Viral Nucleic Acid Research Articles

Oncolytic activity of a coxsackievirus B3 strain in patient-derived cervical squamous cell carcinoma organoids and synergistic effect with paclitaxel

BackgroundCervical squamous cell carcinoma (CSCC) is a prevalent gynecological malignancy worldwide. Current treatments for CSCC can impact fertility and cause long-term complications, underscoring the need for new therapeutic strategies. Oncolytic virotherapy has emerged as a promising option for cancer treatment. Previous research has demonstrated the oncolytic activity of the coxsackievirus B3 strain 2035 A (CVB3/2035A) against various tumor types. This study aims to evaluate the clinical viability of CVB3/2035A for CSCC treatment, focusing on its oncolytic effect in patient-derived CSCC organoids.MethodsThe oncolytic effects of CVB3/2035A were investigated using human CSCC cell lines in vitro and mouse xenograft models in vivo. Preliminary tests for tumor-selectivity were conducted on patient-derived CSCC tissue samples and compared to normal cervical tissues ex vivo. Three patient-derived CSCC organoid lines were developed and treated with CVB3/2035A alone and in combination with paclitaxel. Both cytotoxicity and virus replication were evaluated in vitro.ResultsCVB3/2035A exhibited significant cytotoxic effects in human CSCC cell lines and xenograft mouse models. The virus selectively induced oncolysis in patient-derived CSCC tissue samples while sparing normal cervical tissues ex vivo. In patient-derived CSCC organoids, which retained the immunohistological characteristics of the original tumors, CVB3/2035A also demonstrated significant cytotoxic effects and efficient replication, as evidenced by increased viral titers and presence of viral nucleic acids and proteins. Notably, the combination of CVB3/2035A and paclitaxel resulted in enhanced cytotoxicity and viral replication.ConclusionsCVB3/2035A showed oncolytic activity in CSCC cell lines, xenografts, and patient-derived tissue cultures and organoids. Furthermore, the virus exhibited synergistic anti-tumor effects with paclitaxel against CSCC. These results suggest CVB3/2035A could serve as an alternative or adjunct to current CSCC chemotherapy regimens.

Effect of concentration, temperature, and time on the stability of hepatitis C virus nucleic acid in serum.

Previously, there were few reports about the influence of different concentrations of sample nucleic acid on the stability of samples at various temperatures and times in various literatures. Therefore, it is necessary to analyze the influence of concentration factors on the stability of samples and test results at different storage times and temperatures. This study took the concentration of hepatitis C virus nucleic acid as the research object to further understand the stability of hepatitis C virus nucleic acid test samples under various storage conditions, to provide data reference for the treatment of hepatitis C virus nucleic acid and RNA test samples before clinical laboratory test, and provide guidance and help for the improvement of laboratory quality control.

A-279 Performance Evaluation of the Respiratory Viral for BD MAX™ System in Geriatric Population

Abstract Background Respiratory Viral Infection is one of the most common infection in the geriatric population and cause major concern in the Long-Term Care Facilities. Influenza A and B, SARS-CoV-2, and respiratory syncytial virus (RSV) are responsible for most of the hospitalization and deaths among elderly patients. Rapid identification, treatment, and isolation are among the most important steps to fight the spreading of the infection. Methods The BD Respiratory Viral Panel for BD MAX™ System is an automated multiplexed real-time reverse transcriptase polymerase chain reaction (RT- PCR) test intended for the simultaneous, qualitative detection and differentiation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, influenza B, and/or respiratory syncytial virus (RSV) nucleic acid in nasopharyngeal swab (NPS) and anterior nasal swab (ANS) specimens from individuals with signs and symptoms of respiratory tract infection. The assay is targeting RNA from the nucleocapsid phosphoprotein gene (N1 and N2 regions) of the SARS-CoV-2, a conserved region of the matrix protein M1 gene for influenza A, conserved regions of the matrix protein M1 gene and HA gene for influenza B, conserved regions of the N and M genes for RSV, and the human RNase P gene. The assay was evaluated for: precision, reproducibility, accuracy, and correlation with BioGx for SARS-CoV-2, Solana (Quidel) for influenza A, influenza B and RSV; the percentage agreement for the evaluation steps was calculated. We ran 86 samples collected from patients residing in Long-Term Care Facilities. Statistical analyses were done using Analyse-it. Results The percentage agreement for precision, reproducibility, and accuracy were 100%. The patients’ correlation was 100% when compared to the existing molecular testing in the laboratory. Female patients accounted for 62.8% (54 female and 32 male); 2 patients were positive for both influenza A and RSV, and one patient was positive for both influenza A and SAR-CoV-2. Conclusions The Respiratory Viral panel for BD MAX™ System gave the benefit of a fully automated, high precision, accuracy and acceptable sensitivity assay; in addition, it offers easier collection (one swabs versus multiple swabs), faster turnaround time for four targets in less than three hours which allow for earlier isolation to prevent the spreading of the infection and initiating treatment when needed. As with every molecular assay avoiding contamination is very important to eliminated false positive results.

