ABSTRACT A non-replicating Tiantan strain-based vaccinia virus (NTV) holds significant application prospects for vaccination and gene therapy and has recently entered clinical trials as a novel and safer vaccine candidate against monkeypox. However, optimization is still required, particularly regarding its production capacity and immunogenicity. In this study, a recombinant virus was constructed by modifying the F1L and C7L genes in a non-replicating viral backbone using CRISPR/Cas9-mediated gene editing and homologous recombination. The resulting construct, designated NTV-ΔF1L-C7L, exhibited significantly enhanced replication in vaccine production cell lines, with viral yields increasing by more than 680-fold in MRC-5 cells compared to those of the parental NTV. Its pathogenicity in mice was significantly reduced, showing more than a 10-fold decrease compared to the pathogenicity of the vaccinia virus Tiantan strain (VTT). Following two intramuscular doses, NTV-ΔF1L-C7L elicited high titers of orthopoxvirus-specific IgG and neutralizing antibodies against vaccinia and monkeypox viruses, along with a robust cellular immune response exhibiting a Th1 bias, which was significantly stronger than that induced by either parental NTV or VTT vaccination. Complete protection (100%) against a lethal challenge with the vaccinia virus Western Reserve strain was achieved in mice immunized with either a low dose (10 3 PFU) or a single dose (10⁵ PFU) of NTV-ΔF1L-C7L, comparable to that conferred by VTT. These findings demonstrate that NTV-ΔF1L-C7L combines high safety with enhanced replication and immunogenicity, supporting its value as a novel vaccine vector and its potential application in controlling the current global monkeypox outbreak. IMPORTANCE A highly attenuated NTV exhibits an improved safety profile; however, its production capacity and immunogenicity require optimization for clinical application. In this study, a novel recombinant virus, NTV-ΔF1L-C7L, was developed by targeting the deletion of F1L and the insertion of C7L into the NTV backbone. This attenuated live vaccine, NTV-ΔF1L-C7L, demonstrates several significant advantages. Its robust in vitro replication supports scalability for large-scale vaccine production. It has demonstrated strong immunoprotective efficacy in mice while maintaining a high safety margin, exhibiting reduced pathogenicity in vivo , and inducing robust humoral and cellular immune responses against vaccinia and monkeypox virus. These immune responses confer complete protection against lethal VACV challenge at low-dose or single-dose immunization. These findings establish a solid experimental foundation for the further development of NTV-ΔF1L-C7L as a next-generation VACV-based vector or a candidate vaccine against mpox.

In early 2025, the first cluster of imported clade Ib mpox cases in China was reported. Here we reported the laboratory confirmation of these mpox cases and the isolation of replication-competent MPXV from environmental samples. Seven clinical specimens and six environmental samples (from trimmers, shoe inserts, cloth, bedsheets, carpets, pillows, and bedside tables) were collected for molecular and serological detection. These mpox cases were confirmed to be MPXV clade Ib-positive based on qPCR and MPXV-specific seroconversion. Furthermore, clinical and environmental swabs were selected for virus isolation using Vero cells. Replication-competent viruses were isolated from two mpox clinical samples and an environmental sample (designated as MPXV-Ib-CCDC-Tan-TJ04). MPXV clade Ib strains exhibited smaller plaques than those of clade IIb strains. Neutralization assay indicated a strong correlation between clades Ib and IIb. This study highlights the importance of sustained surveillance in laboratory investigations by combining molecular detection with serological testing. The isolation of infectious MPXV clade Ib from environmental samples emphasizes the importance of environmental monitoring, disinfection practices, and health education for the prevention and control of mpox outbreaks, which could provide critical scientific evidence for formulating public health interventions.

The development of therapeutic vaccines against human papillomavirus (HPV)–associated malignancies remains challenging due to the immunosuppressive tumour microenvironment and the limited efficacy of existing delivery platforms. In this study, we designed and systematically compared two mRNA vaccine strategies based on a core–shell structured lipopolyplex (LPP) delivery system: a codon- and untranslated region-optimised non-replicating mRNA (nr-mRNA) and a self-amplifying mRNA (sa-mRNA). In the TC-1 murine model of HPV-driven cancer, both vaccine formulations effectively activated systemic antitumour immune responses, significantly enhancing the infiltration of functional CD8⁺ T cells and natural killer cells into tumours and promoting the repolarisation of tumour-associated macrophages towards an M1 phenotype. Notably, the sa-mRNA–LPP platform achieved comparable therapeutic efficacy at only one-fifth the dose of nr-mRNA–LPP, highlighting its superior potency and dose-sparing potential. Further analysis revealed that immune response induced by sa-mRNA–LPP was predominantly localised to the tumour site, with no significant immune activation detected in peripheral lymphoid organs such as lymph nodes and spleen, suggesting a stronger capacity for localised immunomodulation within the tumour. In summary, this study validates the high efficiency of the LPP platform in delivering different mRNA vaccines and elucidates the unique mechanism by which sa-mRNA exerts potent antitumour effects at low doses through localised immune remodelling. These findings provide important experimental support for the clinical translation of LPP-based mRNA vaccines targeting HPV-associated cancers.

