus V protein, which is identical 

to the amino terminus of Nipah virus W is sufficient to exert the IFN-antagonist activity 

[ 95]. 

 

Matrix (M) protein: 

 The molecular weight of protein M is 40KDa, it consists of 364 aa and is the most 

abundant protein in the virion [97]. It promotes the virion assembly and budding [98]. It 

also interacts with actin [99]. The protein forms the inner layer of the viral envelope and 

is responsible for maintaining viral structural integrity [98]. It has been observed that a 

fraction of the M protein localizes in the nucleus early in infection, where it associates 

with the nucleoli and remains in the nucleus throughout infection, which is important 

for the inhibition of host cell transcription and protein synthesis [100][101]. It is 

considered to be the central organizer of virion morphogenesis, interacting with the 

cytoplasmic tails of the integral membrane proteins F and HN, the lipid bilayer, and the 



 

 

31 

 

ribonucleocapsid and promoting the transport of viral components to the plasma 

membrane [102].  

 

Fusion (F):  

The F protein is a type 1 surface membrane glycoprotein present on NDV envelope, 

and it mediates entry by fusion of the viral envelope with host cell membrane. It is 

synthesized as an inactive precursor F0 (consisting of 553 amino acids) which is 

cleaved by host proteases into two biologically active disulfide-linked subunits F1 and 

F2 [103]. The cleavage specificity is determined by the amino acid sequence 

composition of the cleavage site which varies in different strains [104, 105]. The cleavage 

site of F0 protein is a major determinant of NDV virulence. The F proteins of virulent 

NDV strains contain lysine (K) and arginine (R) at their cleavage site (112R-R-Q-R/K-

R116), and a phenylalanine at position 117 of F1. This site is recognized by ubiquitously 

expressed intracellular furin-like proteases resulting in fully infectious virus budding 

from the cellular membrane. In addition, other subtilisin-like mammalian proteases like 

PC6 and PACE4 are also the candidates for the cleavage of the F protein [106]. F protein 

fusion activity is functional at neutral pH. Expression of the F protein leads to the 

formation of multinucleate cells (syncytia) which induces tissue necrosis and promotes 

virus spread. Low virulent strains of NDV contain monobasic or dibasic amino acid 

residues at its cleavage site. The presence of one or two basic amino acids renders F 

proteins insensitive to the intracellular proteases and makes the virion maturation 

dependent on extracellular proteases, limiting its tropism to respiratory and alimentary 

tracts. 



 

 

32 

 

Hemagglutinin-Neuraminidase (HN): 

HN protein is a type II integral membrane glycoprotein with molecular wight of around 

74kDa. The protein contains an anchor sequence at the N-terminal cytoplasmic tail and 

a large extracellular C terminal domain connected by hydrophobic transmembrane 

domain. It is a multifunctional protein which is responsible for attachment of virion to 

cell surface receptors, increases the fusion activity of F protein and has neuraminidase 

activity that cleaves sialic acid resulting in the release of progeny virions from the 

surface of infected cells. HN is also one of the major protective antigen of the virus and 

plays important role in pathogenicity and immunogenicity of the virus [107,108,109]. 

 

Large Polymerase protein (L): 

The largest protein encoded in the NDV genome, L protein consists of 2204 aa and has 

a molecular weight of 250 KDa [87]. It is the least abundant protein in infected cells and 

virion, it is included in the  nucleocapsid where it associates with P protein through the 

N-terminus [87]. The L protein functions as homomultimers, and L-L interaction occurs 

through N- terminal self-assembly domain. The L protein also interacts with host cell 

proteins and other viral proteins [87]. It has been reported that L protein modulates the 

virulence of NDV presumably by controlling the rate of viral RNA synthesis during 

replication[110]. L protein is the enzymatic subunit of the viral RNA-dependent RNA 

polymerase and has many functions which include synthesis of viral mRNAs, genomic 

RNA replication, mRNA capping, methylation, and polyadenylation of mRNAs [24, 88]. 

In paramyxoviruses, the N terminus of L protein interacts with P protein. 

 



 

 

33 

 

2.4.5. UTR regions:  

The transcriptional unit of NDV gene contains major ORF with 5’ and 3’ untranslated 

regions (UTRs) of varying lengths flanked by conserved transcription initiation and 

termination control sequences called gene start (GS) or gene end (GE) respectively [24]. 

