Chicken microRNA 26a-5p regulates replication of Newcastle disease virus by direct targeting of the viral polymerase
Abhijeet A Bakre, Megan C Mears
TL;DR
Chicken microRNA 26a-5p can inhibit the replication of Newcastle disease virus by targeting its polymerase gene.
Contribution
This study identifies miR-26a-5p as a regulator of NDV replication through direct targeting of the viral polymerase.
Findings
Upregulation of chicken miR-26a-5p inhibits replication of both lentogenic and velogenic NDV strains.
miR-26a-5p directly targets the NDV polymerase gene, reducing its transcripts upon transfection.
Overexpression of miR-26a-5p downregulates innate immune sensing genes and increases viral titer for a velogenic strain.
Abstract
Newcastle disease (ND), caused by Newcastle disease virus (NDV), is a significant threat to the poultry industry and outbreaks of virulent strains can lead to substantial economic losses. Studies to identify molecular pathways that can be used for intervention or to reduce pathology are critical for mitigating losses due to ND. In this study, we demonstrate that chicken mir-26a-5p upregulation inhibited the replication of both lentogenic and velogenic NDV strains. Computational analysis identified a highly conserved miR-26a-5p binding site in the NDV polymerase gene and transfection of the miR-26a-5p mimic following viral infection demonstrated a direct inhibition of polymerase transcripts while inhibitor transfection led to partial rescue of the miR-26a-5p mediated repression. Alternately, stable overexpression of miR-26a-5p led to the downregulation of multiple genes in the innate…
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Figure 4- —https://doi.org/10.13039/100007917Agricultural Research Service
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Taxonomy
TopicsMicroRNA in disease regulation · Virology and Viral Diseases · Animal Virus Infections Studies
Introduction
Newcastle disease (ND) caused by Newcastle disease virus (NDV) (also known as avian paramyxovirus 1 (APMV-1) or Orthoavulavirus javaense (OAVJ)) is an important disease in poultry owing to its commercial impact on domestic and international trade [1]. The most recent outbreak of virulent NDV in the USA (2018) infected 401 backyard flocks, 4 commercial laying hen farms, 1 live bird market and 2 feed stores [2, 3]. The containment of this outbreak required extensive resources for bird depopulation involving more than 800 federal and state personnel and resulted in a significant economic burden. Recently, an outbreak of virulent NDV in Brazil (July 2024) resulted in the death of 7000 chickens with an additional 7000 birds culled. This outbreak led to export restrictions of ∼50–60,000 metric tons of poultry products from Brazil causing significant economic losses to producers [4]. Developing better and faster diagnostics, understanding host and viral drivers of NDV pathology, and improving current NDV vaccines are critical to respond to ND outbreaks effectively, and reduce economic burdens.
NDV is a prototypical paramyxovirus with a ~ 15 kb negative sense single stranded RNA (-ssRNA) genome that encodes six genes, nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and polymerase (L). NDVs are classified by the degree of their virulence into low virulence (lentogenic), intermediate virulence (mesogenic) or high virulence (velogenic) viruses [5, 6]. A polybasic site in the viral fusion protein that can be cleaved by multiple furin-like proteases results in expanded tissue tropism for virulent NDVs and consequent pathology; in constrast, low virulence NDVs have a monobasic site that is cleaved only by trypsin-like proteases and restricts tropism to the respiratory tract [5].Other viral genes such as M, L and P also contribute to virulence [7, 8]. Following infection of target tissues, dsRNA produced during replication is detected by the interferon induced with helicase C domain 1 (IFIH1) gene (also known as melanoma differentiation antigen 5 (MDA5)) owing to lack of the retinoic acid induced gene (RIG-I) in the chicken genome [9]. This sensing activates the interferon (IFN) response, induction of interferon stimulated genes (ISGs) and establishment of an anti-viral state (both autocrine and paracrine).
