Genomic alterations in persistently infecting oncolytic Newcastle disease virus reveal mechanisms of viral persistence in bladder cancer cells

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Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Genomic alterations in persistently infecting oncolytic Newcastle disease virus reveal mechanisms of viral persistence in bladder cancer cells View ORCID Profile U Ahmad , View ORCID Profile D Chau , View ORCID Profile SC Chan , View ORCID Profile K Yusoff , View ORCID Profile A Veerakumarasivam , View ORCID Profile S Abdullah doi: https://doi.org/10.1101/2025.05.06.652422 U Ahmad 1 Medical Genetics Laboratory, Genetics and Regenerative Medicine Research Centre, Faculty of Medicine and Health Sciences, University Putra Malaysia , 43400 UPM Serdang, Selangor, Malaysia 2 Human Genetics Informatics, Department of Anatomy, Faculty of Medical Sciences, Sa’adu Zungur University , Bauchi State, Nigeria Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for U Ahmad D Chau 1 Medical Genetics Laboratory, Genetics and Regenerative Medicine Research Centre, Faculty of Medicine and Health Sciences, University Putra Malaysia , 43400 UPM Serdang, Selangor, Malaysia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for D Chau SC Chan 2 Human Genetics Informatics, Department of Anatomy, Faculty of Medical Sciences, Sa’adu Zungur University , Bauchi State, Nigeria Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for SC Chan K Yusoff 4 Malaysia Genome and Vaccine Institute, National Institutes of Biotechnology Malaysia , Jalan Bangi, 43000 Kajang, Selangor Darul Ehsan, Malaysia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for K Yusoff A Veerakumarasivam 1 Medical Genetics Laboratory, Genetics and Regenerative Medicine Research Centre, Faculty of Medicine and Health Sciences, University Putra Malaysia , 43400 UPM Serdang, Selangor, Malaysia 3 Department of Biological Sciences, Sunway School of Science and Technology, Sunway University, Jalan Universiti , Bandar Sunway, 47500 Selangor, Malaysia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for A Veerakumarasivam For correspondence: abhiv{at}sunway.edu.my S Abdullah 1 Medical Genetics Laboratory, Genetics and Regenerative Medicine Research Centre, Faculty of Medicine and Health Sciences, University Putra Malaysia , 43400 UPM Serdang, Selangor, Malaysia 4 Malaysia Genome and Vaccine Institute, National Institutes of Biotechnology Malaysia , Jalan Bangi, 43000 Kajang, Selangor Darul Ehsan, Malaysia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for S Abdullah Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstracts Newcastle disease virus (NDV) is a promising oncolytic agent with a non-segmented, negative-sense single-stranded RNA (ssRNA) genome of approximately 15 kb. While NDV selectively replicates in and lyses a wide range of human cancer cells, a subset of these cells develop persistent infections, potentially compromising the therapeutic efficacy of NDV-based treatments. To investigate the molecular basis of persistent infection, we performed transcriptome profiling of TCCSUP bladder cancer cells persistently infected with the NDV AF2240 strain. Deep sequencing using Illumina HiSeq 2000 was conducted in triplicate, and the resulting viral and host reads were separated and analyzed. Using Integrative Genomic Viewer (IGV) software, we identified several nucleotide variants linked to persistence. Specifically, nucleotide alterations included a deletion at 359A→C and a substitution at 1653C→T within the nucleoprotein (NP) gene, as well as an insertion at 3338C→T in the matrix protein (M) gene. Additionally, a GGG base insertion was detected at position 2290 in the phosphoprotein (P) gene. Crucially, we observed truncations in the hemagglutinin-neuraminidase (HN) gene (nt 8263–8390) and the large polymerase (L) gene (nt 6203–6342). These mutations and truncations suggest significant disruptions in viral replication, assembly, and host cell attachment, potentially facilitating viral persistence in bladder cancer cells. Understanding these genomic alterations provides valuable insights into the mechanisms driving viral persistence and could inform strategies to optimize the oncolytic efficacy of NDV in cancer therapy. 