The non-translated leader region of 55 nucleotides and trailer regions of variable length 

of the NDV genome contain promoters for viral replication [60]. The transcription of 

NDV starts at a single promoter site located at 3’ leader end, and all six genes of NDV 

are copied into individual mRNAs by start-stop mechanism in a sequential manner 

guided by GS and GE signals [87]. Previous studies have shown that 5’ UTR modulates 

level of transcription and translation of downstream gene affecting viral replication and 

pathogenicity, and that complete deletion of NDV HN UTRs decreased mRNA level 

and protein content in virus particles [111]. 

 

2.4.6. Life cycle of NDV: 

2.4.6.1. Viral adsorption and entry:  

NDV initiates infection by attaching to the epithelial cells of respiratory and enteric 

tracts by binding to sialic acid-containing receptors like N- glycoproteins and 

gangliosides on cell surface by its surface glycoprotein HN. The virus enters host cells 

by direct fusion of the viral membrane with the host cell membrane and via endocytosis. 

The viral entry results in the release of helical nucleocapsids into the cytoplasm. The 

acidic pH of the endocytic pathways promotes NDV entry through disruption of matrix 

protein oligomeric structure for the nucleocapsid release [112]. Previous studies 

revealed that NDV enters HeLa and avian fibroblast cells (DF1) by cell fusion through 



 

 

34 

 

receptor-mediated and dynamin-dependent endocytosis [113], furthermore it infects 

DF1 cells via micropinocytosis and clathrin-mediated endocytosis [114]. The virus 

spreads from infected to uninfected cells by release into extracellular space and by 

syncytia formation. 

 

2.4.6.2. Viral RNA synthesis:  

Viral transcription and replication are mediated by viral RNA-dependent RNA 

polymerase. The replication of the genome starts when the polymerase initiates RNA 

synthesis at the first nucleotide at 3’ end of the genome followed by synthesis of 

complete positive sense copy of the genome called antigenome. Both the genome and 

antigenome are encapsidated by the nucleocapsid protein. The processes of 

transcription and replication are tightly regulated in NDV. The switch from 

transcription to replication is controlled by the intracellular concentration of 

nucleocapsid protein. When unassembled nucleocapsid protein is limiting, the viral 

RNA polymerase is preferentially engaged in mRNA synthesis, raising the intracellular 

levels of unassembled nucleocapsid protein and all other viral proteins. When 

nucleocapsid protein levels are sufficient for encapsidation of nascent RNA chain, viral 

RNA polymerase activity switches from transcription to replication mode. A study by 

Horikami et al, showed that the replication mode of the polymerase is favored when 

the concentration of N  protein is high enough to allow encapsidation of the nascent 

RNA chain [115]. The 3’ end of the antigenome contains complement of trailer called 

antigenome promoter, which signals the polymerase to synthesize progeny genomes 

using antigenome as template. The transcription of paramyxovirus follows the “start-



 

 

35 

 

stop” mechanism, The polymerase stops at the upstream GE and reinitiates synthesis 

of the next mRNA at the next GS. The intergenic sequences (IGS) are not transcribed. 

This “start-stop” mechanism of transcription results in a gradient level of mRNA with 

higher levels of mRNAs transcribed from genes located at the 3’end of the genome. 

The RNA genome template is copied without dissociation of N from viral nucleocapsid 

core during transcription and replication. 

 

2.4.6.3. Virion assembly and release: 

The first step in viral assembly is the encapsidation of genome RNA into nucleocapsid 

in the cytoplasm of infected cell. First, the free N proteins are tightly associated with 

the genomic RNA to form the ribonucleocapsid protein (RNP) core structure; secondly, 

the P and L proteins bind to RNP core, forming a complex [88]. The membrane 

glycoproteins, F and HN, along with M protein and RNPs are needed for virion 

assembly. The F and HN glycoproteins are synthesized in the endoplasmic reticulum 

(ER) and undergo stepwise conformational maturation during transport through the 

secretory pathway. The mature F and HN proteins are transported to the plasma 

membrane. The M protein plays a coordinating role by interacting with cytoplasmic 

surface of the plasma membrane via hydrophobic interactions with lipid bilayer and 

binds to plasma membrane where the viral surface glycoproteins are concentrated [116] 

and brings the assembled viral RNPs to the plasma membrane to form budding virions. 

