Genetic Resistance to Newcastle Disease in Poultry: A Narrative Review

Abstract

Newcastle Disease (ND) is an important and notable disease among the avian infectious diseases, because of its high contagiousness, and the most virulent strains of ND virus (NDV) have impacted poultry breeders all over the world. Immunization and biosecurity measures are used to reduce ND; however, vaccination has been shown to offer protection against clinical signs but not against virus proliferation and shedding, which could have an adverse effect on the environment. The genetic basis for inherent resistance to NDV has been established, and genetic selection on existing resistance-related genetic variation can help to mitigate virus propagation. Further, understanding the genes and processes that drive the response to NDV will lay the groundwork for genetic improvement in poultry. The majority of studies on NDV susceptibility make use of phenotypic indicators such as body weight, morbidity, mortality, antibody response, and viral load. According to recent advancements in molecular genetic research, many different genes are diversely regulated in different chicken lines to NDV infection, which might be used in the future to establish disease-resistant breeding approaches. It is possible that many more genes linked to illness and resistance are still to be discovered, because the precise mechanism of resistance is not entirely understood. The enhanced genetic knowledge of chickens and the development of more advanced transgenic techniques would lead to pathogen resistance. Hence, this paper summarizes the current understanding of genetic resistance to Newcastle Disease, and we additionally highlight a few possible genes/markers connected with NDV that may improve chicken resistance to NDV infections and can be used to produce NDV-resistant chicken breeds/strains in the near future.

1. Introduction

Newcastle Disease (ND) is one of the WOAH (i.e., World Organisation for Animal Health formerly known as the Office International des Epizooties-OIE) notifiable diseases among avian infectious diseases because of its extremely contagious nature. It is caused by the virulent ND virus (NDV), which is highly contagious and can induce respiratory, neurological, and intestinal lesions [1]. The different strains of virulent NDV are endemic to poultry raised in different parts of the world and the poultry raised in some nations (viz., USA and Canada) are not infected; these nations maintain the status quo by imposing restrictions on the import of live poultry, hatching eggs, and raw poultry products from countries where virulent NDV (vNDV) is endemic and also by conducting thorough surveillance and monitoring, and culling sick as well as vulnerable birds from contaminated areas [2,3,4,5]. Immunization and biosecurity are the two main ways that ND is controlled and nonetheless, studies have demonstrated that vaccination offers defense against clinical symptoms but not against the multiplication and shedding of viscerotropic velogenic NDV (vvNDV), which can contaminate the environment [6,7,8,9]. Early detection in the field is difficult due to the wide range of clinical symptoms and lack of pathognomonic lesions, which are comparable to those of many other poultry diseases [10]. Both naturally occurring and experimentally induced NDV infections resulted in decreased egg production [11,12,13].

In general, disease resistance in chickens is a polygenic characteristic involving multiple genes that provide resistance to pathogens. The disease resistance generally refers to an ability to fight off disease or weakened susceptibility [14,15] and under the guidance of multiple genes and impacted by environmental factors, general resistance exhibits immunity and defensive qualities against the majority of infections. Special disease resistance has strong resistance to a limited number of specific illnesses and pathogens and the genome contributes to an individual breed’s distinctiveness by allowing various qualities to be passed down from generation to generation [14,15,16,17]. The global demand for poultry products has led to increased interest in developing disease-resistant chickens and breeding disease-resistant chicken strains could help to combat infections and advance our understanding of host genetics in the battle against contagious diseases.

Genetic resistance to disease provides several advantages over conventional disease control methods, such as immunization, antibiotics, and management strategies. In general, genetic resistance can provide a long-term solution to disease problems, eliminating the need for continual interventions [18,19,20,21] and the genetic resistance can result in healthier birds, and lowering morbidity and mortality rates caused by infectious diseases. The genetic resistance to illness is significant not only for diseases against which vaccinations or medications are available, but also for diseases that have been effectively eradicated, unless in situations when no other effective measures of control exist. Hence, enhancement of survival capacity is an essential element of every pragmatic breeding initiative, irrespective of the principal production characteristic or traits being targeted for selection [22,23,24,25,26,27] and prior to considering other selection factors, it is necessary to look into the existence and significance of genetic variability [23,24].

In genetic resistance studies, choosing the most accurate trait related to resistance for measurement is arguably the most critical question and recording resistance variations throughout lines or breeds is typically the first step; it is also possible to forecast what could be anticipated from selection by estimating heritability. The final stage is to identify the resistance genes related to susceptibility that are resistant to diseases in various poultry lines [22,25,27]. The genetic mechanism underlying innate resistance to NDV is one of the oldest documented links between poultry genetics and disease resistance, having been studied for more than 50 years. It has been conclusively demonstrated by earlier research that the genetic responses of turkeys and hens to NDV have variations [20,21,28,29,30,31,32,33,34,35,36,37,38]. The previous research has provided compelling evidence for the potential of identifying the underlying genotypes in chickens that determine sensitivity and resistance to NDV. However, it is not until now that the genetic basis of resistance can be accurately and fully characterized, as well as efficiently used to improve poultry health [27,39]. A number of genes and markers have been identified as essential components of poultry resistance to NDV and all play a role in host defense [17,23,40,41,42,43,44]. Understanding these genes can help to improve breeding practices and disease management and the development of resistant breeds/strains will also be aided by cutting-edge molecular technologies [23,27,45].

The disease resistance research against NDV is crucial for chicken health, poultry industry support, and food security. As a result, this review aims to undertake a comprehensive analysis of existing knowledge on host genetic factors that influence chicken resistance or susceptibility to NDV. By understanding mechanisms, the work will help us understand the production of genetically modified poultry lines with improved NDV resistance. Such advances could significantly reduce economic losses in the poultry industry which could lead to sustainable prevention of the disease.