Predicting malignant potential of solitary pulmonary nodules in patients with COVID-19 infection: a comprehensive analysis of CT imaging and tumor markers

ObjectiveTo analyze the value of combining computed tomography (CT) with serum tumor markers in the differential diagnosis of benign and malignant solitary pulmonary nodules (SPNs).MethodsThe case data of 267 patients diagnosed with SPNs in the First Affiliated Hospital of Zhengzhou University from March 2020 to January 2023 were retrospectively analyzed. All individuals diagnosed with coronavirus disease 2019 (COVID-19) were confirmed via respiratory specimen viral nucleic acid testing. The included cases underwent CT, serum tumor marker testing and pathological examination. The diagnostic efficacy and clinical significance of CT, serum tumor marker testing and a combined test in identifying benign and malignant SPNs were analyzed using pathological histological findings as the gold standard. Finally, a nomogram mathematical model was established to predict the malignant probability of SPNs.ResultsOf the 267 patients with SPNs, 91 patients were not afflicted with COVID-19, 36 exhibited malignant characteristics, whereas 55 demonstrated benign features. Conversely, within the cohort of 176 COVID-19 patients presenting with SPNs, 62 were identified as having malignant SPNs, and the remaining 114 were diagnosed with benign SPNs. CT scans revealed statistically significant differences between the benign and malignant SPNs groups in terms of CT values (P<0.001), maximum nodule diameter (P<0.001), vascular convergence sign (P<0.001), vacuole sign (P = 0.0007), air bronchogram sign (P = 0.0005), and lobulation sign (P = 0.0005). Malignant SPNs were associated with significantly higher levels of carcinoembryonic antigen (CEA) and neuron-specific enolase (NSE) compared to benign SPNs (P < 0.05), while no significant difference was found in carbohydrate antigen 125 (CA125) levels (P = 0.054 for non-COVID-19; P = 0.072 for COVID-19). The sensitivity (95.83%), specificity (95.32%), and accuracy (95.51%) of the comprehensive diagnosis combining serum tumor markers and CT were significantly higher than those of CT alone (70.45%, 79.89%, 76.78%) or serum tumor marker testing alone (56.52%, 73.71%, 67.79%) (P < 0.05). A visual nomogram predictive model for malignant pulmonary nodules was constructed.ConclusionCombining CT with testing for CEA, CA125, and NSE levels offers high diagnostic accuracy and sensitivity, enables precise differentiation between benign and malignant nodules, particularly in the context of COVID-19, thereby reducing the risk of unnecessary surgical interventions.

Next Generation Sequencing and Genetic Analyses Reveal Factors Driving Evolution of Sweetpotato Viruses in Uganda.

Sweetpotato (Ipomoea batatas L.) is an essential food crop globally, especially for farmers facing resource limitations. Like other crops, sweetpotato cultivation faces significant production challenges due to viral infections. This study aimed to identify and characterize viruses affecting sweetpotato crops in Uganda, mostly those associated with sweetpotato virus disease (SPVD). Infected leaf samples were collected from farmers' fields in multiple districts spanning three regions in Uganda. MiSeq, a next-generation sequencing platform, was used to generate reads from the viral nucleic acid. The results revealed nine viruses infecting sweetpotato crops in Uganda, with most plants infected by multiple viral species. Sweet potato pakakuy and sweet potato symptomless virus_1 are reported in Uganda for the first time. Phylogenetic analyses demonstrated that some viruses have evolved to form new phylogroups, likely due to high mutations and recombination, particularly in the coat protein, P1 protein, cylindrical inclusion, and helper component proteinase regions of the potyvirus. The sweet potato virus C carried more codons under positive diversifying selection than the closely related sweet potato feathery mottle virus, particularly in the P1 gene. This study provides valuable insights into the viral species infecting sweetpotato crops, infection severity, and the evolution of sweet potato viruses in Uganda.