Study on NDV virus-like particle (VLP) suggests that M-HN and M-NP interactions 

are responsible for incorporation of HN and NP proteins into VLPs and the F protein 

is incorporated indirectly due to interactions with NP and HN protein [98]. The virions 



 

 

36 

 

assembled at the plasma membrane are released by budding from host cell membrane.  

Studies suggested that the assembly and release of infectious NDV particles like that 

of other enveloped RNA viruses take place at the membrane lipid rafts [117].  

 

2.4.7. Molecular Basis of NDV virulence: 

NDVs are divided into three major pathotypes based on virulence in chickens: (i) 

velogenic strains cause severe respiratory, enteric, and neurological disease with high 

mortality; (ii) mesogenic strains cause disease that resembles that of velogenic strains 

but is less severe with mortality only in very young birds; (iii) lentogenic strains cause 

mild or asymptomatic infections limited to respiratory or enteric tract, and are often 

used as live vaccines against velogenic strains [81]. The  velogenic strains are further 

divided into viscerotropic (causing hemorrhagic lesions in intestines ) or neurotropic 

(neurological diseases without any intestinal lesions) [118]. The major determinant of 

virulence of NDV strains is the amino acid sequence at F protein cleavage site but the  

degree of virulence is also influenced by other envelope-associated proteins (M, and 

HN) and proteins forming the ribonucleoprotein complex with the genome RNA (N, P, 

L) [119, 120,110]. Among all the viral proteins, F and L play major roles in determining 

virulence of NDV [121]. The envelope-associated proteins promote virulence by 

functioning in the entry and spread of the virus while the internal proteins cause 

virulence by determining the rate of replication of the virus. The primary determinant 

of NDV virulence is the amino acid sequence in the F protein cleavage site which 

differentiates the virus into these three strains[122, 123]. Velogenic NDV strains contain 

multiple basic residues (112R/K-R-Q-R/K-R117), a favored recognition site of host 



 

 

37 

 

protease furin while the F protein cleavage site of avirulent or lentogenic strains have 

a monobasic amino acid motif (112G-R/K-Q-G-R117) which lacks furin recognition 

motif and is cleaved by trypsin-like extracellular proteases (present in secretions of 

respiratory and  enteric tracts of chickens).The furin motif R-X-(R/K)-R (X is any 

residue) of F protein cleavage site in virulent strains of NDV is responsible for the 

replication of the virus in wide range of tissues [124]. 

 

2.4.8. Reverse Genetics of NDV: 

Reverse genetics is a technique of manipulation of genome of RNA viruses encoded as 

cDNA. Genome manipulation of negative-sense RNA viruses is more difficult 

compared to that of positive-sense RNA viruses since the negative-sense RNA viruses 

require the virion RNA to be assembled into an active transcriptase-replicase complex 

to initiate viral infection. Rescue of recombinant NDV needs co-transfection with four 

plasmids: 1.  Full length NDV antigenome, and three support plasmids coding for, 2. 

NDV N gene, 3. NDV-P gene, 4. NDV-L gene. The positive strand RNA will be 

encapsidated by N protein. The P and L proteins synthesize the negative strand viral 

genomic RNA by binding to 3’antigenome trailer thereby initiating the first NDV 

infection cycle. The plasmids used in NDV recovery system are usually under control 

of T7 RNA polymerase promoter. The T7 RNA polymerase is usually expressed by 

coinfecting the cell with a recombinant vaccinia virus expressing the enzyme (Modified 

Vaccinia strain Ankara (MVA-T7) [125].  The reverse genetics technique is now 

available for all three pathotypes of NDV strains [126,125,121,127]. This technique has 

improved our understanding of NDV molecular biology and pathogenesis. 



 

 

38 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 4. Plasmid-based recovery of recombinant NDV: HEp-2 cells are co-

transfected with the antigenome plasmid and expression plasmids encoding the N, P 

and L proteins of NDV. The T7 RNA polymerase is provided by the recombinant 

vaccinia MVA/T7 strain. 

 

2.4.9. NDV as a vector vaccine: 

NDV, an avian paramyxovirus is a promising viral vector for rational design of live 

attenuated vaccines for both human and animal use. It has many advantageous 

characteristics: 

1. NDV infects via the intranasal route inducing robust mucosal and systemic immune 

responses and is useful to deliver protective antigens derived from respiratory 

pathogens. 



 

 

39 

 

2. A wide range of NDV strains (lentogenic LaSota, Hitchner B1) exists that can be used 

as vaccine vectors. 