2. Review Methodology

This review systematically evaluates published material on chicken genetic resistance to NDV. A thorough and organized search was carried out across many credible electronic databases, including Scopus, PubMed, Web of Science, ResearchGate, and Google Scholar, spanning papers over several decades (from 1920 to date). The official online portals of these databases as well as personal visits to libraries were used to ensure access to genuine and high-quality peer-reviewed literature. We used a keyword-based search strategy that included particular terms like “genetic resistance,” “Newcastle disease,” “NDV,” “chickens,” “molecular breeding,” and “innate immunity genes.” Boolean operators and filters for publication year, article type, and subject area were used to narrow the search. In addition to electronic database searches, manual screening of relevant journals and article references was conducted to uncover studies that were not caught by automated searches. The study inclusion criteria were as follows: (1) original research publications, (2) studies concentrating on genetic or molecular mechanisms of NDV resistance in hens, (3) articles written in English, and (4) studies using proven molecular techniques or genomic tools. Reviews, editorials, and research that did not address NDV resistance mechanisms were omitted. This review’s two main goals were to summarize the most recent scientific developments in the creation of NDV-resistant chicken breeds by molecular and genomic approaches, and to investigate the genetic and immunological responses of chickens after they were exposed to NDV.

3. Newcastle Disease Overview

The NDV was originally reported in Java, Indonesia, in 1926 [46] and then in Newcastle-on-Tyne, England, in 1927 [47,48,49] as well as in India, and is also known as Ranikhet disease due to its place of emergence in India [50]. The International Committee on Virus Taxonomy (ICTV) recently established three genera within the new subfamily Avulavirinae of the Paramyxoviridae family. According to the latest unified phylogenetic classification system and revised nomenclature for NDV, avian orthoavulavirus 1 (AOAV-1) is the causal agent of ND and belongs to the genus Orthoavulavirus, subfamily Avulavirinae, family Paramyxoviridae, and order Mononegavirales [39]. The ND virions are pleomorphic; however, they can seem spherical with a diameter ranging from 100 to 500 nm or filamentous with a diameter of about 100 nm and with varying length [51].

The NDV features a lipid bilayer envelope embedded with two distinct glycoproteins, the haemagglutinin-neuraminidase (HN) and fusion (F) proteins, which appear as small spikes roughly 8 nm long [52]. A layer of non-glycosylated matrix (M) protein, which is comparatively hydrophobic and helps with the packing and release of freshly assembled viruses, lies beneath this lipid membrane [53]. It is thought that the M protein interacts with the nucleocapsid (NP), which has a shape that is similar to the conventional herringbone and is visible when the viral membrane is split or removed. About thousands of NP subunits make up the herringbone structure and are closely linked to multiple copies of large protein (L) and phospho-protein (P). The middle hollow of the herringbone-like nucleocapsid contains the non-segmented, single-stranded negative-sense RNA genome [54,55,56] and the viral transcriptase complex is made up of the RNA genome with three RNA-associated proteins. The NDV genome contains six genes which encode six structural proteins, namely the nucleoprotein (NP), RNA polymerase or large protein (L), phosphoprotein (P), matrix (M), fusion (F), and hemagglutinin-neuraminidase (HN), and two non-structural proteins, V and W proteins (the P gene encodes two non-structural proteins V and W) [57].

The NDV strains are divided into three major pathotypes: lentogenic, mesogenic, and velogenic, based on virulence and tissue tropism, which influence disease severity and host response. The lentogenic strains have low virulence, generating minor respiratory symptoms or subclinical infections, and are commonly employed as live attenuated vaccines due to their safety profile. The mesogenic strains are moderately virulent, causing respiratory distress and, in some cases, neurological indications such as tremors or incoordination, earning them the name neurotropic mesogenic; while death is normally low, these viruses can nonetheless have an impact on flock health. In contrast, velogenic strains are very virulent, causing severe systemic disease with significant mortality rates via viscerotropic (hemorrhagic intestinal lesions) or neurotropic (neurological damage) pathways [1,2,3,51]. Direct contact between susceptible and infected birds is the primary mode of transmission for NDV. Other probable transmission vectors include contaminated clothing, drink, food, and equipment. Furthermore, farmed poultry may contract the virus from wild birds that serve as carriers [1,3]. The NDV must be detected and treated as soon as possible using appropriate management strategies like as immunization, isolation of affected birds, and stringent biosecurity measures.

The severity of clinical manifestations of NDV varies with the virus’ innate virulence as well as host-related parameters such as age, route of infection, immunological status, secondary infections, and environmental stress [1,58]. Non-vaccinated chickens infected with virulent viscerotropic isolates become listless and depressed two days after infection, with complete mortality by the third or fourth day [59]. The respiratory indications were infrequent, with only a few birds displaying open mouth breathing, and the neurotropic velogenic strains of NDV predominantly cause respiratory distress, followed by neurological symptoms such as head twitching, tremors, opisthotonus, paralysis, and decreased egg production [60]. Morbidity with virulent neurotropic NDV strains frequently reaches 100 percent, and death is typically 50 percent but can reach 100 percent in young chicks [61,62]. Infection with mesogenic strains can cause nonfatal respiratory sickness, decreased egg production, and neurological symptoms such as head tremors, torticollis, and paralysis [60].

The NDV is generating substantial economic losses and jeopardizing chicken industries in both high- and low-income countries [63,64,65,66,67]. The global epidemiological burden is significant, with outbreaks documented in more than 100 countries [1], especially in areas with dense poultry populations and insufficient biosecurity. In Southeast Asia, NDV causes 80–100 per cent mortality in unvaccinated flocks [2]. In Nepal, the NDV [67] causes significant economic loss through mortality and illness, estimated at NPR 75 million per year (USD 535,729). In Chad [68], NDV outbreaks resulted in a total of XAF 35 billion losses (USD 62,682,312) in the poultry business, with 55 percent mortality. The NDV outbreaks also caused significant economic losses in Southeast Asia’s commercial poultry [69]. Despite regular vaccination regimens and biosecurity measures, commercial poultry farms infected by NDV in Pakistan had economic losses totaling USD 200 million between 2011 and 2013 [70]. Despite strict NDV vaccination, commercial poultry farmers in India have also been suffering tremendous economic losses. The economic losses among a total of thirteen flocks of eleven-layer farms were documented throughout the period of January 2013 to July 2014 [71] and the total economic loss for all layer flocks was INR 3,719,223 (USD 42,403). Mortality contributed INR 2,998,105 (USD 34,181) to the overall economic loss. The highest 80.61 percent and the lowest 0.70 percent of total economic loss were observed as a result of mortality and biosecurity measures, respectively.