1. Introduction Newcastle disease virus (NDV), a member of the Avian paramyxovirus (APMV) family, has demonstrated significant oncolytic potential against various cancer types. NDV contains a non-segmented, negative-sense single-stranded RNA (ssRNA) genome of approximately 15– 16 kb in size, which encodes six structural proteins arranged as 3’-NP-P-M-F-HN-L-5’[ 1 , 2 ]. These structural proteins include the nucleoprotein (NP, 55 kDa), phosphoprotein (P, 53 kDa), and large polymerase protein (L, 200 kDa), which form the nucleocapsid[ 3 , 4 ]. The external membrane of the virion is composed of the haemagglutinin-neuraminidase (HN, 74 kDa) and fusion glycoprotein (F, 67 kDa), crucial for viral attachment and fusion with host cells[ 5 , 6 ]. The inner layer of the viral envelope is made of the matrix protein (M, 40 kDa), which plays an essential role in viral assembly and budding[ 7 ]. NDV’s ability to selectively replicate in and kill cancer cells has been well-established in both preclinical and clinical studies[ 8 , 9 ]. However, it has been observed that a subset of cancer cells can develop persistent infections, enabling them to resist NDV-mediated oncolysis[ 10 ]. This phenomenon of viral persistence has also been noted in other oncolytic viruses, such as Reovirus[ 11 ] and the Edmonston Measles virus[ 12 ]. Persistent infection suggests that a subpopulation of cancer cells may harbor genetic or molecular mechanisms that allow them to evade viral lysis[ 10 ]. This poses a significant challenge to the therapeutic efficacy of NDV, as tumors contain genetically heterogeneous cell populations with various aberrations[ 13 ]. In heterogeneous tumors, the differential response to NDV could be attributed to the diverse genetic profiles of the cancer cells[ 14 ]. Persistent infection is particularly concerning as it may reduce the overall effectiveness of NDV-based oncolytic virotherapy[ 15 ]. Although recombinant NDV strains with enhanced oncolytic potential have been developed[ 16 ], the risk of viral persistence remains. Understanding the molecular mechanisms that allow cancer cells to resist NDV-induced cytotoxicity is essential for improving therapeutic outcomes. In this study, we employed bioinformatics tools to investigate the genomic profile of the persistently infecting NDV strain (NDVpi) isolated from TCCSUP bladder cancer cells. Using RNA-Seq data, we identified several nucleotide variations, including deletions, insertions, and truncations in key viral proteins. These mutations likely contribute to viral persistence in cancer cells. By deciphering these genomic alterations, we aim to gain insights into the mechanisms driving viral persistence and suggest strategies to enhance NDV’s oncolytic efficacy. 2. Materials and Methods 2.1. Cell Culture and Virus Infection The TCCSUP human bladder cancer cell line was used as the in vitro model for persistent infection studies. Cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% L-glutamine, under standard conditions at 37°C with 5% CO □. The cells were persistently infected with the oncolytic Newcastle disease virus (NDV) AF2240 strain at a multiplicity of infection (MOI) optimized for persistent infection studies[ 10 ]. Infections were monitored over time to ensure establishment of persistent infection without full cytolysis. At the point of harvest, cells were washed with PBS, lysed, and total RNA was extracted for transcriptomic analysis. 2.2. RNA Extraction and Quality Control Total RNA was extracted from both the persistently infected TCCSUP (TCCSUPPi) cells and uninfected TCCSUP control cells using the Qiagen RNeasy Micro Kit (Qiagen, Germany), following the manufacturer’s protocol. RNA concentration and purity were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific), while RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Only RNA samples with an RNA Integrity Number (RIN) greater than 7.0 were considered suitable for sequencing to ensure high-quality transcriptomic data. 2.3. RNA Sequencing RNA-Seq libraries were prepared from 1 µg of total RNA using the TruSeq RNA Library Preparation Kit (Illumina) following the manufacturer’s protocol. Briefly, mRNA was isolated from total RNA using poly(A) selection, followed by fragmentation and reverse transcription into cDNA. The cDNA was then end-repaired, A-tailed, and ligated with Illumina adapters. The libraries were amplified by PCR and purified using AMPure XP beads (Beckman Coulter). Library quality was validated using the Agilent 2100 Bioanalyzer, and libraries were quantified using qPCR. The prepared libraries were sequenced on an Illumina HiSeq 2000 platform, generating paired-end reads of 100 bp in triplicate for each sample. Approximately 30 million reads were generated per sample to ensure sufficient coverage for both host and viral transcriptome analysis. 2.4. Bioinformatics Analysis Raw sequencing reads were first subjected to quality control using FastQC[ 17 ]. Low-quality reads and adapter sequences were trimmed using Trimmomatic. The high-quality filtered reads were then mapped to a combined human (hg19) and NDV reference genome (GenBank: JX012096 .20) using HISAT2 aligner[ 18 ]. After mapping, BAM files for viral and human reads were separated for downstream analysis. 