3. This virus can be safely used for human vaccination due to its natural host range 

restriction. 

4. NDV can be grown with high titers in embryonated chicken eggs or cell culture, thus 

making it a cost-effective vaccine. 

5. In contrast to adeno, herpes or pox virus vectors whose genome encodes many proteins, 

NDV encodes only seven proteins and thus has less competition for immune responses 

between vector proteins and the expressed foreign antigen [23]. 

6. NDV is an RNA virus that replicates in the cell cytoplasm thus there is no risk of its 

integration into the host genome. 

7. Recombinant NDV expressing foreign antigen shows high and stable expression of 

foreign protein after many passages both in vitro and in vivo. 

8. NDV-vectored vaccines can be administered by mass vaccination approaches such as 

by water or aerosol application, thus reducing the cost of administration, major part of 

cost of vaccination. 

9. In humans, infection by NDV appears to be limited and benign based on clinical studies 

using NDV as an oncolytic agent [24]. 

 

2.4.9. The rationale behind using NDV as a vector for my project: 

Apart from all the above discussed advantages of NDV as a vector, the main rationale 

for selecting this vector over other vectors is mainly depend on the quantity of antigen 

expressed by this vector upon infection of the cells by NDV vector. Research led by 



 

 

40 

 

Nakaya et al. showed about the first NDV-vectored vaccine which was generated by 

expressing the major protective antigen, HA protein of human influenza A/WSN/33 

(H1N1) [ 128]. This approach of NDV as a vector was further evaluated for poultry 

vaccination against HPAI viruses. NDV is an ideal vaccine vector for prevention of 

HPAI virus infections in poultry since its infection route is intranasal as the same 

route of infection with AIV, thus capable of inducing both local and systemic immune 

responses at the respiratory tract [7, 129, 130]. The presence of neutralizing antibodies 

specific for the HA protein at systemic or mucosal sites of infection provides 

immediate protection against AIV. In addition to that, NDV vector can be used to 

update protective antigens with replaceable vaccine cassettes of currently circulating 

virus in the field.  NDV also grows to high titers in cell cultures (108 pfu/ml) and in 

embryonated eggs (109 pfu/ml), thus making it cost-effective [131]. Since my project 

involves with protective antigens for H7 and H5 avian influenza strains, there’s no 

alternative as best fitted vector like NDV. 

 

 

 

 

 

 



 

 

41 

 

Chapter 3: Evaluation of protective efficacy of NDV vectored 

vaccine candidates against H7 HPAIV in broiler chickens and 

turkeys. 

3.1. Introduction: 

Highly pathogenic avian influenza (HPAI) virus causes a devastating disease in 

poultry and is an economically important worldwide pathogen of poultry. The natural 

reservoirs of the virus are  wild aquatic birds including ducks, gulls, and shorebirds 

[132]. In their natural host, avian influenza viruses typically cause asymptomatic 

infection and little pathology. In contrast, HPAI virus causes severe disease in 

terrestrial birds. Avian influenza virus is divided into subtypes based on combination 

of two viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) (16 

HA types and 9NA types) [1]. The HA protein is responsible for virus attachment to 

the host cell and is the major target of the humoral immune response [2]. The NA 

protein plays a role in release and spread of progeny virions by removing sialic acid 

from glycoproteins. The NA antibodies have been shown to play a role in reducing 

clinical signs and shedding [3]. Low pathogenic avian influenza (LPAI) viruses 

contain an HA cleavage site which can only be cleaved by proteases produced in the 

intestinal and respiratory tracts [4]. In contrast, HPAI viruses contain multiple basic 

amino acids at the HA0 cleavage site, resulting in cleavability of HA by ubiquitous 

intracellular proteases. Therefore, HPAI viruses can cause systemic infection and 

high mortality in chickens and other terrestrial poultry. Two subtypes (H5 and H7) of 

LPAIVs can naturally change to a highly pathogenic phenotype by acquisition of 

basic amino acids in the cleavage region of the HA protein through insertion or 



 

 

42 

 

substitution and recombination with another gene segment(s) or host RNAs 

[6].Research done by Horimoto etal showed after three consecutive passages in 14-

day-old eggs, avirulent H5 viruses with K/R-K-K/T-R sequence at the HA cleavage 

site became virulent in chickens acquiring high HA cleavability [6].  