Bangladesh sustained average yearly losses of BDT 2,561, with national consequences of USD 288 million [63]. In addition, Newcastle Disease has had a significant influence on Saudi Arabia’s commercial chicken sector. Poultry producers have faced significant financial losses as a result of outbreaks, decreased egg production, and higher mortality rates [72]. These repercussions include food security, the availability of chicken commodities on the local market, and the poultry industry’s financial sustainability. The economic impact of NDV on impacted commercial broiler farms [65] found that NDV had a significant impact on 23 of the 25 commercial broiler farms. Furthermore, the emergence of NDV in these fields caused SAR 8.01 million (USD 92,594) in economic losses. It clearly shows that once the ND outbreak began on the farm, it had a significant negative economic impact throughout the flock’s lifespan. A study of 74 ND-infected farms [66] indicated average losses of SAR 58.71 per bird and SAR 4.74 million (USD 54,033) per farm. Morbidity-related expenditures (e.g., reduced output) accounted for 90% of losses, whereas death accounted for just 10 per cent.

4. Genetic Research on Resistance to NDV

The existence of a genetic underpinning for intrinsic resistance to NDV has been recognized for about 50 years and the initial research (from the 1950s to the 1960s) focused on the impact of NDV on poultry and the identification of genetic resistance variations. The early breeding programs aimed to discover chickens with some level of resistance to NDV, paving the path for greater understanding of genetic resistance. Iyer and Dobson [28] were the first to attempt to generate resistance to NDV and they proposed that the mode of inheritance was by multiple genetic factors, regardless of the limited number of chickens utilized. Similar research was conducted by Teklinski, although he did not perform serological testing to ascertain the immunization status of the birds under study and proposed the existence of genetic resistance against NDV [29]. However, earlier findings did not offer compelling reason in favor of genetic variations in ND resistance and their observations were probably confounded by egg-borne passive immunity, something they were unaware of at the time but which is present for this disease [28,29]. In addition, it was found that the different strains of NDV might differ in their capacity to agglutinate red blood cells using the conventional NDV [73,74]. Later, it was discovered that two strains of the NDV differed significantly in their ability to reach high concentrations in the central nervous system [75], and Beaudette’s review includes several descriptions of infected flocks with varying degrees of inherited or natural resistance to NDV and the majority of these reports deal with disease observations during natural outbreaks rather than experiments designed as genetics studies [76]. Further, Godfrey also found that the variations in the duration of a pause in egg production following an epidemic of ND among various bird stocks suggested difference in susceptibility [30]. In another investigation, Francis and Kish attempted to select NDV resistance, and the New Hampshire family lines demonstrated variability in mortality in response to a typical NDV challenge dose, and also reported that the sire families differed in their resistance [31].

Cole and Hutt’s work considerably improved the study of NDV and its genetic resistance in chickens and provided an explanation for the distinct differences in mortality between the K (Kansas strain) and C (Cornell strain) strains following vaccination against ND, which are thought to be proof that these strains have different genetic makeups [38]. The study indicated that resistance to the mild form (or forms) of ND may be selectively bred to a point where losses would be negligible. Another investigation in an Athens random bred subpopulation that were exposed to the GB strain of the NDV found that the S (Susceptible) line consistently had a higher average death rate than the R (Resistant) line and the heritability estimates ranged from 0.07 to 0.17, indicating that there is little genetic variability for resistance or susceptibility to ND in this population [32]. Subsequently, genetic variations in the immunological response to NDV were noted by Peleg et al. in broilers that had received an attenuated (live) or inactivated NDV vaccination and significant differences were observed across the sire families, with reported heritability estimates between 0.31 and 0.60 [33]. In addition, genetic and phenotypic variations in immunological responses to inactivated NDV and Escherichia coli vaccines in commercial poultry strains were investigated. The researchers found significant variation in immunological response to both vaccines amongst sire families and reported heritability of 0.41 for immunological response to NDV [77].

In another study, divergent selection was used to ascertain whether a population of broiler chickens would respond favorably or adversely to vaccinations against NDV, and E. coli and after four selection rounds of divergent selection, they discovered that the titer index viability was higher in the early-high line than in the early-low line and recommended that the antibody responses at 18 days of life might be selected [78]. In another study, Heller et al. developed two lines (early and late line) of meat-type hens chosen for antibody responsiveness to vaccination with inactivated E. coli and they identified different levels of sensitivity to NDV [37]. Subsequent research found that highly inbred Fayoumi lines are less susceptible to NDV infection than highly inbred White Leghorn hens [79]. Significant variation against NDV infection has also been documented between ecotypes in backyard settings [80].

According to earlier findings, local Egyptian breeds are genetically resistant to NDV, with the Mandarah breed emerging as a resistant breed (with only 20 percent mortality), while the Sina, Dandrawi, and Gimmizah varieties were sensitive with 100, 85, and 100 percent mortality, respectively [81]. In further research, it was discovered that sensitive Leghorn lines and resistant Fayoumi lines that were exposed had unique sensitivities to NDV, providing insight into the probable pathways behind both sensitivity and resistance [20,21]. In order to test the NDV response characteristics, Ghanaian and Tanzanian chickens were exposed to velogenic NDV strains and observed a moderate level of heritability, indicating that selective breeding could enhance these local chicken ecotypes’ responsiveness to NDV [82]. In addition to chickens, genetic diversity in antibody responses to NDV vaccinations has been seen in turkeys [34,35,36,83]. These studies have mostly demonstrated that different genetic lines respond differently to the virus, and these significant research investigations highlighted the need of both genetics and immunization in the fight against NDV.

Decades of research into NDV resistance has resulted in three essential facts [28,29,30,73,74,75,76,77,78,79,80,81,82,83]. First, resistance is polygenic in nature, with several genes regulating immune response, viral replication dynamics, and host–pathogen interactions [28,29,73,74,75,76]. This quantitative genetic architecture needs genome-wide selection tactics over single-gene alterations. Second, disease consequences are dependent on both the viral genotype and the host’s genetic background. Distinct breed-specific resilience patterns are seen, particularly in Fayoumi and Mandarah chickens, which have innate resistance, but other commercial lines remain very vulnerable [20,21,81]. Parallel findings in turkeys and spatially suited village ecotypes underscore the importance of co-evolution in generating resistance [34,35,36,83]. Third, despite moderate heritability estimates for survival variables (0.07–0.17), antibody response after vaccination has significantly greater heritability (up to 0.60), indicating a possible channel for genetic improvement when combined with vaccination programs [32,33,77]. These findings support multidimensional control strategies that combine genotype-informed breeding, strain-matched vaccination protocols, and enhanced biosecurity measures, particularly in regions endemic to velogenic NDV strains, to reduce economic losses and promote long-term poultry health.