Rabies in equids in Sudan

Rabies is endemic in Sudan with continuing outbreaks occurring annually, the most common animals affected are dogs, followed by goats and equids. This work focused on equid rabies, to elucidate the current situation of the disease through analysis of reports of equid rabies outbreaks in Sudan during 2010–2022 supported by laboratory confirmation of the disease. During the study period, 66 animals were affected during 35 equid rabies outbreaks. The highest incidences were found in Al Gezira (30.3%), followed by Darfur (24.2%) and Kordofan (15.2%). The highest incidence rate was observed during 2018 (33.3%), followed by 2015 (16.7%). Within seasons, the highest incidence rate was reported during October – December (33.3%), followed by July – September (30.3%). Chi-square analysis revealed a significant correlation between rabid animals and year, season, and state. Wald statistics demonstrated that year and season had a significant association with the disease. Virus antigen was identified (72.2%) in brain tissues using the fluorescent antibody test. Viral nucleic acid was amplified (n = 6) with a reverse transcriptase polymerase chain reaction assay.ContributionAs equids are kept in close contact with humans and other animals in the country, according to the present investigation, equid rabies in Sudan is a potential public health concern, emphasising the importance of implementing effective control measures.

Quantum Dot Reporters Designed for CRISPR-Based Detection of Viral Nucleic Acids.

Diagnostic methods based on CRISPR technology have shown great potential due to their highly specific, efficient, and sensitive detection capabilities. Although the majority of the current studies rely on fluorescent dye-quencher reporters, the limitations of fluorescent dyes, such as poor photostability and small Stokes shifts, urgently necessitate the optimization of reporters. In this study, we developed innovative quantum dot (QD) reporters for the CRISPR/Cas systems, which not only leveraged the advantages of high photoluminescence quantum yield and large Stokes shifts of QDs but were also easily synthesized through a simple one-step hydrothermal method. Based on the trans-cleavage characteristics of Cas12a and Cas13a, two types of QD reporters were designed, the short DNA strand and the hybridization-based QD reporters, achieving the detection of DNA and RNA at the pM level, respectively, and validating the performance in the analysis of clinical samples. Furthermore, based on the unique property of QDs that allowed multicolor emission under one excitation, the application potential for simultaneous detection of diseases was further investigated. Taken together, this work proposed novel QD reporters that could be applied to the various CRISPR/Cas systems, providing a new toolbox to expand the diagnosis of bioanalytical and biomedical fields.

Molecular analysis of canine distemper virus H gene in the golden jackal (Canis aureus) population from Serbia

Canine distemper virus (CDV) is a highly contagious and often fatal disease affecting wild and domesticated carnivores. The virus is a single-stranded RNA virus from the genus Morbillivirus and the family Paramyxoviridae. While domestic dogs are the most common hosts, the virus poses a significant threat to endangered wildlife due to its broad host range. This study aimed to characterize the CDV Haemagglutinin (H) gene in golden jackals and explore the molecular evolution of the virus in an underrepresented host. A total of 88 brain samples from hunted golden jackals were tested for the presence of CDV viral nucleic acid, and the H gene of positive samples was amplified and sequenced using the Sanger method. Phylogenetic analysis, conducted using maximum likelihood methods, revealed that all Serbian sequences clustered within the Arctic lineage. Notably, the analysis identified a tyrosine (Y) at position 549 of the H protein, a mutation commonly associated with wildlife hosts, instead of the histidine (H) typically found in domestic strains. Additionally, a mutation at position 310 was observed, which could potentially affect the protein’s function and virus-host interactions. These findings provide valuable insights into the genetic diversity and evolutionary dynamics of CDV in golden jackals, with broader implications for understanding the virus’s adaptability to different hosts. Further research is needed to investigate the functional impact of these mutations, particularly their role in vaccine efficacy and disease transmission across wildlife and domestic species.

