1. Introduction
Newcastle disease (ND) has been globally distributed and the causative agent, Newcastle Disease virus (NDV) belongs to the
Paramyxoviridae family, genus
Avulavirus and is designated as
Avian Paramyxovirus-1 (
APMV-
1). NDV is a non-segmented, negative sense, single-stranded enveloped RNA virus with approximately 15kb genome encodes namely, hemagglutinin–neuraminidase (HN), nucleoprotein (NP), fusion (F), phosphoprotein (P), matrix (M), RNA-dependent RNA polymerase (L) [
1]. ND virus has a wide range of hosts and infection was reported in 250
Avian species in the world by either natural or experimental mechanisms [
2]. Depending on the viral pathotype, the incidence of ND that affects poultry manifests as gastrointestinal, respiratory and neurological conditions that can result in up to 100% mortality [
3,
4]. ND has a high impact on the poultry industry through heavy economic loss aroused due to heavy mortality and production loss and also due to extensive attention to the prevention and treatment of this disease such as standard vaccination protocols and biosecurity measures [
5]. It was calculated that from thirteen layer farms in the Gujarat state of India suffered a total economic loss of
$4,588 (₹ 37,19,223) per year [
5]. Various disease preventive strategies and vaccination are not effective due to the complex genetic diversity of viruses. So, it is necessary to develop a chicken population with disease resistant naturally.
During viral infection
Type I interferon defense mechanism are triggered to express a set of genes against viral infections known as interferon-stimulated genes (
ISGs) [
6,
7]. The
IFITM (interferon-inducible transmembrane) gene is one of these
ISGs and has been shown to prevent the propagation of several highly virulent viral pathogens, such as the coronavirus responsible for the severe acute respiratory syndrome (
SARS), the filoviruses Marburg and Ebola, the influenza A viruses (
IAVs), and flaviviruses (dengue virus) [
8,
9,
10]. Scientists discovered that the
chIFITM gene expression has a negative correlation with the emergence of influenza virus and its titre in
in vitro study, indicating that
chIFITMs
1 ,2 and
3 had a functional role in the management of viral infections [
11,
12]. In chicken,
IFITM genes were located in Chromosome 5 and found in two loci, one containing the various numbers of immune-related (
IR)-
IFITM (
IFITM 1, 2, 3) genes and
IFITM5 gene and
IFITM10 gene in another loci [
13,
14]. Lanz et al. found that
swIFITMs (swine IFITMs) had a dose-dependent restriction against
IAV after infecting porcine
HEK293-T cells with
IAV A/WSN/33 (WSN) for 24 or 48 hours [
15]. sw
IFITM2 and -3 were expressed at late endosomes and have most potent antiviral activity against
IAV in porcine cells. Furthermore, no sw
IFITM5 expression was detected in any of the tested cell lines and concludes that the
IFITM5 do not have significant role in immune function [
15]. Further, the knockdown of
IFITM3 in DF-1 cells by siRNA increased the infectivity of a vesicular stomatitis virus G protein-pseudo-typed lentiviral vector [
16].
Hence this study was undertaken to examine the chIFITM gene expression pattern in two chicken breeds of India, Aseel and Kadaknath, whose levels of viral resistance are high. So, we selected the model of chicken embryo fibroblasts (CEFs) to observe chicken IFITM gene expression in vitro following infection with NDVs. We compared the expression of IFN γ and Mx gene in response to NDV infection by quantitative real-time polymerase chain reaction (qRT-PCR).
In addition to providing prospects for a deeper understanding of viral resistance, analysis of these genes in chickens offers potential strategies for preventing viruses in poultry farming. It may be possible to do a selective breeding program in poultry breeds for increased resistance against viral infections. So it needs to discover characteristics of resistance and understand how they operate. Further, The observation may have useful implications in terms of vaccine production. Many vaccines are produced in embryonated hen’s eggs or continuous avian cell lines. However, it is well established that the rate-determining step in the manufacture of numerous vaccines is the induction of antiviral immune responses that prevent the replication of vaccine viruses and the high cost involved in maintain and producing specific pathogen free (SFP) eggs from chicken.