Depopulation of infected flocks is commonly used to control the spread of avian 

influenza viruses (AIV) in poultry. However, the use of vaccines in poultry has 

increased during the past two decades with the increase in the number of countries with 

endemic AIVs [133]. Vaccine development has become a critical tool to control AIV 

infection in poultry and to prevent transmission of these viruses from birds to 

humans[18,19, 131]. Currently, effective vaccines and efficient vaccination methods are 

required for a better protection of poultry.  

Live Newcastle disease virus (NDV) vectored vaccines have shown promising results. 

However, maternal antibodies to vaccine vectors have previously hampered the 

efficacy of NDV vectored vaccines [134]. To overcome this limitation, a chimeric NDV 

vector (NDV strain Beaudette C) was generated by replacing the ectodomains of F and 

HN proteins with those of serologically distinct avian paramyxovirus serotype-2 

(APMV-2) (Fig.5) [21]. This additional chimeric vector was constructed due to 

inefficient replication of avirulent APMV-2. This chimeric virus was attenuated and 

safe for administration to chickens but replicated efficiently in vivo. We further 

established a heterologous prime and boost immunization approach for protection of 

broiler chickens against H5 HPAI viruses [130,98]. In this study, we generated NDV 

vectored vaccine candidates against H7 HPAIV. To provide broadly reactive H7 

subtype-specific avian influenza vaccine, consensus sequence of the HA protein was 



 

 

43 

 

expressed by NDV vectors as the protective antigen. Based on the previously vaccine 

improvement challenge research, we aimed to explore the protective efficacy of our 

vaccine candidates in broiler chickens and turkeys against highly pathogenic H7 AI 

virus.  

 

 

 

 

 

 

Figure:5. Representation of antigenically distinctive chimeric NDV vaccine 

vector: Mesogenic NDV strain BC is on the top of the figure. A chimeric NDV vector 

is generated by replacing the ectodomains of NDV F and HN proteins with those of 

avirulent avian paramyxovirus serotype-2 (APMV-2). 

 

Strategy to address the problem: 

H7 viruses are classified into two major genetic lineages, the American and Eurasian 

lineages [7]. Hence, we propose to use a consensus HA sequence covering both North 

American and Asian isolates to generate a broadly effective H7 vaccine. Viral vectors 

offer several advantages over traditional vaccines. Viral vector vaccines induce 

outstanding antibody and cytotoxic lymphocyte responses (CTL), which is important 

for control of viral infections [133]. The use of NDV as a vaccine vector for HPAIV 

has multiple advantages over other viral vaccines [23]. The NDV-vectored vaccines 



 

 

44 

 

are highly restricted for replication in the respiratory tract and provide protection 

against challenge with HPAI viruses. Various strategies have been used previously to 

enhance protective immunity by modifying NDV vector and trying diverse ways of 

immunization. In previous studies, a heterologous prime and boost immunization 

approach was established for protection of broiler chickens against H5 HPAI viruses 

[130]. In an AIV infection, antibodies are produced against both surface proteins, HA 

and NA. Antibodies against HA inhibit the attachment to the sialic acid-containing 

host cell receptor and inhibit fusion between viral and host cell membranes[2]. 

Antibodies to the NA protein impede its receptor-destroying function reducing virus 

replication by inhibiting virus release from infected cells. Therefore, HA and NA 

proteins of target influenza strains have been expressed simultaneously in various 

vaccine development studies [135]. This led us to simultaneously express the HA 

protein as a major protective antigen and the NA protein as a minor antigen by taking 

an advantage of the nature of polar gradient of NDV transcription[136]. Previous 

research result  showed that boosting 2-week-old chickens with LaSota/ H5 can also 

serve as a dual vaccination approach against HPAIV and highly virulent NDV [ 

130].  Based on this hypothesis, we aimed to follow the heterologous scheme using 

H7HA consensus sequence and N8NA as antigens to develop an improved protective 

response against H7-HPAIV infection in poultry. 