Many host genes and pathways regulating viral replication have been identified by genome-wide RNA interference (RNAi) studies for several DNA [10–13] and RNA viruses [14–19], including NDV. RNAi targeting the NP, P, M, F, and L genes of the LaSota strain of NDV expressing a velogenic fusion protein were shown to delay viral replication [20, 21]. Specifically, siRNAs against NP were shown to also promote cellular viability while reducing viral replication [20]. Additionally, host encoded microRNAs (miRs) have been demonstrated to regulate viral replication for multiple virus families. Data from recent studies demonstrates altered miR and mRNA expression during infection with different NDV strains. Direct impact of miRs on the chicken inflammatory response during infection has been demonstrated by miR-203a regulation of transglutaminase 2 (TGM2) [22]. In chicken fibroblasts (DF-1), gga-mir-451 and miR-199a were demonstrated to support NDV replication; the former by suppression of the NDV induced inflammatory response [23]. In contrast, chicken miR-19b-3p and gga-mir-29a-3p inhibited NDV replication [23]. In chicken macrophages, LaSota infection led to differential miRNA expression predicted to target immune related pathways such as the retinoic acid induced gene (RIG-I) and nucleotide oligomerization domain (NOD)-like receptor pathway [24]. Wang et al. showed that gga-miR-455-5p suppressed NDV replication by targeting the cellular suppressors of cytokine signaling (SOCS3) gene [25].
In this study, we characterize one such chicken miRNA, gga-miR-26a-5p, which was found to modulate NDV replication by two distinct mechanisms. In the first mode, transient over- expression of miR-26a-5p reduced NDV replication by targeting the viral polymerase via a highly conserved binding site. Alternatively, constitutive miR-26a-5p over-expression led to downregulation of viral sensing, the ensuing IFN and ISG response, and potentially other cellular pathways, culminating in a small but significant increase in viral titer. These data demonstrate the complex nature of miRNA function during NDV infection and replication.
Materials and methods
Cell culture
Chicken fibroblast cells (DF-1) (CRL-3586) obtained from the American Type Culture Collection (ATCC, VA, USA) were grown in DMEM containing high glucose, 1x antibiotic-antimycotic mix, 1mM Sodium pyruvate, 1X Glutamax and 10% heat inactivated fetal bovine serum (FBS)(Biowest, USA) (referred to as complete medium hereafter) at 39 °C with 5% CO_2_ as recommended by ATCC. The cells were confirmed to be mycoplasma free using mycoplasma specific primers.
DF-1 cell lines overexpressing gga-miR-26a-5p were constructed by stable transfection of DF-1 cells with PiggyBac plasmids expressing miR-26a-5p under the control of a moderately active mouse phosphoglycerate kinase promoter. Stable transfectants were selected using hygromycin B (Roche, USA) selection, visualization of fluorescent marker expression (mCherry), and limiting dilution to select single cell colonies that were expanded under selection and then used for infection. The overexpression of miR-26a-5p was validated by qPCR.
MiRNA mimic transfection
Forward transfections were performed by plating 12–15,000 DF-1 cells per well in 96-well plates excluding the outermost columns and rows that were filled with medium only or phosphate buffered saline (PBS). Cells were incubated overnight to achieve 70–85% confluency. The following day, the medium was gently aspirated, and the monolayers were gently washed twice with serum-free medium. Custom miRNA mimics of gga-miR-26a-5p were obtained commercially (Horizon Discovery, UK). All miRNA control, inhibitor, or mimic transfections were carried out using Dharmafect 1 (Horizon Discovery, UK) according to the manufacturer’s recommended conditions at a final concentration of either 25nM or 100nM. In some experiments, transfections (at 25nM final concentrations) were carried out immediately following a 1-hour infection with a red fluorescent protein (RFP) expressing LaSota strain of NDV (MOI = 3.0) or non-fluorescent CA18 (MOI = 1.0).
Cytotoxicity was measured using the Cell Titer Blue (CTB) assay (Promega, USA) according to manufacturer’s recommendations. Briefly, 48 h post-transfection, media in the wells was replaced with fresh media containing CTB reagent at a final 10% volume, incubated for 1.5 h, and then absorbance was measured on a Synergy HTX plate reader (Biotek, USA) using the manufacturer’s recommended settings. Wells with no cells or completely reduced media with CTB were used as the negative and positive controls respectively. The reduction in absorbance was plotted relative to the controls to calculate the percent cytotoxicity. The cell density and incubation periods for DF-1 cells in this assay were optimized prior to the experiments.