5. Advances in Molecular Genetics Research on Resistance to ND

5.1. Genomic and Transcriptomic Approaches in Studying Resistance to NDV

Genomic studies are critical for comprehending and combating resistance to NDV. It assists in identifying the genetic diversity of NDV strains, which is critical for tracking outbreaks and studying how various strains might avoid immune responses in host species. Researchers can find specific NDV resistance genes by studying the genomes of resistant and susceptible poultry breeds. This knowledge can be used to guide breeding strategies aiming at increasing disease resistance. Hence, genomic research is crucial for increasing our understanding of NDV and developing practical solutions to manage resistance as well as to improve chicken health, thereby providing assured food security.

5.1.1. Candidate Genes Associated with Resistance to NDV

Identifying candidate genes associated with resistance to NDV can help to guide breeding strategies aiming at improving poultry immune responses and thereby making them less susceptible to NDV, and these candidate genes can also be used as genetic markers for selective breeding, which enables chicken breeders to choose individuals with favorable genetic profiles as well as enhance flock resistance over time. Studies on the NDV have shown a number of potential genes linked to susceptibility and resistance in chickens.

The genome-wide association studies (GWAS) to identify genes related to antibody response to NDV following immunization with NDV in two broiler lines with varying resistance and the association study revealed that the genes viz., ROBO1 and ROBO2 genes (roundabout guidance receptor 1 and 2), which are located in the chicken chromosome 1, have a considerable effect on the antibody response to NDV and this discovery sets the path for further research into the host immune response to NDV [43]. In another study, it was found that the CCL4 (chemokine C-C motif ligand 4) gene, which encodes the chemokine CCL4, plays a crucial role in the immune response to NDV [84]. In addition, it was also found that CCL4 is differently expressed in susceptible lines, and it was reported that the inbred and outbred lines exhibit distinct expression patterns for various innate immune genes, thereby indicating susceptibility or resistance [85]. Based on this observation, they concluded that CCL4 was found to be positively linked with viral load in all ecotypes.

The Myxovirus resistance gene (Mx) is an important candidate gene for studying NDV and its resistance mechanisms in chickens. There is a correlation between the polymorphisms in the Mx gene promoter and the ability of chicken embryos to withstand a severe NDV challenge [86]. They found a link between the presence of this gene in specific chicken breeds and resistance to ND. At the genotypic level, the SNP, i.e., G > A mutation inside the IFN-stimulating response region, was linked to sensitivity to NDV challenge and the allele “G” frequency was higher in the less susceptible cohort, while the allele “A” frequency was higher in the highly susceptible group. Further, the Mx-knockout DF-1 cells were generated by using the CRISPR/Cas9 gene editing approach and it was found that the absence of the Mx gene reduced the cells’ resistance to viral invasion following NDV infection, indicating that the Mx gene was necessary for both innate immunity and anti-virus invasion [87].

The Interferons (IFNs), including Type I (IFN-α and IFN-β) and Type II (IFN-γ), play an important role in the immune response to NDV and the Interferon Regulatory Factor (IRF) genes in chickens such as IRF3, IRF7, and IRF8 are critical for responding to NDV infection. Earlier research on IRF genes has shown that genetic polymorphisms in the expression levels of IRF genes can affect the immune response to NDV and the infection with NDV triggers a significant immunological response, as evidenced by the overexpression of several cytokines and chemokines [88]. The IFNα and β have distinct antiviral actions, the IFNα may have the most antiviral efficacy against NDV infection in chickens, and the IFNβ may primarily be involved in signaling and immunological regulation [89]. Chickens with higher expression of IRF7 or IRF3 may have a stronger interferon response, leading to better resistance to NDV, and hence genetic selection for increased IRF activity may improve resistance to NDV in commercial flocks [26,90]. The increased production of Th1 and cytotoxic T cells was confirmed by the high production of IFN-g in chickens resistant to ND and other diseases [91].

The enzyme known as OAS (2′,5′-oligoadenylate synthetase) impedes viral replication by activating RNase L and destroying viral RNA, and after viral infection, the OAS gene was shown to be elevated in chickens. In addition, the eukaryotic translation initiation factor 2 (eIF2) gene family, which is considered a strong contender for viral replication control, is the translation machinery of the host cell and is required for NDV replication. Studies of the transcriptomes of two distinct inbred chicken lines, viz., Leghorn and Fayoumi, when challenged with NDV revealed a role for the eIF2 signaling pathway [20,21,92]. Further, the Fayoumi breed has a higher level of antibody during NDV infection, and this breed clears the virus more quickly than the Leghorns, which makes them relatively more resistant to the virus [20,21,93].

A pathway analysis of transcriptome data [20,92] suggested that eIF2 signaling might represent one of the pathways linked with enhanced resistance to NDV. A study utilizing two inbred lines found that the OAS gene is critical to the host response to NDV and discovered that the NDV-infected Fayoumis birds expressed less of the EIF2B5, EIF2S3, EIF2B4, and EIF2S3 genes than the Leghorn-infected lines [25]. They showed that different genetic lines exhibit varying expressions of host translation initiation factor-2 linked genes. Subsequent studies demonstrated that OASL knockdown raised the amount of ND viral RNA and decreased the antiviral host gene expression response [94].

Another study on Leghorn hens revealed that this breed had greater modifications in gene expression than the Fayoumi breed and also observed seven differentially expressed genes (DEGs) playing a crucial role in regulating the immune response to NDV [95]. Regarding DEG and detectable NDV, the Harderian glands of two genetic lines reacted to NDV quite differently [20]. The Fayoumis were able to clear themselves of the disease more quickly than the Leghorns and provided the groundwork for future research into the special role of Harderian glands in host defense. Further research involving Leghorn and Fayoumi breeds noticed that a total of 552 genes and 1580 lncRNAs had differential expression, with almost 52 genes categorized with immunological pathways and gene ontologies [96]. They also found that the Leghorn has a higher number of differentially expressed genes, with the bulk of them downregulated at various stages of the disease. Based on their findings, they concluded that immune-related gene downregulation and co-expression of lncRNAs may play a role in the Leghorn breed’s sensitivity, being comparable to the Fayoumi breed.