Development of targeted whole genome sequencing approaches for Crimean-Congo haemorrhagic fever virus (CCHFV)

Crimean-Congo haemorrhagic fever (CCHF) is the most prevalent human tick-borne viral disease, with a reported case fatality rate of 30 % or higher. The virus contains a tri-segmented, negative-sense RNA genome consisting of the small (S), medium (M) and large (L) segments encoding respectively the nucleoprotein (NP), the glycoproteins precursor (GPC) and the viral RNA-dependent RNA polymerase (RDRP). CCHFV is one of the most genetically diverse arboviruses, with seven distinct lineages named after the region they were first reported in and based on S segment phylogenetic analysis.Due to the high genetic divergence of the virus, a single targeted tiling PCR strategy to enrich for viral nucleic acids prior to sequencing is difficult to develop, and previously we have developed and validated a tiling PCR enrichment method for the Europe 1 genetic lineage.We have developed a targeted, probe hybridisation capture method and validated its performance on clinical as well as cell-cultured material of CCHFV from different genetic lineages, including Europe 1, Europe 2, Africa 2 and Africa 3. The method produced over 95 % reference coverages with at least 10x sequencing depth. While we were only able to recover a single complete genome sequence from the tested Europe 1 clinical samples with the capture hybridisation protocol, the data provides evidence of its applicability to different CCHFV genetic lineages.CCHFV is an important tick-borne human pathogen with wide geographical distribution. Environmental as well as anthropogenic factors are causing increased CCHFV transmission. Development of strategies to recover CCHFV sequences from genetically diverse lineages of the virus is of paramount importance to monitor the presence of the virus in new areas, and in public health responses for CCHFV molecular surveillance to rapidly detect, diagnose and characterise currently circulating strains.

Clinical efficacy of Fufang Yinhua Jiedu (FFYH) granules in mild COVID-19 and its anti-SARS-CoV-2 mechanism by blocking autophagy through inhibiting the AKT/mTOR signaling pathway.

Fufang Yinhua Jiedu (FFYH) granules are recommended for treating coronavirus pneumonia (COVID-19) in China. However, its anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) activity and clinical efficacy against COVID-19 remain to be confirmed. Our study aimed to investigate the anti-SARS-CoV-2 effect and potential mechanism of FFYH. The activity of FFYH against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was evaluated via cell pathogenic effects, immunoblotting, immunofluorescence staining, and qRT-PCR. The potential mechanism of FFYH against SARS-CoV-2 was investigated by immunoblotting. One head-to-head randomized controlled trial was designed to evaluate the clinical efficacy of FFYH in mild COVID-19. Two hundred patients were randomly recruited to receive either FFYH or LHQW (Lianhua Qingwen) granules. The in vitro results indicated that FFYH effectively inhibited SARS-CoV-2 replication by suppressing CPE and decreasing viral RNA and protein expression. A time-of-drug-addition assay confirmed that FFYH mainly targeted the binding and replication stages of the SARS-CoV-2 life cycle. Mechanistic studies revealed that blocking SARS-CoV-2-triggered autophagy may be the primary mechanism by which FFYH protects against SARS-CoV-2 infection by regulating the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway. Clinical results confirmed that FFYH effectively shortened the recovery time of clinical symptoms and viral nucleic acid negativity, improved abnormal hematology parameters, and controlled excessive cytokine responses in mild COVID-19 patients. Subgroup analysis revealed that FFYH improved the recovery time of clinical symptoms, improved hematological parameters, and controlled excessive cytokine storms to a greater extent in the mild COVID-19 male subgroup, abnormal hematology subgroup, and 32-42-year-old subgroup than in the corresponding LHQW subgroup (P < 0.05). No patients progressed to severe or critical cases. Our results indicate that FFYH not only has good anti-viral activity against SARS-CoV-2 but also has significant efficacy against COVID-19, indicating that FFYH may be a novel complementary option for treating COVID-19.

Analysis of serum IgM antibody and viral nucleic acid test results in measles and rubella cases in China from 2014 to 2023