Viruses
Chicken/California/D1806566/2018 (CA18) virus [26], LaSota virus, or LaSota-RFP virus (SEPRL in-house stocks) were grown and harvested from embryonated chicken eggs (ECEs) as described previously [27]. Viral titers were determined as 50% egg infectious doses (EID_50_) and HA assays were performed according to the OIE manual [6]. All infections with CA18 viruses were performed in a BSL3E laboratory as per the institutional SOPs.
qRT-PCR
Total RNA from transfected and CA18 infected cells was isolated using Trizol (Invitrogen, USA) according to the manufacturer’s recommendations. However, 1–2µL of Glycoblue (Cat # AM9516, ThermoFisher, USA) was used during precipitation for easy pellet visualization. RNA was quantified using a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific, USA). First-strand synthesis was carried out using equal amounts of RNA across all samples with the miRNA cDNA synthesis kit (Agilent, USA) according to the manufacturer’s recommendations. Briefly, total RNA was polyadenylated with E. coli Poly A polymerase at 37 °C for 30 min, then polyadenylated RNA was annealed to a proprietary adaptor oligo at 25 °C for 15 min, reverse transcribed at 42 °C for 60 min followed by inactivation of reverse transcriptase at 95 °C for 5 min. First-strand cDNA was diluted 1:10 in molecular grade water then used as template for qPCR.
All qRT-PCR reactions contained 1 µl of 1:10 diluted cDNA, 2X Lunascript Universal SYBR master mix (New England Biolabs (NEB), USA) and gene specific forward and reverse oligos (250nM final concentration) with or without probe. Amplifications were performed on a Quantstudio 5 thermocycler (Applied Biosystems, USA) using the following program: 95 °C for 1 min, 40 cycles of 95 °C for 15 s, annealing at 56 °C for 30 s and extension at 60 °C for 30 s, followed by melt curve analysis. Data were analyzed using the ΔΔCt methodology using 18S rRNA as the housekeeping gene [28]. All qPCR setup and data analysis were in conformance with the MIQE 2.0 guidelines [29]. The expression of miR-26a was determined by qPCR using an miR-26a-5p specific forward primer (5’-TTCAAGTAATCCAGGATAGGC-3’) and a universal polyT primer (5’-GCGAGCACAGAATTAATACGAC-3’) under the following conditions: Initial denaturation at 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 s, annealing at 46 °C for 30 s and extension at 60 °C for 30 s followed by melt curve analysis. Primer sequences used for all other gene expression assays are listed in Supplementary File 1.
Hemagglutination assay
Supernatants from cells transfected with controls or miRNA mimic, and mock infected or LaSota/Ca18 virus infected cells were freeze-thawed three times before being used in an HA assay. For the HA assay, 50 µl of supernatant was serially diluted 2-fold across a 96-well plate with an equal volume of PBS with consistent mixing between dilutions. Equal volumes of freshly prepared 1% specific pathogen free chicken red blood cells were added to each well, and the plates were sealed and then mixed for 30 s at room temperature followed by incubation for 30 min before reading. A row of wells containing only PBS + erythrocytes was used as a negative control whereas wells containing PBS + LaSota of known titer and erythrocytes were used as a positive control. Hemagglutination was verified by lack of tear drop formation in the tilted plates.
Computational analyses
The sequence of gga-miR-26a-5p was compared to the chicken/California/D1806566/2018 (CA18) (accession number MK040373.1) or LaSota (accession number JF950510/AF077761) sequences using BLASTN-short with a gap open/extend penalty set to + 1/−3 which corresponds to > 90% identity between sequences [30]. Only hits spanning the entire miRNA seed site in the anti-sense orientation were considered for the analysis.
To identify additional core pathways that are potentially regulated by mir-26a-5p, we first mined all predicted and/or validated gene targets of miR-26a-5p in humans and chicken using Targetscan and miRTarbase [31, 32] respectively. Gene lists were then annotated and analyzed for gene functional enrichment using FLAME ver 2.0 [33] which combines gene ontology (GO) annotations from the Kyoto encyclopedia of genes and genomes (KEGG) to identify over represented processes and pathways using three different algorithms: gProfiler [34–37], WebGestalt and aGOtool. Pathways were filtered using a significance threshold of 0.05.
Statistical analysis
All statistical analyses was performed using GraphPad Prism ver. 9.6.4 (build 681) (GraphPad Software LLC, USA). Statistical analyses were performed using either 2-tailed unpaired t-tests or repeated-measures one-way ANOVA with Dunnett’s or Sidak’s multiple comparisons test. Data represent mean±standard error of the mean of three or more independent experiments (biological replicates) with three technical replicates.