Numerous genes and immunological pathways interact intricately to control the host’s genetic resistance to the Newcastle Disease virus (NDV) in chickens, according to decades of research. These results show that NDV resistance arises from a combination of antiviral defense mechanisms, immune modulation, and viral recognition systems rather than being based on a single gene [43,84,85,88,89,90,91,92,93,94,95,96]. Several important genetic components have been identified, including the Mx gene [86,87], whose promoter polymorphisms directly affect survival rates, as demonstrated by CRISPR/Cas9 knockout studies; ROBO1 and ROBO2 [43] on chromosome 1, which control antibody production after vaccination and are useful markers for selective breeding; and CCL4, a chemokine whose expression patterns correlate with viral load control in early infection stages [84,85]. Furthermore, through Th1 and cytotoxic T-cell responses, interferon regulatory genes (IRF3, IRF7, and IRF8) improve viral clearance, but resistant breeds like Fayoumi are better able to activate viral replication restriction pathways (OAS, eIF2) than susceptible lines like Leghorn [88,89,90,91].

The transcriptomic investigations reveal further breed-specific strategies: Fayoumi chickens exhibit quick virus clearance, powerful antibody responses, and effective immune activation, whereas Leghorns exhibit broad gene repression and delayed defense mechanisms, making them more vulnerable. These genetic findings have important practical implications, as they provide molecular markers for genetic breeding programs and guide the development of genotype-tailored vaccinations that augment innate host immunity [20,25,92,94]. Poultry producers can improve flock resilience, particularly in areas where velogenic NDV strains are prevalent, by incorporating these findings into breeding plans—especially those that aim to develop high-heritability antibody traits—as well as by using improved vaccination and biosecurity protocols. It is still difficult to translate these genetic insights into commercial breeding programs, though, as disease resistance must be carefully balanced with other crucial productivity qualities. In the future, developments in genomic selection and gene editing could hasten the creation of NDV-resistant poultry lines, thereby promoting sustainable poultry production worldwide.

5.1.2. Major Histocompatibility Complex (MHC) and Resistance to NDV

The MHC aids in the immune response to NDV by promoting the presentation of viral antigens to T cells. Variations in MHC genes can affect how well an individual’s immune system detects NDV, resulting in variation in resistance or susceptibility across bird groups. In MHC studies, the sheep red blood cells (SRBCs) are commonly utilized in research that can emulate immunological reactions, particularly those produced by NDV. Chickens from a hybrid of White Leghorn populations selected for high or low antibody response to SRBC antigens were segregated for MHC haplotypes B13 and B21. Following an NDV vaccine, B13 haplotype hens developed higher antibodies than B21 [97]. Furthermore, other scientists employed a lentogenic NDV strain to challenge hens with different MHC haplotypes, and the results revealed that CD4+ T cell responses varied and the T cells from hens with the B12 haplotype exhibited typically low antigen-specific responses, with just a small percentage of each haplotype responding strongly [98].

The transcriptional response of innate immunity genes in chicken embryos infected with NDV, the MHC-locus, may play a genetic role in regulating the innate immune response of chicken embryos to NDV [99]. The transcriptional responses in inbred sublines of the Fayoumi and Leghorn breeds were then examined and it was discovered that five genes—Mx1, STAT1, IRF1, IRF7, and SOCS1—are up-regulated in all sublines and that the expression of these genes varies in accordance with the breed and subline; it was also stated that the Fayoumi breed pro-inflammatory response varies by subline and is likely controlled by the MHC [100]. In MHC genes, the relationship between MHC-B alleles and microsatellite alleles in chickens in relation to antibody response to NDV was found, and 10 alleles were discovered, four of which were from the MHC-B marker, i.e., LEI0258, and six of them from five QTL markers associated with NDV resistance/tolerance [101]. These findings indicated MHC-B’s role in the immunological response to NDV. In another study, it was discovered that hens with high NDV-antibody titers had a higher frequency of MHC-B alleles and also reported that diverse genetic variants of the LEI0258 marker may be associated with different chicken MHC-B haplotypes, resulting in variance in disease resistance or susceptibility, which can be used in breeding programs to improve chicken disease resistance [102,103].

In general, the basic assumption of candidate gene research generally holds that genetic variations near or inside a gene affect its function, which directly affects the variance in individual phenotypes. The most convincing candidate genes are those whose genetic variant (often a single-nucleotide polymorphism, or SNP) has been demonstrated to have a discernible impact on the structure or abundance of its protein product, and which influence the trait at many biological levels. To enhance the overall health and survival of chickens, expedite the breeding process, and shorten the time required to produce resistant breeds; and the breeding program may incorporate the significant candidate genes linked to NDV (

Table 1. Important candidate genes related to NDV in chickens.

Sl. No	Name of the Candidate Gene	Role	References
1	Roundabout guidance receptor 1 and 2 (ROBO1 and ROBO2 genes)	These genes play a role in tissue organization and immune cell migration. ROBO1/2 has been linked to antiviral defense mechanisms and regulates the immune response.	[43]
2	Myxovirus resistance gene (Mx)	The Mx gene produces a GTPase that inhibits the replication of RNA viruses, including NDV. It is a well-known antiviral gene with polymorphisms associated with illness resistance in chickens.	[86,87,100]
3	Chemokine C-C motif ligand 4 (CCL4) gene	The CCL4 is a pro-inflammatory chemokine that attracts immune cells such as T cells and macrophages to infection sites. Elevated expression is connected with increased resistance.	[85]
4	Interferon Regulatory Factor (IRF) genes—IRF3, IRF7, and IRF8	These transcription factors are required for the induction of interferons and subsequent antiviral genes. They orchestrate the early innate immune responses to NDV infection.	[87,90,91,100]
5	Eukaryotic translation initiation factor 2 (eIF2) gene family	The eIF2 gene is involved in both protein synthesis and stress response. During viral infection, it alters host translation to promote antiviral responses and prevent viral protein synthesis.	[20,21,92,95,100]
6	Oligoadenylate synthase-like (OASL),	The OASL is a member of the interferon-stimulated gene family. It stimulates RNase L, which degrades viral RNA and contributes to the suppression of NDV replication.	[25,94]
7	Major Histocompatibility Complex (MHC)-B13 and B21	These MHC class I and II alleles are related with improved antigen presentation and immunological response. B21 is commonly associated with NDV resistance.	[97]
8	Major Histocompatibility Complex (MHC)-B12	Unlike B21, B12 is frequently linked to vulnerability to NDV. Comparison with resistant haplotypes helps to understand host–pathogen interactions.	[98]
9	Major Histocompatibility Complex (MHC) and LEI0258 marker linked with MHC	The LEI0258 is a microsatellite marker that is significantly related with the chicken MHC region. It functions as a genetic marker for identifying MHC haplotypes associated with disease resistance.	[100,101,102,103]

).