Objective: To analyze the results of serum IgM antibody and viral nucleic acid testing in measles and rubella cases in China from 2014 to 2023. Methods: Surveillance data on measles and rubella during 2014-2023 were obtained from the Chinese Disease Prevention and Control Information System, and cases that underwent measles/rubella IgM antibody testing and viral nucleic acid testing were included in the study. Information on the number of cases, vaccination status, and laboratory test results was collected, and laboratory test results were compared among cases with different times of rash or onset and different doses of vaccination. Results: From 2014 to 2023, the total number of measles and rubella surveillance cases was 581 746, and the number of measles and rubella cases that underwent both IgM antibody and viral nucleic acid testing was 39 124 and 21 766, respectively, with a double-positive rate of IgM antibody and viral nucleic acid testing of 63.73% (for measles) and 41.68% (for rubella). The single-positive rate for IgM antibody was 21.12% (for measles) and 27.15% (for rubella). The single-positive rate for viral nucleic acid tests was 13.84% (for measles) and 28.19% (for rubella). According to different days of rash or onset (0-3, 4-5, 6-10 and >10), samples of 24 629 (62.95%) measles cases and 15 785 (72.52%) rubella cases were from within 3 days of rash or onset, and the single positive rate of viral nucleic acid detection was 16.90% (for measles) and 32.07% (for rubella). The single positive rate of IgM antibody increased gradually with the time of onset (measles: χ2trend=314.098, P<0.001, rubella: χ2trend=763.846, P<0.001), and reached 31.66% (for measles) and 53.12% (for rubella) after 10 days of rash or onset. Depending on the number of vaccination doses (1, 2,≥3), the single positive rate of viral nucleic acid detection ranged from 12.93% to 20.29% (for measles) and from 25.30% to 27.88% (for rubella). The single positive rate of IgM antibody detection ranged from 36.89% to 47.47% (for measles) and from 22.81 to 41.15% (for rubella). Conclusions: Combined testing of serum IgM antibody and viral nucleic acids could facilitate laboratory confirmation of measles and rubella cases and was also important for measles and rubella elimination efforts.

Association between FTO polymorphism and COVID-19 mortality among older adults: A population-based cohort study

Association between FTO polymorphism and COVID-19 mortality among older adults: A population-based cohort study

Paper‐Based RNase Digestion toward Viral Nucleic Acid Self‐Tests

Abstract Home‐based Nucleic Acid Amplification Tests (NAATs) for viral infections would be an important step to improve public health, but sample preparation remains an important obstacle, particularly for the protection of target RNA from RNases in samples. Here, a new method for RNase deactivation in saliva samples is presented. This method uses Proteinase K (PK) immobilized on nitrocellulose membrane to store and deliver the enzyme, capable of digesting nucleases present in the sample. The immobilized PK is also separated from the amplification reagents, so that it does not disrupt DNA amplification, thus omitting the need for heat‐deactivation or dilution steps. Treatment by PK nitrocellulose at 50 °C dramatically decreases the RNase activity of RNase A, in diluted saliva samples, and even shows promising results at 42 °C. The potential of this method to protect RNA from digestion is further demonstrated, by pretreating diluted saliva samples spiked with Influenza Virus A (IVA) genomic RNA, which allows its amplification and colorimetric detection by lateral flow strips. In the absence of PK pretreatment, the RNA is digested by RNase, which leads to false negative results. These findings show that immobilized PK enables the integration of sample preparation of viral samples toward home‐based NAATs.

Validation of a direct multiplex real-time reverse transcription PCR assay for rapid detection of African swine fever virus using swine field samples in Vietnam

ObjectiveThis study validates a direct multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay which was previously established for enabling rapid and simultaneous detection of African swine fever (ASF) virus (ASFV) and classical swine fever virus. The assay eliminates the need for viral nucleic acid purification using a buffer system for crude extraction and an impurity-tolerant enzyme. However, the assay had not yet been validated using field samples of ASFV-infected pigs. Therefore, to address this gap, we tested 101 samples collected from pigs in Vietnam during 2018 and 2021 for validation.ResultsThe rRT-PCR assay demonstrated a diagnostic sensitivity of 98.8% and a specificity of 100%. Remarkably, crude samples yielded results comparable to those of purified samples, indicating the feasibility of using crude samples without compromising accuracy in ASFV detection. Our findings emphasize the effectiveness of the rRT-PCR assay for the prompt and accurate diagnosis of both swine fever viruses, which is essential for effective disease prevention and control in swine populations.

New dimension in viral hepatitis research

Chronic hepatitis B is the leading cause of hepatocellular carcinoma and a significant global health issue, affecting over 296 million people worldwide, with 15 million people coinfected with hepatitis delta...

222.2: Drop in peripheral blood Epstein-Barr virus nucleic acid loads after post-transplant lymphoproliferative disorders associates with better 5-year event-free survival: results from the multicenter PTLD-MSMS study.

222.2: Drop in peripheral blood Epstein-Barr virus nucleic acid loads after post-transplant lymphoproliferative disorders associates with better 5-year event-free survival: results from the multicenter PTLD-MSMS study.