 

 

 

 

https://www.sciencedirect.com/topics/medicine-and-dentistry/membrane-protein
https://www.sciencedirect.com/topics/medicine-and-dentistry/cell-receptor


 

 

45 

 

3.2. Materials and Methods: 

3.2.1. Generation of chimeric NDV vectored vaccine candidates: 

HA protein sequences were obtained from NIAID Influenza Research Database (IRD) 

(PMID 27679478) for designing the HA consensus sequence. A multiple sequence 

alignment (MSA) was generated using the MAFFT program, version 7 (PMID 

23329690). The MSA was analyzed by a custom Perl program to generate the 

consensus sequence which has the cleavage site sequence of HA protein of LPAIV 

(PENPKTR; GLF) (Fig.6A). The coding sequence for the HA gene was synthesized 

with codon optimization since this enhanced the levels of protein expression in our 

previous invitro replication studies [134]. In addition, co-expression of HA and NA 

genes of avian influenza virus enhanced protective efficacy of vaccines according to 

previous research [134]. Hence, both HA and NA genes were cloned in the NDV vectors. 

Since HA protein is a major protective antigen, we inserted HA gene into upstream and 

the NA gene into downstream of NDV genome by taking an advantage of polar gradient 

of NDV transcription (Fig.6.B) [130]. The HA gene was placed between the P and M 

genes of each NDV vector using the restriction enzyme site of PmeI (Fig.6.B). 

Subsequently, the N8 of NA gene was placed between M and F genes of NDV strain 

LaSota using restriction enzyme site of PacI. Each ORF of HA and NA gene was 

flanked by gene-start and gene-end signals of respective NDV vector following the 

‘‘rule of six.” Therefore, three novel vaccine candidates were generated : 1. Chimeric 

NDV/HA, 2. LaSota/HA and LaSota/HA-NA. Infectious viruses were rescued using 

NDV reverse genetics following standard protocol of NDV [137]. 

  



 

 

46 

 

 

 

 

 

6A.                                                                   6B. 

 

 

 

 

 

 

Figure:6. Generation of chimeric NDV and LaSota vectored vaccine viruses: 

A. A phylogenetic tree of HA sequences of the North American and Asian isolates was 

generated to determine whether the consensus sequence aligned within the clusters 

from the original sequences (indicated with red color in circle in fig. 6A).  

B. The H7HA and N8NA genes were placed between the P and M genes and between 

the M and F genes, respectively. Ectodomains of the F and HN genes derived from 

APMV-2 are shown as orange rectangle. 

 

3.2.2. In vitro experiments: 

Expression of HA and NA proteins in virus-infected chicken embryo fibroblast cell line 

(DF1) was confirmed by Western blot and immunofluorescence analyses. Each virus 

was further purified by using a 30% sucrose cushion. Incorporation of the HA and NA 



 

 

47 

 

proteins into NDV particles was analyzed by Western blot. The efficiency of in vitro 

replication of the vaccine candidates was determined in virus infected DF1 cells in 

duplicate [134]. 

 

3.2.3. In vivo experiments: 

3.2.3.1. Protective efficacy of heterologous NDV vectored vaccines in broiler 

chickens:  

For vaccination groups, twenty-day-old broiler chickens (one-day old) were 

immunized with chimeric NDV/HA by intranasal route. At 2 weeks post prime, the 

chickens were subdivided into two groups (10 for each group) for booster 

immunization with either LaSota/HA (group 1) or LaSota/HA-NA (group 2). Before 

prime immunization, eight one-day old chicks were randomly selected and remained 

unimmunized as a control group. Prior to boost and challenge experiments, serum 

samples were collected to monitor the immune responses in chickens. The antibody 

titers in serum samples were determined by hemagglutinin inhibition assay (HI) using 

NDV strain LaSota, chimeric NDV or influenza H7N8 virus as individual antigens to 

evaluate the induction of vector-specific and H7-specific antibody responses. The 

detailed illustration of vaccine scheme and challenge day for broiler chicken is shown 

below in Fig.7. 

 

 

 

 



 

 

48 

 

Fig.7. Schematic model of animal experiments for broiler chicken. In same way 

turkey immunization is done. 

 

Hemagglutination Inhibition Assay (HI) assay: 

The hemagglutination inhibition (HI) assay is used to titrate the antibody response to a 

viral infection. The HI assay takes advantage of the ability of some viruses to bind red 

blood cells and thus preventing the red blood cells from clumping. In the HI assay, 

twofold dilutions of the sera to be tested are made in 96 well plates. A known titer of 

the virus is added, and the plate is incubated for 30 minutes at room temperature. Red 

blood cells of chickens are then added, and the plate is incubated for 30 minutes at 

room temperature. If antibodies are present in the sera sample that cross-react with the 

virus, the antibodies will bind to the virus and prevent the virus from hemagglutinating 

the red blood cells. In this way, the titer of the antibodies in the sera can be determined. 