The results of research findings support the idea that MHC variation plays a crucial role in determining the innate and adaptive immune responses to NDV [97,98,100,101,102,103]. Genetic variation within or close to a gene can directly impact its function, altering phenotypes like disease resistance. Poultry breeding programs can benefit greatly from the identification of such candidate genes, especially those containing functional single nucleotide polymorphisms (SNPs) that change the production or structure of proteins [43,92,93,94,95,96,97,98,99,100,101,102,103,104]. The evolution of NDV-resistant chicken breeds might be accelerated by incorporating these genes into selection processes, which would improve flock health, increase production, and lower financial losses.

5.1.3. Quantitative Trait Loci (QTL) Linked to NDV in Chickens

Breeders can more efficiently select for NDV resistance by identifying specific QTL associated to the trait, which can result in the evolution of more resistant poultry strains. To uncover DNA markers linked to QTL associated with immunological response, researchers divergently selected for high or low antibody (Ab) response to E. coli using 25 microsatellite DNA in a cross between two meat-type lines [104]. They observed that out of the 25 markers, two are related with Ab to SRBC and NDV viz., ADL0146 on Chromosome 2 and ADL0290 on Linkage Group 31. Later, a GWAS study [105] in Jinghai yellow chickens identified six single-nucleotide polymorphisms (SNPs) associated with antibody levels against NDV and reported five genes viz., Plexin B1, LRRN1, TRIM27, and PDGFC related to production of antibodies against NDV. Subsequently, scientists examined a commercial egg-laying line with a lentogenic strain of NDV, discovering six QTL associated with NDV response and/or growth [106]. Three SNPs in two QTLs were shown to be suggestively linked with antibody levels prior to challenge and the strongest relationship was found on chromosome 3. In addition, the second QTL for antibody pre-challenge on chromosome 10 included two SNPs and antibody levels post-infection were linked to one SNP on chromosome 21. Another study revealed suggestive QTL locations on chromosomes 1 and 24, as well as many candidate genes that may play a significant role in the chicken’s response to NDV during heat stress [107].

Furthermore, identification of six QTL linked with growth and/or response to NDV in three Tanzanian chicken ecotypes [108]. The strongest QTL region was detected on chromosome 24 and included important candidate genes viz., EST1 and TIRAP, which may be important in response to NDV infection. Subsequent study also demonstrated QTL for NDV response characteristics in three Ghanaian and Tanzanian indigenous chicken ecotypes, as well as brown commercial laying birds in the USA and based on moderate heritability estimates, they proposed that selective breeding can improve NDV resistance and vaccine efficacy [109]. In addition, SNPs were also discovered that are crucial in the antibody response to NDV in Rwandan indigenous chickens [110]. Four significant SNPs on chromosome 1 were shown to be strongly linked with the chicken antibody response to NDV. MX1, CDC16, ZBED1, and GRB2 were the genes with the strongest associations with these four SNPs, and they provide a solid foundation for identifying the genes responsible for substantial QTL impacting NDV in chicken lines. In addition, another researcher carried out a GWAS on Sasso chicken that were naturally exposed to infectious diseases and found five SNPs were located on chromosomes 1, 5, and 13 in genomic areas containing multiple genes that affect the immune response [111].

In general, the QTL mapping for resistance to ND is a significant area of study in poultry genetics, and by pinpointing the exact genes and loci responsible for immune responses to NDV, breeders can considerably improve their tactics and raise flocks of chickens that are more resilient. The aforementioned research showed that a large number of QTL associated (

Table 2. QTL linked to NDV in chickens with probable candidate genes.

Chr/LG *	Probable Candidate Genes	Species/Breed/Type	QTL/SNP	References
Chr 1	CDC123, CAMK1d, and CCDC3.	Commercial Brown laying chicken	rs316767446	[107]
	FAT3, SPRY2, MIR17	Ghanaian Local Chickens	AX-75376474, AX-75297835, AX-75297834	[109]
	CDC16, ZBED1, MX1, GRB2	Indigenous Chicken in Rwanda	rs314787954, rs13623466, rs13910430, rs737507850	[110]
	FOXP2	Sasso Chickens in Ethiopia	rs316795557	[111]
Chr 2	-	Commercial broiler chicken	ADL0146	[104]
	CHORDC1, JAZF1	Ghanaian Local Chickens	AX-76076573	[109]
	CWC22, MIR7474, FAM133B, CDK6		AX-76057582
	ITGA9		AX-76103078
Chr 3	B3GALNT2, GPR137B, NTPCR	Commercial egg laying chicken	AX-76468260	[106]
Chr 4	PDGFC	Jinghai Yellow Chicken	rs420701988	[112]
	POF1B	Ghanaian Local Chickens	AX-76755933	[109]
Chr 5	GALC	Ghanaian Local Chickens	AX-76842268	[109]
	CEP170B	Sasso Chickens in Ethiopia	rs313761644	[111]
Chr 7	CCDC141	Ghanaian Local Chickens	AX-76984929	[109]
	GTF2A1, PIK3CA, TBL1XR1, STK17B, STAT4,		AX-77054277
Chr 9	ZC3H14	Ghanaian Local Chickens	AX-80796104	[109]
	APOOL, MIR6704, LEKR1, PTX3, SSR3		AX-77162202
Chr 10	ACTB	Commercial egg laying chicken	AX-75608938	[106]
	LINGO1, HMG20A		AX-75605132
Chr 12	LRRN1	Jinghai Yellow Chicken	rs1218289310	[112]
	Plexin B1		rs1211307701, rs1211307711
Chr 13	-	Sasso Chickens in Ethiopia	rs733628728	[111]
Chr 15	SNRPD3	Ghanaian Local Chickens	AX-75846279	[109]
Chr 17	VAV2		AX-75874883	[109]
Chr 21	TARDBP, APITD1, CASZ1	Commercial egg laying chicken	AX-76244799	[106]
	SPRY2, MIR17, VAMP3, PHF13	Ghanaian Local Chickens	AX-76244242	[109]
Chr 24	TIRAP ETS1	Tanzanian Local Chicken Ecotypes	AX-76312211, AX-76312344, AX-76311970	[108]
Chr 24	TIRAP (4), SRPRA, KIRREL3 (4), ST3GAL4, EI24 (4), FAM118B (5), DCPS (3), STT3A, CHEK1, FOXRED1	Commercial Brown laying chicken	rs14292128	[107]
	KIRREL3 (4), ST3GAL4		rs315615997
	KIRREL3		rs16192874, rs315836090, rs315093440, s314396434, rs318146300, s316420264, rs318146300, rs316424273
	ETS1		rs314541596, rs14292586, rs312349782
	ARHGAP32		rs16193617
	ETS1, FLI1, KCNJ1, 5S_rRNA		rs316089514
	ETS1, FLI1, KCNJ1, KCNJ5, 5S_rRNA		rs315752152, rs315834610
	-		rs312459125, rs315334251, rs14292586, rs314510760, rs315406121, rs317888742, rs313717240, rs318216384, rs316424273, rs316882370, rs15212279, rs15212295, rs312419519, rs316420264, rs316132202, rs315406121, rs313717240
Chr 27	PJA2, FER, SLC25A46, EFNA5,DDX42	Ghanaian Local Chickens	AX-76359725	[109]
Chrs 30, 33	-	Sasso Chickens in Ethiopia	Two unidentified SNPs	[111]
LG 31	ADL0290	Commercial broiler chicken	-	[104]
Chr Z	SETBP1	Jinghai Yellow Chicken	rsZ2494661, rsZ2494710	[112]
	TBX6	Ghanaian Local Chickens	AX-77227276	[109]