4. Discussion
To measure the level of
chIFITM gene expression against NDV in the current work, we employed the
velogenic genotype XIII of NDV strain that was used to infect CEF cells. The ND viral load increases, prompting the chicken embryo fibroblast (
CEFs) cells to express more
chIFN-
γ significantly [
25]. In Aseel's CEF cells, there was noticeable upregulation starting at 6 hpi and continuing until 48 hpi. In contrast, in Kadaknath CEF cells,
chIFN-γ expression began to increase significantly at 3 hpi and peaked (13.9±0.49 log
2 fold) like Aseel CEF at 48 hpi. Further, it was significantly (p<0.001) several folds higher than Aseel. However, interferons (IFNs), a vital component of innate immune signaling, serve as the first line of defense against invading viruses [
26]. Therefore, it consistently and strongly expressed against the ND virus [
27].
chIFITMs and
Mx are members of the interferon-stimulating gene (
ISG) group (Mycovirus resistant gene) [
28,
29]. Chicken has five members of the
IFITM family:
chIFITM1,
chIFITM2,
chIFITM3,
chIFITM5, and
chIFITM10. These protein genes are activated and made to express themselves by type I and
type II IFNs, signifying the start of the innate host response [
13,
30]. Recent studies confirm that
STAT/IRF signaling pathways activate
IFITM gene expression together with other
ISGs during infection and inflammation [
31]. This study examines the relative mRNA expression profile of chicken
IFITMs after Newcastle disease virus (NDV) infection
in vitro with a focus on how the cells react during the early stages of NDV infection.
We observed significant upregulation of
chIFITMs,
chIFN-γ and
Mx in the CEF cells of both breeds. A significant viral load indicated the presence of a replicating virus in the CEF cells.
chIFITM1, 2, 3, and
5 are noticeably and gradually upregulated in both breeds of CEFs after NDV infection. In Kadaknath CEF cells,
chIFITMs and
IFN-γ expressions were relatively high with statistical significance (p<0.001) from 3 to 48 hours post-infection compared to control uninfected. Researchers also found that high quantities of
chIFITM1,
2, and
3 are expressed in CEFs from 4 to 24 hours after H9N2 infection.[
32]. While CEF cells from Aseel took longer to exhibit strong mRNA expression of
chIFN-γ and
chIFITM genes strongly expressed at 6 hours post-infection. In Kadaknath CEF, among the
IR-IFITM family members,
chIFITM3 (11.68 log
2 folds) has the greatest expression observed at 6 hours post-infection, followed by
chIFITM2 (8.89 log
2 folds) at 48 hours post-infection,
chIFITM1 (7.79 log
2 folds) and
chIFITM5 (7.04 log
2 folds) at 24 hours post-infection [
13,
15]. But in Aseel,
IFITM1 (6.00 log
2 folds) came first, then
chIFITM3 (3.46 log
2 folds), and then
chIFITM2 (3.25 log
2 folds) at 6hpi, lastly
chIFITM5 (3.82 log
2 folds) at 24hpi. In our finding that Kadaknath expressed large levels of
IFN-γ and stimulated high levels of
chIFITMs compared to Aseel is supported by studies from other scientists that
IFN treated CEFs expressed high levels of
chIFITMs [
13,
30,
32]. It is known that
chIFITM 1, 2, and
3 prevent the replication of a variety of RNA viruses that enter the host cell through endocytosis [
8]. Infected CEF of Kadaknath showed a significant (p<0.01) and robust overexpression of all
chIFITMs started from 3hpi when compared to the control. Whereas similarly in Aseel
chIFITM genes upregulation starts from at 3 hours post-infection, compared to the control. A Significant and high level
Mx gene response has been observed at 3 hours post-infection (4.18 log
2 folds) and then decreased level of expression similar to H9N2 infected CEFs [
32] and it was delayed in Kadaknath at 24 hours post-infection (3.57 log
2 folds) [
31].
Mx gene was used as positive control gene because it is a one of the well-known
IFN-stimulating and it highly expressed as restriction factor of influenza A viral infection [
33].
Results from other publications' findings support in a similar way that the expression of
chIFITM-2, 3, and
Mx significantly increased after H3N8 infection, and this increase started at 6 hours after infection. Although there was a reduction in
Mx expression at 12 hours after infection and both
chIFITM2 and 3 were significantly elevated. At 6 hours after infection,
chIFITM1 2, 3, and
5 and
Mx expression significantly increased and persisted for 24 hours in H5N3-infected CEF cells [
32]. Like this,
IFITMs were constantly and significantly upregulated in Kadaknath CEF cells throughout the study, which may be related to the increased level of
IFN-γ gene expression. In contrast, Aseel CEFs expressed low levels of
IFN-γ and
chIFITM genes investigated. This is supported by the findings of other papers. Thus, it was hypothesized that interferons would activate and upregulate the expression of
chIFITM in CEFs based on evidence from researchers Whitehead and Smith et al. [
14,
32]. It was further demonstrated that it is possible to inhibit virus replication by simply preventing access into a cell as evidenced by the production of the
IFITMs following
IFN treatment [
26,
30].