2.5. Viral Genome Analysis The viral BAM file was analyzed using the Integrative Genomic Viewer (IGV)[ 19 ] to visualize aligned reads and identify mutations in the NDV genome. To facilitate this, a custom viral genome annotation file was generated using the NDV reference genome and corresponding GFF3 and FASTA files. Viral variants, including single nucleotide polymorphisms (SNPs), insertions, deletions, and truncations, were manually identified and recorded. Supplementary workflow details are provided in Supplementary Figure 1 . 3. Results and Discussion Genomic Variations in Persistently Infecting NDV (NDVpi) To investigate the genomic alterations in the Newcastle disease virus (NDV) strain AF2240 responsible for persistent infection in bladder cancer cells, RNA-Seq data from TCCSUPPi cells were analyzed. The viral genome was aligned to the NDV reference sequence using the Integrative Genomic Viewer (IGV) to identify mutations, insertions, deletions, and truncations. Throughout the viral genome (3’-NP-P-M-F-HN-L-5’), spanning regions revealed several nucleotide mismatches and low-quality bases (often semi-transparent or faint), which were detected at key coding regions in the viral genome ( Table 1 ). These alterations suggest mutations that may contribute to the establishment and maintenance of persistent infection by the virus. View this table: View inline View popup Download powerpoint Table 1: Putative variants detection 1. Nucleoprotein (NP) Variants The nucleoprotein (NP) gene displayed two distinct mismatches, including a deletion at position 359A→C (2.4% of the reads) and a substitution at position 1653C→T (11.0% of the reads). NP plays a critical role in encapsidating viral RNA, a process essential for viral replication and transcription[ 1 ]. Mutations in this region may impact the stability of the ribonucleoprotein complex, potentially impairing viral replication and allowing a subset of infected cells to survive, thereby contributing to viral persistence ( Figure 2 ). In other studies of RNA viruses, similar NP mutations have been linked to altered replication efficiency and immune evasion[ 9 ]. Download figure Open in new tab Figure 1. IGV alignment of deep sequencing reads of NDVpi obtained from cancer RNA-Seq data. The various proteins encoded by the corresponding regions of the genome are shown above the alignment. The horizontal bars represent read alignments with the genome; gray bars are properly aligned and placed read pairs, and coloured bars are read pairs that differ from the expected insert size or read orientation. The vertical coloured lines in the coverage plot represent nucleotide positions that were different from the reference genome sequence. Download figure Open in new tab Figure 2. Putative variant in NP protein of NDVpi. Changes in nucleotide sequence of NP protein is observed at 359AdelC (deletion) and 1,653C>T positions. 2. Matrix Protein (M) Variants A single insertion at 3338C→T was identified in the matrix protein (M) gene, accounting for 22.3% of the viral reads ( Table 1 ). The M protein is involved in viral assembly and budding from the host cell membrane[ 7 ]. Mutations in this protein could reduce the efficiency of virion assembly or release, limiting cytopathic effects and allowing infected cells to remain viable, which can facilitate persistent infection ( Figure 3 ). Previous research has shown that alterations in matrix proteins can significantly affect viral budding efficiency, promoting persistent infections[ 8 ]. Download figure Open in new tab Figure 3. Putative variant in M protein of NDVpi. Changes in nucleotide sequence of M protein is observed at 3,338CinsT (insertion). 3. Phosphoprotein (P) Variants An insertion of three guanine nucleotides (GGG) was detected at position 2290 in the phosphoprotein (P) gene, contributing to 15.3% of the aligned reads. The P protein is essential for the viral RNA-dependent RNA polymerase complex, working as a cofactor in replication and transcription[ 11 ]. Mutations in this region could affect the functionality of the viral polymerase, leading to inefficient viral RNA synthesis, which can contribute to viral persistence through suboptimal replication dynamics ( Table 1 ). Changes in polymerase cofactor proteins are known to be crucial in maintaining persistent infections by modulating viral replication and host immune response[ 5 ]. 