* Chromosome (Chr)/Linkage Group (LG).

) with ND resistance are associated with immune system-related genes. The chicken industry can develop breeds that are more resistant to NDV and reduce the economic burden of the disease by combining QTL data with other breeding procedures, such as genomic selection.

Cutting-edge research over the decades in poultry genetics has transformed our understanding of the genetic basis for NDV resistance in chickens. Comprehensive QTL analyses and GWAS [105] have revealed that NDV resistance is mediated by several genetic variables scattered throughout the avian genome. Initial successes in marker-assisted selection were made by research correlating microsatellite markers ADL0146 and ADL0290 to immunological responses against both NDV and sheep red blood cells, laying the framework for advanced genomic breeding approaches [105]. Modern GWAS on Jinghai yellow chickens have identified six significant SNPs associated with NDV antibody production, highlighting important immune-related genes such as Plexin B1, LRRN1, TRIM27, and PDGFC [105]. These findings highlight the intricate network of biochemical processes involved in avian immune defense mechanisms. The study identifies both population-specific and generally conserved genetic elements that regulate disease resistance. This dual nature of resistance genetics leads to more precise genomic selection strategies that can improve disease protection while preserving productivity. Genetic resources identified in African native chicken populations [108,109,110,111] are of special relevance, since their adoption into commercial breeding lines may improve NDV resistance while conserving tropical adaptation features. The development of sophisticated SNP arrays for breeding applications, the analysis of genetic–environment interactions in diverse farming systems, the harmonization of genetic and traditional breeding techniques, and broad-spectrum genomic evaluations across numerous breeds should be the top priorities for future research. These genomic applications offer tremendous potential to boost global poultry production and reduce economic losses associated with NDV.

6. Breeding Strategy for Disease Resistance to NDV

Selective Breeding

In the poultry industry, chicken breeding companies concentrate on elite birds in the breeding nucleus at the top of the breeding pyramid (Figure 1) and due to the highly structured breeding sector, genetic advancements have occurred at the fastest pace in the chicken industry [113]. It is possible to use selective breeding to increase chicken resistance to specific infections, including NDV. The initial process in selective breeding is to identify which birds are innately more resistant, followed by arranging suitable mating to produce chicks that are more resistant to infectious diseases. When adding disease resistance traits into breeding programs, poultry producers must consider genetic diversity, heritability, economic traits, and potential costs. However, long-term strategies are required to advance resistance breeding, which is hampered and delayed through a shortage of techniques that allow for general resistance as well as the unintentional amplification of susceptibility to a disease through selection for specialized resistance to another disease.

The researchers identified significant genetic variation in chickens, as well as the importance of specific genomic regions in the chicken genome for immunological and disease resistance attributes. In addition to direct selection, indirect selection for resistance to NDV can be an effective alternative method in breeding operations, especially when direct approaches are difficult. By focusing on associated qualities that contribute to general health and immune competence, breeders can increase the possibility of producing NDV-resistant poultry while simultaneously increasing other performance features. Several markers related to immune responses have been identified (Table 1 and Table 2) and future direction related to identifying more genetic markers associated with NDV resistance can facilitate indirect selection. Individuals can be selected through breeding programs using these markers, which are associated with resistance qualities.

Genomic selection becomes more prevalent in livestock and poultry breeding due to its time and cost advantages over traditional methods. A DNA-based selection technique eliminates the necessity for challenge tests and natural epidemics. Identifying fundamental genes or QTL for resistance, as well as establishing accurate SNP-chip-based genomic predictors, can help to attain this objective. New and innovative gene mapping techniques are being developed specifically for the discovery of complex disease loci [45,115], and microarray technology is rapidly improving, allowing the linkage of animal DNA with human and mouse DNA [116]. The marker-aided backcrossing method can introduce genes that explain breed variations in economically significant traits and disease resistance, boosting genetic variety that can be used in selection processes. Despite tremendous progress, transgenic breeding for disease resistance has not yet been applied in the chicken sector. There are still concerns regarding the safety of transgenic animals and hence, in order to completely benefit from transgenosis, more efficient vectors need to be found [117,118]. Figure 2 shows the comprehensive strategic design to support native chicken production, with an emphasis on resilience against Newcastle Disease, and based on the design, they concluded that because of the low to medium heritability of the survival time, numerous generations of selection are needed to detect a commercially viable genetic improvement on the survival time in the face of velogenic NDV infection [119]. Moreover, natural exposure to velogenic NDV challenges are necessary for each generation of selection to validate genomic selection panels.