Results
MicroRNA gga-miR-26a-5p reduces NDV replication
Upregulation or inhibition of miRNA activity via transfection of miRNA mimics or inhibitors can be used to identify cellular pathways that are critical for viral replication. In a primary screen, DF-1 cells were transiently transfected with a miRNA mimic or inhibitor for 48 h then tested for cytotoxicity. In parallel, cells infected with either LaSota-RFP or CA18 isolates of NDV were also transfected to identify miRNAs that modified the viral replication.
Transfection of the chicken miRNA-26a-5p mimic reduced replication of the LaSota-RFP reporter virus at 48 h post-transfection (Fig. 1a) and led to a significant reduction in the HA titer for the CA18 strain of NDV (Fig. 1b). HA titers were significantly reduced relative to infection in untreated cells but not from cells transfected with the mimic negative control (MNC), suggesting that transfection itself also affected viral replication. Therefore, real time PCR analysis of the CA18 infected cells was used to determine the impact of the miR on the matrix (M) (Fig. 1c) and polymerase (L) (Fig. 1d) viral genes. There was a significant reduction in L transcripts, but not M, relative to 18 S rRNA and control transfection. Together, these data support the hypothesis that miR-26a has an anti-viral effect on replication of NDV viruses.
Fig. 1miR-26a-5p regulates replication of two different NDV strains. a) Transfection of miR-26a-5p mimic (filled bars) reduces fluorescence from a LaSota red fluorescence protein reporter strain compared to control transfection (clear bars). b) Mir-26a-5p mimic transfection reduces CA18 HA titers. Matrix (c) and polymerase (d) gene expression following miR-26a-5p mimic transfection is graphed relative to 18 S rRNA and control. *=p < 0.05, **=p < 0.01, ***=p < 0.005, ****=p < 0.0001. Data represent the mean ± SEM of six independent replicates. MNC = Mimic Negative Control
Gga-miR-26a-5p targets the NDV RNA polymerase to reduce viral replication
Since direct targeting of many viral genomes by host miRNAs has been previously demonstrated [38–42], we hypothesized that reduction in NDV L (Fig. 1d) might be mediated by miR-26a-5p binding to the viral genome or transcripts to modulate replication. To identify potential binding sites, we analyzed sequence complementarity, if any, between miR-26a-5p and the genomes of the LaSota and CA18 strains of NDV using BLASTN-short with parameters optimized to find short sequence matches (gap open/extend penalty set to + 1/−3) [30]. Hits were shortlisted as those that contained the miRNA seed site which has been shown to be essential for miRNA function [43]. The longest complementarity was identified between nucleotides 2–13 of miR-26a-5p and positions 8253–8264 of the CA18 polymerase gene with only 1 nucleotide mismatch (Fig. 2a). Other potential binding sites for miR-26a-5p were predicted for both LaSota and CA18 (Supplementary File 1), however these sites did not encompass the miRNA seed site and were excluded from further studies. The minimal free energy predictions of this complementarity as per RNAhybrid [44] also suggested a stable binding (mean free energy = −18.1 Kcal/mol) of mir-26a-5p to CA18 L gene (Fig. 2a).
The conservation of this binding site was evaluated across all available class II polymerase sequences (n = 1052; Supplementary File 1). Sequence logo analysis showed that except for three positions (8260, 8261, and 8263), all other positions were nearly 100% identical in the alignment, demonstrating a strong conservation of this motif (Fig. 2b). The nucleotide frequency at position 8263 demonstrated that most sequences (76.8%) had a G and 23% had an A at this position. It is important to note that A: U and G: U pairing have comparable thermodynamic stabilities [45] and are thus thermodynamically equivalent. Position 8261 had a U in 53.7% of sequences and a C in 46.3% of sequences, whereas position 8260 had a C in 76% of the sequences. These data suggest that miR-26a-5p is likely to bind to the NDV L gene for a majority of class II NDV viruses, but binding efficiency may be affected in some strains as these positions are located within the seed site of the miRNA.