Phagosome Isolation with Magnetic Beads and Purification of Toll-Like Receptor (TLR) Complexes in Phagosomes.

Phagocytosis is a central process by which macrophage cells internalize and eliminate microbes as well as apoptotic cells. The nascent phagosome undergoes a complex maturation process involving sequential fusion with endosomal compartments. The endosomal TLRs, including TLR3, -7, -8, and -9, play a critical role in innate immunity by sensing bacterial or viral nucleic acids and are preferentially transported to the phagosomal membrane of innate immune cells upon activation. Therefore, phagosome isolation is helpful for studies on pathogenic invasion and the functions of phagosome proteins, including endosomal TLRs.

3D-printed devices for optimized generation of cold atmospheric plasma to improve decontamination of surfaces from respiratory pathogens

Three-dimensional (3D)-printing technology is instrumental in creating devices for biological applications, including the exploitation of cold atmospheric plasma (CAP). CAP, a partially ionized gas that functions at ambient temperatures, serves as a safe, inexpensive, and effective tool for the inactivation of various pathogens on different surfaces. In this study, we compared three different 3D-printed devices with respect to their ability to provide optimized CAP compositions effective against select respiratory viruses (SARS-CoV-2, influenza virus, adenovirus, and rhinovirus) and the bacterium Pseudomonas aeruginosa, which is associated with serious lung diseases. The transmission of respiratory pathogens via surface contamination may pose a serious health threat, thus highlighting the biological importance of the current study. The properties of a prototype 3D-printed CAP-generating device and two optimized versions were characterized by detecting reactive oxygen and nitrogen species (RONS) in a gaseous environment via infrared spectroscopy and analyzing the composition of the reactive compounds. The virucidal effects of CAP were examined by determining virus infectivity and particle integrity. The bactericidal effect was documented by viability testing and visualization via transmission electron microscopy. The findings indicate that optimization of the 3D-printed devices for CAP production yielded an environment with relatively high amounts of RONS (O3, N2O, NO2, and H2O2), reducing the exposure time required for inactivation of respiratory pathogens by approximately 50%. In addition to reducing infectivity and viability, CAP treatment led to the destruction of viral nucleic acids and physical damage to bacterial cells. Owing to its flexibility and easy implementation, optimized CAP generated by 3D-printed devices provides an attractive inactivation method adaptable for different biological applications, including surface decontamination from viral and bacterial pathogens. &amp;nbsp;&amp;nbsp;

Efficient concentration of viral nucleic acid in wastewater through surfactant releasing and a two-step magnetic bead extraction and purification

Efficient concentration of viral nucleic acid in wastewater through surfactant releasing and a two-step magnetic bead extraction and purification

Molluscum contagiosum virus protein MC089 inhibits interferon regulatory factor 3 activation.

Molluscum contagiosum virus (MCV) is a human-specific poxvirus that causes a highly common but mild infection characterized by distinctive and persistent papular skin lesions. These lesions can persist for long periods without an effective clearance response from the host. MCV, like all poxviruses, encodes multiple known immunosuppressive proteins which target innate immune signalling pathways involved in viral nucleic acid sensing, interferon production and inflammation which should trigger antiviral immunity leading to clearance. Two major families of transcription factors responsible for driving the immune response to viruses are the NF-κB and the interferon regulatory factor (IRF) families. While NF-κB broadly drives pro-inflammatory gene expression and IRFs chiefly drive interferon induction, both collaborate in transactivating many of the same genes in a concerted immune response to viral infection. Here, we report that the MCV protein MC089 specifically inhibits IRF activation from both DNA- and RNA-sensing pathways, making it the first characterized MCV inhibitor to selectively target IRF activation to date. MC089 interacts with proteins required for IRF activation, namely IKKε, TBKBP1 and NAP1. Additionally, MC089 targets RNA sensing by associating with the RNA-sensing adaptor protein mitochondrial antiviral-signalling protein on mitochondria. MC089 displays specificity in its inhibition of IRF3 activation by suppressing immunostimulatory nucleic acid-induced serine 396 phosphorylation without affecting the phosphorylation of serine 386. The selective interaction of MC089 with IRF-regulatory proteins and site-specific inhibition of IRF3 phosphorylation may offer a tool to provide novel insights into the biology of IRF3 regulation.