 

3.2.3.2. Standard Pathogenicity Test:  



 

 

49 

 

To assess any pathology associated with vaccination we evaluated intracerebral 

pathogenicity index (ICPI) test. The vaccine candidates were subjected to the standard 

intracerebral pathogenicity index (ICPI) test in 1-day-old specific-pathogen free (SPF) 

chicks. Fresh infective allantoic fluid with an HA titer greater than 24 (1/16) was 

diluted 1/10 in sterile isotonic saline with no additives. 0.05mL of the diluted vaccine 

preparation was injected intracerebrally into each of 10 one-day-old chicks hatched 

from an SPF flock. We confirmed that the expression of the HA and NA proteins did 

not affect the avirulent nature of the vaccine candidates. All infected chickens were 

healthy during the 8 days of infection period (ICPI value of 0.00), thus indicating these 

chimeric viruses are safe for vaccination of chickens. All the animal experiments were 

conducted following the guidelines and approval of the Animal Care and Use 

Committee (IACUC) and Institutional Biosecurity Committee (IBC), University of 

Maryland. 

 

 

Fig 8. Illustrative of ICPI test 

 

3.2.4. Statistical analysis: 



 

 

50 

 

Statistically significant differences in serological analysis of different immunized 

groups were evaluated by one-way analysis of variance (ANOVA) using the Turkey’s 

multiple comparison test. The survival rate was compared using the log-rank test and 

chi-square statistics. All the results were analyzed by using Prism 5. (GraphPad 

Software Inc., San Diego, CA) with a significance level of P < 0.05. 

 

3.3. Results: 

3.3.1. Expression of a consensus H7HA protein by NDV vectors:  

For induction of a broad protective immunity, we designed a consensus sequence of 

the HA protein based on the sequences from North American isolates (a total of 186 

sequences, dated from 2013 to 2018). The amino acid sequences of the North 

American HA have 84.8% of identity with those of Asian isolates. A generated 

phylogenetic tree showed that the consensus sequence aligned with the clusters from 

the sequences of North American isolates (indicated with red color in North America 

in extreme left side, Fig. 6A). Further codon optimized HA gene was cloned into two 

NDV vectors. Chimeric NDV vector containing the HA gene (chimeric NDV/HA) 

was generated for prime immunization (Fig. 6B). LaSota vector was used to generate 

boost vaccine candidates: LaSota with HA (LaSota/HA) and LaSota with HA and NA 

(LaSota/HA-NA). After recovery of the vaccine viruses, RT-PCR and sequence 

analysis were performed to confirm proper insertion of HA and NA genes and the 

absence of mutations. Expression of HA and NA proteins in virus-infected chicken 

embryo fibroblast cell line (DF1) was confirmed by Western blot and 

immunofluorescence analyses (IFA). The HA protein (70kDa) in DF1 cell lysates of 



 

 

51 

 

three vaccine candidates was detected by monoclonal antibodies against H7HA 

proteins, A/Netherlands/219/2003 (H7N7) (HA-a in Fig 9A) and 

A/Anhui/1/2013(H7N9) (HA-b in Fig 9A), indicating a broad reactivity of the 

consensus HA protein. Expression of the NA protein (60 kDa) was detected in 

infected DF-1 cells with LaSota/HA-NA using polyclonal antibodies against N8 NA 

protein (Fig.9A). Further, expressed HA and NA proteins were incorporated into 

NDV particles (Fig. 9A). The HA protein was further detected as uncleaved HA0 (70 

kDa) and cleaved HA1 proteins (55 kDa). Parental samples (chimeric NDV and 

LaSota viruses without HA) showed no HA expression as expected. IFA showed that 

all three vaccine candidates expressed HA protein on the surface of infected DF-1 

cells (Fig. 9B). Thus, these experiments suggest that the consensus sequence HA 

protein can be a good immunogen. 

 

 

 

 

 

 

 

 

 

 

 



 

 

52 

 

9A. 

 

 

 

 

 

 

 

 

 

 

 

 

Fig:9. A) Expression of H7 HA and N8 NA proteins by NDV vectors were 

analyzed by Western blot. DF1 cells were infected with each virus at MOI 1. The 

HA protein in cell lysates was detected by using a monoclonal antibody against H7 

HA