To determine the impact of miR-26a-5p on polymerase transcripts, wild-type DF-1 cells were transfected with either miR-26a-5p mimic or inhibitor one hour post-infection with either LaSota (MOI = 3.0) or CA18 (MOI = 1.0) followed by the analysis of polymerase transcripts at 24 h post-infection. In transfected but uninfected controls, mimic transfection upregulated miR-26a-5p levels as expected, while the inhibitor reduced miR-26a-5p expression, although miR-26a-5p expression was not completely abolished (Fig. 2c). This is likely because miR-26a-5p is expressed at a moderately high level in chicken tissues [46, 47]. Transfections with the miR-26a-5p mimic significantly reduced polymerase transcripts whereas transfections with the inhibitor partly relieved miR-26a mediated repression, only in the case of LaSota virus infection but not in the case of CA18 (Fig. 2d). This could potentially be due to the different replication kinetics of these viruses, as LaSota is a lentogenic pathotype of NDV whereas CA18 is a velogenic pathotype. Overall, these data suggest that miR-26a-5p can directly target the NDV polymerase and can regulate polymerase transcript levels.
Fig. 2miR-26a targets and regulates NDV L expression. a) Sequence alignment of NDV- L gene (8253–8264) with miR-26a-5p as predicted by RNAhybrid is shown. Straight lines indicate Watson-Crick base pairing. Mean free energy for this interaction as predicted by RNAhybrid is indicated. b) Sequence logo demonstrates conservation of miR-26a-5p binding site in NDV L sequences (n = 1052) analyzed. Heights of letters corresponds to degree of conservation. Frequency of each nucleotide with variants, if any, is tabulated below. The predominant nucleotide is highlighted in red. Black arrows and coordinates indicate three positions where the conserved base differs from CA18. c) Transfection of miR-26a-5p mimic increases (solid black bar) miR-26a-5p levels while miR-26a-5p inhibitor reduces miR-26a-5p levels d) Expression levels of NDV L gene in cells transfected with miR-26a-5p mimic (solid bars) or 26a-5p inhibitor (hatched bars) are shown. Data represent ± SEM of three independent replicates. **=p < 0.005
Mir-26a-5p regulates multiple host genes and alters viral replication
Stable DF-1 cell lines that constitutively overexpress miR-26a-5p were constructed to overcome variations associated with transient transfection. As a control, DF-1 cells that stably overexpress miR-67 from Caenorhabditis elegans (Cel-67), which are computationally not predicted to target any chicken gene were also developed. Stable DF-1 cells overexpressing miR-26a-5p or Cel-67 were selected using mCherry expression and hygromycin B resistance to obtain single cell derived clones over several passages. qRT-PCR analysis of miR-26a-5p in two of these clonal lines (26aE5 and 26aF6) demonstrated ~ 1000-fold overexpression of miR-26a-5p in 26aE5 cells and ~ 640-fold induction in 26aF6 cells compared to the control Cel-67 cell line (Fig. 3a).
Recently, Vu et. al demonstrated that chicken miR-26a-5p could directly regulate the expression of IFIH1 and downstream effectors such as MAVS, NFκβ, IRF7, and MAPK11 in HD11 chicken macrophage cells during highly pathogenic avian influenza (HPAI) infection [48]. Computational analysis predicted two miR-26a-5p binding sites in the 3’-UTR of IFIH1 (at nucleotides 451 and 602) and a single site in chicken interferon gamma (IFNγ) (Fig. 3b), suggesting that miR-26a-5p may directly regulate the expression of IFIH1 and IFNγ. No binding sites were identified for other mediator genes (MAVS, NFκβ, STAT1, IRF7, Mx1, IFIT5 and MAPK11) or anti-viral interferons (IFNα, IFNβ, or IFNλ). To investigate whether miR-26a-5p overexpression alters the expression of these genes in these DF-1 derived cells, qRT-PCR with gene specific primers was performed using total RNA from uninfected Cel-67, 26aE5, and 26aF6 stable cells. The data demonstrated that the overexpression of miR-26a-5p led to a consistent and statistically significant repression of IFIH1, NFκβ, STAT1, IRF7, MX1, IFNα, IFNβ, IFN-λ, IFNγ, MAPK11, and IFIT5 in the 26aE5 cell line relative to the Cel-67 control (Fig. 3c). This was not, however, observed consistently in the 26aF6 cell line (Fig. 3c), and could be presumably due to lower miR-26a-5p expression in this line. These data suggest that 26a-5p overexpression regulates these IFNs and ISGs via either direct targeting (in the case of IFIH1 and IFNγ) or other mechanisms that are not presently well-understood. Additionally, treatment of wild type DF-1, Cel67 and 26aE5 cells with poly I: C (20 µg for 12 h), a known inducer of the interferon response demonstrated a statistically significant downregulation of IFIH1 and concomitant downregulation of both type I and II IFNs (Supplementary Fig. 1).