Altogether, selective breeding in poultry, particularly for resistance to Newcastle Disease virus (NDV), is a viable long-term method for improving flock health and production. The structured breeding pyramid in commercial chicken production allows for the rapid spread of improved traits, and advances in genomic technologies such as SNP-chip-based genomic selection, marker-assisted selection, and QTL mapping have accelerated the discovery of genetic markers associated with disease resistance. Both direct and indirect selection methods can be beneficial, with indirect selection enhancing overall immunological competence and a variety of performance characteristics.

Indigenous breeds provide chances to protect genetic variety while increasing resistance to local NDV strains. However, because survival time against velogenic NDV has a low-to-moderate heritability, significant genetic gains take numerous generations of selection, which frequently necessitates natural exposure to validate genomic prediction panels. Furthermore, there are possible trade-offs, as selecting for resistance to one illness may mistakenly increase susceptibility to another, and a lack of broad-spectrum resistance strategies limits applicability. Transgenic breeding has theoretical potential, but it has yet to be realized in the commercial chicken industry due to technological, safety, and acceptance concerns. Despite the high initial costs, genomic techniques’ precision and time-saving benefits outperform traditional challenge-based selection, making resistance breeding a viable but long-term investment for the chicken sector.

7. Challenges and Opportunities for Disease Resistance Against NDV

The breeding program for chicken disease resistance has come a long way, but there are still many obstacles to be addressed. For example, breeding for disease resistance is costly; additional research is needed to ascertain the connection between economic characteristics and disease-resistant selection; producing a chicken line resistant to multiple infections is difficult due to pathogen interactions; and precisely identifying genetic resistance candidates through phenotypes is another challenge facing researchers. In a nutshell, the challenges facing resistant breeding are daunting and hence more study in this area is necessary to improve the prospects for resistant breeding in the future [23,120,121].

The NDV evolution poses a significant danger to the long-term viability of genetically modified resistance measures in chickens. The NDV exhibits high genetic variability due to the error-prone nature of its RNA-dependent RNA polymerase and numerous recombination events [1,2]. These pathways lead to the ongoing creation of novel NDV genotypes and variants with different virulence, host range, and antigenicity [3]. Such viral evolution is usually accelerated by selective pressure from vaccination, biosecurity measures, and host immune responses, including genetically acquired resistance. As a result, if viral variants develop escape mutations that bypass these defenses, resistance features effective against currently circulating NDV strains may become obsolete [4,5]. This issue is especially important when pushing genetically based, long-term poultry control techniques, which must be able to survive viral variety over time. To mitigate the risks posed by NDV’s evolutionary plasticity, resistance breeding should employ polygenic selection models that focus on immune-related gene networks and biological pathways (such as interferon signaling and antigen processing), rather than single major-effect loci, which are more vulnerable to viral adaptation [6]. Continuous molecular surveillance of field NDV strains is also essential to track genetic drift and detect new escape variants early [2]. Furthermore, the combination of genomic selection, epidemiological monitoring, and functional immunogenetics results in a dynamic and responsive framework for long-term disease resistance. Future genetic resistance approaches must be adaptive, taking into account feedback from ongoing viral evolution, to ensure long-term efficacy and relevance in NDV control efforts.

The critical analysis of the various studies revealed that, while candidate gene studies have identified promising immune-related markers [43,86,87,100,101,102,103], their small sample sizes and narrow focus on known genes limit broader applicability, emphasizing the importance of multi-breed validation and regulatory variant functional characterization. Genome-wide association studies (GWAS) have successfully found [105] novel quantitative trait loci, but their extensive use in commercial breeds and lack of integration with transcriptome data highlight a fundamental gap in our understanding of causative pathways across varied genetic backgrounds. While transcriptome studies [20,92] have identified important immune pathways activated during NDV infection, their reliance on inbred lines and cell cultures reduces translational applicability, emphasizing the importance of single-cell techniques and multi-omics integration. Indigenous breeds [110,119] offer valuable inherent resistance, but their genetic basis remains poorly understood due to a lack of controlled challenge tests and established phenotyping methods. Although marker-assisted selection [17,23,40,41,42,43,44] has shown promise in improving resistance, trade-offs with productivity traits and limited field testing highlight the importance of comprehensive breeding techniques that balance many objectives. To build long-term genetic solutions for NDV resistance, future research should focus on systems genetics approaches that include multi-omics data, functional validation by gene editing in diverse genetic backgrounds, and extensive studies of underutilized indigenous breeds. Addressing these gaps will need collaborative efforts to establish standardized phenotyping methodologies, conduct long-term field trials, and devise cost-effective implementation strategies, particularly in smallholder agricultural systems with the highest NDV burden. By combining these findings, this study provides a road map for improving both scientific understanding and practical uses of poultry genetic resistance to NDV, resulting in more sustainable disease control in the worldwide poultry sector.

Despite the challenges involved, breeding for disease resistance in chickens remains an achievable approach for addressing issues such as treatment tolerance and pathogen mutation. The employment of cutting-edge approaches in resistance breeding will significantly hasten the process [23]. In near future, resistance breeding research may find new applications for more recent molecular technologies, such as CRISPR/Cas9 systems, sperm-mediated gene transfer, zinc-finger nucleases (ZFNs), pronuclear injection, transcription activator-like effector nucleases (TALENs), somatic cell nuclear transfer, recombinases, transposons, and viral vectors [45,122,123]. Additionally, as the cost of gene chips and sequencing continues to drop, more candidate genes and QTL will become available, which might lead to amazing advancements in the breeding of disease resistance and higher societal profits.

8. Conclusions

Genetic resistance to disease is appealing because it provides a reliable, cost-effective, and ecologically friendly disease-control method. Numerous QTL and genes related with disease resistance have been found in poultry; however, the present understanding of the relationship between resistant genes and disease remains limited. Furthermore, there may be many more genes associated with disease resistance that are still yet to be discovered, and the precise mechanism of resistance is unexplained. As a result, more study into disease resistance genetics, epigenetics, and quantitative trait loci could potentially help in the identification of resistance markers and the development of disease-resistant breeds, thereby boosting chicken production.