We measured the log
10 viral copies of NDV at 3-, 6-, 12-, 24- and 48-hours post-infection in CEF cells and contrasted both breeds. The viral load steadily increased from 3 hours post-infection itself in both breeds and the viral load was significantly (p<0.01) lower in Kadaknath when compared to Aseel from 3- to 12- hours post-infection and also overall load through the infected period. The outcomes showed that the ND viral load at 12 hours post-infection in Kadaknath CEF (2.15 log
10) cells was reduced by 60% in comparison to Aseel CEF (5.38 log
10) cells. Inversely proportional to viral load, Kadaknath showed significantly (p<0.01) high expression of all the
chIFITM genes at all the periods than Aseel. Like this, Blyth et al. found that overexpressing
chIFITM3 reduces influenza H6N2 and H1N9 strain infection in DF-1 cells by 30 to 40% [
34]. Similarly to this,
in vitro overexpression of
chIFITM3 limits the multiplication of the influenza virus by 55% [
14]. Infectious Bursal viral (IBV) strains of QX, M41-CK and Beaudette infection significantly upregulates all
IR-chIFITM genes at 24 hpi [
13]. Scientist concluded that
chIFITM2 and 3 greatly decrease the lyssavirus infection [
13,
14].
However, by altering the characteristics of cellular membranes and blocking the cell surface receptors to restrict viral entry, the Interferon-inducible transmembrane proteins (
IFITMs) prevent many harmful viruses from infecting cells and causing infection [
35,
36,
37]. This ultimately prevents viral fusion [
36]. Several reports confirm that the
IFITMs effectively control the RNA viruses such as Avian influenza A virus (IAV), Lyssaviruses [
14], Infectious Bronchitis virus (IBV) [
13] and Avian Reovirus multiplication (ARV) [
30], which follows the endosomal pathway to enter the host cell membrane for multiplication.
Microscopic analysis of the infected cells showed that the monolayer remained intact, much like in mock-infected cells, and that no CPE occurred until 3 h post-infection (Aseel) to 6 h post-infection (Kadaknath). However, after 6 to 12 hpi, the light-microscopy analysis revealed morphological changes indicative of CPE, including rounding, the fusing of infected cells to form syncytia, and the detachment of cells from the monolayer followed by cell death. Cellular rounding, membrane blebbing, cytoplasm vacuolation, nuclear condensation, and nuclear envelope collapse are among the morphological changes brought on by NDV infection. This result is congruent with what has been documented in other publications [
38]. The CPE in the current investigation, however, showed that CEF cells of both breeds began exhibiting CPE sooner than had been previously reported. According to Li
et al., overexpression of
IFITM3 inhibited the inflammatory response of PF15 cells and is crucial to the
TLR4-
NF-
B signalling pathway, which is implicated in the inflammatory response [
39]. In Kadaknath,
chIFITM3 expression levels are consistently high (p<0.01), with a log
2 fold range of 10.08 to 11.68 in a contrast to 1.00 to 3.46 in Aseel. As a result, compared to Aseel, the Kadaknath CEF cells had a delayed cytopathic effect and cell death. Elevated
IFITM gene expression inhibits the spread of infections by restricting host cell proliferation. It is also involved in inhibiting cell adhesion and controlling cell growth [
40]. Additionally, Anjum et al. noticed a decrease in cytopathic effects in
chIFN-treated CEF cells when they were infected with ND and AIV [
41]. Similarly, in Kadaknath CEF cells expressed high level of
IFN-γ and delayed cytopathic changes was observed.
Author Contributions
Conceptualization, M.M., A.K.T. and A.R; methodology, M.M., and A.K.T.; software, M.M., and V.B.R.; validation, N.M., K.S., and M.S.; formal analysis, A.K.T.; investigation, M.M.; resources, V.G.; data curation, M.M. and A.K.T.; writing—original draft preparation, M.M., and A.K.T.; writing—review and editing, N.M., M.S., A.R., S.O.P and K.S.; supervision, A.K.T., N.M., M.S., and K.S; project administration, N.M., K.S., and M.S.; funding acquisition, N.M., and M.S.: All authors have read and agreed to the published version of the manuscript.