These data suggest that under constitutive miR-26a-5p expression, replication of NDV viruses may increase owing to the inhibition of the IFNs and ISGs. To evaluate this hypothesis, 26aE5 cells constitutively overexpressing miR-26a-5p were infected with either the LaSota (lentogenic pathotype) or CA18 (velogenic pathotype) strains of NDV for 48 h before total RNA was collected for evaluation of the expression of IFNs, ISGs, and viral genes by qRT-PCR, and overall viral titer was evaluated by HA test. The data demonstrated significant downregulation of STAT1, Mx1 and IFIT5 following LaSota and CA18 infection (Fig. 4a). The expression of IFIH1, MAVS, NFκβ, IRF7, IFNα, IFNβ, IFNγ and MAPK11 did not change significantly. IFNλ expression increased slightly relative to the Cel-67 control cell line, but the differences were not statistically significant. The expression levels of both the M and L transcripts of NDV increased but were not statistically significant compared to the control. However, the HA titer from infected cell supernatants showed a small but significant increase from infection with CA18, but not LaSota (Fig. 4b), suggesting that 26a-5p may regulate NDV replication differently for different pathotypes, or via additional mechanisms.
Fig. 3. Gga-mir-26a-5p regulates multiple immune related genes. a) Fold change in miR-26a-5p expression in two clonal cell lines, 26aE5 (filled bar) and 26aF6 (hatched bar), relative to Cel-67 control (clear) cell line is graphed. b) Sequence alignment demonstrates complementarity between miR-26a-5p (blue) and the 3’UTRs of IFIH1 (top) and IFNγ (bottom) in black. Arrows and nucleotide coordinates are indicated above each alignment. Straight lines indicate Watson-Crick base pairing. c) Changes in expression of multiple immune related genes in uninfected 26aE5 or 26aF6 clonal cell lines relative to 18 S rRNA and Cel-67 control cells are indicated. *=p < 0.05, **=p < 0.01, ****=p < 0.0001. Data represent mean ± SEM of three biological replicates. Statistical comparisons were performed using a 2-way ANOVA with Geisser-Greenhouse correction on a full model and Tukey’s multiple comparisons test with individual variances computed for each comparison in GraphPad 10.5.0 (build 774)
Fig. 4. Overexpression of mir-26a-5p alters viral replication via modulation of multiple immune-related genes and viral titer. a) Change in expression of immune related genes in miR-26a-5p overexpressing cell line 26aE5 following a 48-hour infection with either LaSota (dark filled bars) or CA18 (light filled bars) relative (indicated) to Cel-67 control (clear) cell line is graphed. b) HA titers of LaSota or CA18 (filled bars) relative to Cel67 (clear bars) from 48-hour infection are plotted. *=p < 0.05, **=p < 0.01, ****=p < 0.0001. Data represent mean ± SEM of three biological replicates. Statistical comparisons were performed using repeated measures two-way ANOVA with Geisser-Greenhouse correction on a full model and correcting for multiple comparisons using Sidak’s testing as recommended in GraphPad Prism 10.5.0 (build 774)
Discussion
Newcastle disease remains a challenge for backyard and commercial poultry farmers because virulent strains can cause severe mortality as seen in the 2018 outbreak in California and the 2024 outbreak in Brazil [2, 26]. The central hypothesis of this work was that targeting host genes such as miRNAs that have anti-viral activity would potentially both reduce viral replication and prevent evolution of escape mutant viruses. Previous studies have shown that some miRNAs (miR-203a [22], miR-19b-3p and miR-29a-3p [24], and miR-455-5p [25]) can regulate host genes and alter NDV replication.