4. Hemagglutinin-Neuraminidase (HN) and Large Polymerase (L) Truncations The IGV analysis also revealed significant truncations in the hemagglutinin-neuraminidase (HN) and large polymerase (L) genes. A truncation between nucleotide positions 8263 and 8390 in the HN gene was detected in 55.1% of the viral reads ( Table 1 ). The HN protein is vital for viral attachment and entry into host cells by binding to sialic acid receptors on the cell surface[ 16 ]. Truncations in this region may impair the virus’s ability to efficiently infect new host cells, limiting its spread and contributing to viral persistence ( Figure 4 ). Similarly, the L gene, which encodes the viral RNA polymerase, exhibited a truncation between nucleotide positions 6203 and 6342 in 41.4% of the reads. Truncations in the L gene can significantly reduce viral replication capacity[ 10 , 20 ]. Such genome truncations have been observed in other RNA viruses as well, where they contribute to persistent infection by limiting the cytolytic potential of the virus[ 12 ]. Download figure Open in new tab Figure 4. NDVpi aligned to the reference sequence showing genome truncation. Alignment of deep-sequence reads obtained from NDVpi. The alignment shows genome truncations (indicated by arrows), the majority of which were mapped to nucleotide position from 8263 to 8390 in HN protein. Download figure Open in new tab Figure 5. Alignment of NDVpi to the reference with truncated genome. Truncation of the viral genome is shown by the arrows. The truncated area was mapped to nucleotide position from 6203 to 6342 in L protein. Implications for NDV Persistence in Cancer Cells The various genomic mutations and truncations identified in NDVpi provide insight into the molecular mechanisms that facilitate persistent infection in bladder cancer cells. The mutations in the NP, M, and P genes likely contribute to reduced replication efficiency and viral assembly, while the truncations in the HN and L genes may impair the virus’s ability to effectively attach to and replicate within host cells. This allows the virus to maintain a persistent, low-level infection, evading complete clearance by the host’s immune system. Similar mechanisms of persistence have been reported in other oncolytic viruses, such as vesicular stomatitis virus (VSV) and reovirus[ 10 , 14 ]. Persistent infection poses a challenge for the use of NDV as an oncolytic virus in cancer therapy. While persistent infection may allow NDV to remain within the tumor microenvironment, it can reduce the cytolytic effects needed for tumor clearance. Previous studies have suggested that targeting the specific genomic regions involved in persistence could enhance NDV’s oncolytic potential[ 8 ]. The development of engineered NDV strains that minimize the likelihood of persistence, while maintaining robust cytolytic activity, represents a promising approach to overcoming this limitation[ 9 ]. 4. Conclusion This study identified key genomic alterations in the persistently infecting oncolytic Newcastle disease virus (NDVpi) in bladder cancer cells, specifically within the NP, M, P, HN, and L genes. These alterations include truncations, deletions, and insertions, suggesting that defects in these critical viral proteins may contribute to viral persistence by impairing viral replication, assembly, and host cell attachment. The mutations in the NP, M, and P genes, along with the truncations in the HN and L genes, appear to significantly affect the virus’s ability to effectively complete its replication cycle, potentially allowing the virus to evade immune detection and establish long-term persistence in the host cells. However, the precise mechanisms by which these mutational changes lead to persistent infection remain unclear. Further investigation is needed to determine how these mutations arise and how they influence viral behavior, as well as to explore strategies to prevent persistence. Understanding these mechanisms is crucial for improving the therapeutic potential of NDV as an oncolytic agent in cancer therapy, enabling the development of targeted approaches to mitigate viral persistence and enhance treatment efficacy. Author Contributions A.V., U.A., C.S.C., C.D., S.A., and Y.K., designed the study. U.A. performed the wet and dry lab work as well as analysed the data. U.A wrote the paper. All authors reviewed the manuscript. Funding This study was supported by the Ministry Energy, Science, Technology, Environment and Climate Change (MESTECC) Malaysia Flagship Fund, reference number: FP0514B0021-2(DSTIN). Data Availability All data supporting the results in this article are included in the present and supplementary files except the raw and processed sequencing data that have been submitted to the NCBI Sequence Read Archive with accession number: PRJNA543209 ( https://www.ncbi.nlm.nih.gov/bioproject/PRJNA543209 ) and Gene Expression Omnibus (GEO) with accession number GSE140902 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE140902 ). Conflicts of Interest The authors declare no conflict of interest. Abbreviations Supplementary Download figure Open in new tab Supplementary 1. Workflow and bioinformatics pipelines used for the analysis Acknowledgments None Funder Information Declared Ministry Energy, Science, Technology, Environment and Climate Change (MESTECC) Malaysia , FP0514B0021-2(DSTIN) Footnotes https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE140902 References 1. ↵ Seal , B.S. , D.J. King , and H.S. Sellers , The avian response to Newcastle disease virus . 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