In this study, the initial screenings of miRNAs for anti-viral activity against NDV identified miR-26a-5p as a potential inhibitor of NDV replication. Upregulation of miR-26a-5p levels by the transfection of miR-26a-5p mimic reduced the fluorescent reporter signal from the lentogenic LaSota strain of NDV (Fig. 1a). Reduction in viral replication was independently tested against the velogenic NDV CA18 strain from the California ND outbreak, where significantly reduced HA titers were observed (Fig. 1b). Molecular analysis revealed statistically significant downregulation of NDV L transcripts (Fig. 1d) following miR-26a-5p mimic transfection. Computational analysis revealed a highly conserved mir-26a-5p binding site in the NDV polymerase gene (Fig. 2a and b). To overcome off-target effects and demonstrate a more direct effect of miR-26a-5p on NDV L, wild-type DF-1 cells were transfected with the mir-26a-5p mimic or inhibitor immediately following LaSota or CA18 infection; these experiments demonstrated reduction in NDV polymerase upon mimic transfection which was partly relieved by LaSota by the miR-26a-5p inhibitor (Fig. 2d). Luciferase assays incorporating either the wild type or mutated polymerase binding site along with miR-26a-5p mimic transfections would likely yield conclusive evidence; however, these were not included in the present study. Reduction in polymerase levels would correlate with lower transcript abundance and possibly lower virulence as has been demonstrated earlier [49].
Since prior published data suggested that miR-26a-5p regulated IFIH1, we analyzed the expression of IFIH1 and other IFNs and ISGs in cells constitutively overexpressing miR-26a-5p. These studies demonstrated that miR-26a-5p regulated expression of the RNA sensor IFIH1, transcription factors (NFκβ, STAT1, IRF7, Mx1), interferons (IFNα, IFNβ, IFNλ, and IFNγ) and ISGs (MAPK11 and IFIT5) either directly (IFIH1 and IFNγ) or indirectly (Fig. 3).
Following the infection of a miR-26a-5p overexpressing cell line (designated 26aE5) with either LaSota or CA18, only the expression of STAT1, Mx1 and IFIT5 was significantly reduced (Fig. 4a). On the other hand, there was a small, but not statistically significant increase in expression of all other genes, suggesting that the anti-viral response induced was insufficient to curb viral replication. This was supported by an increase in both M and L transcripts as well as an increase in the HA titer for the CA18 virus (Fig. 4a and b).
The above data suggest two distinct mechanisms through which miR-26a-5p functions to regulate NDV replication; an anti-viral role when miR-26a is transiently overexpressed and a pro-viral role when miR-26a-5p is expressed constitutively. It is likely that other mechanisms are also involved in the miR-26a-5p regulation of NDV replication. Indeed, computational analyses of genes predicted to be miR-26a-5p targets identified four key pathways that may play a role in the interaction between miR-26a-5p and NDV replication: cell cycle, cellular senescence, the Forkhead box O (FoxO) signaling pathway, and the p53 signaling pathway. This is consistent with previous studies showing that NDV viruses alter the cell cycle; NDV viruses preferentially infect cells in the G2/S phase [50] and subsequent infection arrests cells in the G0/G1 phase of the cell cycle [51]. Since miRNAs target multiple pathways, it is likely that other pathways that are important for viral replication are also affected by miR-26a-5p and were not explored in this manuscript. This study thus adds to our current understanding of the role of miRNAs in regulating NDV replication and highlights the complexity of host-pathogen interactions.
Supplementary Information
Supplementary Material 1.
The reference list from the paper itself. Each links out to its DOI / PubMed record.
- 1Mano A. Brazil halts some poultry exports after Newcastle disease case. Reuters 2024 [cited 2024 07/19/2024]; Available from: https://www.reuters.com/world/americas/brazil-declares-animal-health-emergency-rio-grande-do-sul-after-newcastle-2024-07-19/
- 2OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 2021.
- 3Bustin SA et al. MIQE 2.0: Revision of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments Guidelines. Clin Chem, 2025.10.1093/clinchem/hvaf 04340272429 · doi ↗ · pubmed ↗
- 4Karatzas E et al. Flame (v 2.0): advanced integration and interpretation of functional enrichment results from multiple sources. Bioinformatics 2023;39(8). 10.1093/bioinformatics/btad 49010.1093/bioinformatics/btad 490PMC 1042303237540207 · doi ↗ · pubmed ↗