In 2022, a global outbreak of mpox was anticipated, with several cases reported in non-endemic countries in early May. Given the challenge of distinguishing the mpox virus (MPXV) from other pathogens based solely on symptoms, there is an urgent need for prompt and reliable MPXV detection methods. In this study, we developed assays using recombinase-aided amplification (RAA) to identify MPXV and evaluated their applicability with clinical samples. The assays were designed to detect the N4R gene of MPXV. All assays demonstrated detection limits of 1 copy/µL within the reaction system and exhibited no cross-reactivity with ectromelia or the TianTan strain of vaccinia virus, confirming their high specificity. Our established assay provides results in less than 50 min. Furthermore, we evaluated our assay using clinical samples from laboratory-confirmed mpox patients and demonstrated that the RAA-based assay is valuable for diagnosing MPXV infections in field and clinic settings, especially in areas with limited laboratory resources. Overall, three RAA-based nucleic acid assays for MPXV were established, providing a powerful tool for efficient, rapid, and specific detection of MPXV infection.

Current COVID-19 vaccines can effectively reduce disease severity and hospitalisation; however, they are not considerably effective in preventing infection and transmission. In this context, mucosal vaccines are pertinent to prevent SARS-CoV-2 infection and spread. In this study, we generated a replication-competent recombinant chimeric influenza A virus (IAV) expressing the receptor-binding domain (RBD) of a SARS-CoV-2 prototype in the C-terminus of the neuraminidase (NA) of A/Puerto Rico/08/1934 H1N1 (PR8). The remaining seven segments from A/WSN/1933 H1N1 (WSN) were named PR8NARBD/WSN. We observed that the recombinant virus with the WSN backbone demonstrated improved expression of NA and RBD. A single intranasal dose of PR8NARBD/WSN(103PFU) in mice generated robust mucosal immunity, neutralising antibodies, cellular immunity, and tissue-resident memory T cells specific to SARS-CoV-2 and IAV. Importantly, immunisation with PR8NARBD/WSN viruses effectively protected mice against lethal challenges with H1N1, H3N2 IAV, and SARS-CoV-2 Beta variant and significantly reduced lung viral loads. Overall, our research demonstrates the promising potential of PR8NARBD/WSN as an attractive vaccine against emerging SARS-CoV-2 variants and influenza A virus infections.

Background Several COVID-19 vaccines are in widespread use in China. Few data exist on comparative immunogenicity of different COVID-19 vaccines given as booster doses. We aimed to assess neutralizing antibody levels raised by injectable and inhaled aerosolized recombinant adenovirus type 5 (Ad5)-vectored COVID-19 vaccine as a heterologous booster after an inactivated COVID-19 vaccine two-dose primary series. Methods Using an open-label prospective cohort design, we recruited 136 individuals who had received inactivated vaccine primary series followed by either injectable or inhaled Ad5-vectored vaccine and measured neutralizing antibody titers against ancestral SARS-CoV-2 virus and Omicron BA.1 and BA.5 variants. We also measured neutralizing antibody levels in convalescent sera from 39 patients who recovered from Omicron BA.2 infection. Results Six months after primary series vaccination, neutralizing immunity against ancestral SARS-CoV-2 was low and neutralizing immunity against Omicron (B.1.1.529) was lower. Boosting with Ad5-vectored vaccines induced a high immune response against ancestral SARS-CoV-2. Neutralizing responses against Omicron BA.5 were ≥ 80% lower than against ancestral SARS-CoV-2 in sera from prime-boost subjects and in convalescent sera from survivors of Omicron BA.2 infection. Inhaled aerosolized Ad5-vectored vaccine was associated with greater neutralizing titers than injectable Ad5-vectored vaccine against ancestral and Omicron SARS-CoV-2 variants. Conclusions These findings support the current strategy of heterologous boosting with injectable or inhaled Ad5-vectored SARS-CoV-2 vaccination of individuals primed with inactivated COVID-19 vaccine.

Objective:To develop a recombinase-aided amplification (RAA)-clustered regularly interspaced short palindromic repeats(CRISPR)/Cas12a-based nucleic acid assay for monkeypox virus with high specificity and sensitivity.Methods:RAA primers and CRISPR RNA (crRNA) were designed based on the known conserved regions of the monkeypox virus gene and synthesized, and specific crRNAs were screened using fluorescence detection. The sensitivity and specificity of the detection system were evaluated.Results:An RAA-CRISPR/Cas12a-based nucleic acid assay for monkeypox virus was developed with a sensitivity of 2.5 copies/reaction and high specificity without cross-reactivity with ectromelia virus and vaccinia virus.Conclusions:An RAA-CRISPR/Cas12a-based nucleic acid assay for monkeypox virus was established, which would provide a powerful tool for efficient, rapid and specific detection of monkeypox virus.

West Nile virus (WNV) is a mosquito-transmitted flavivirus distributed globally for decades and can cause dis-ease in humans and animals. So far, no WNV vaccine has been licensed for human use. Therefore, the devel-opment of novel candidate vaccines and the improvement of vaccination strategies is imperative. As the WNV envelope (E) glycoprotein plays an important role in mediating viral binding to cellular receptors and virus-cell membrane fusion, it is a critical target for the host humoral response. Here, we prepared a recombinant trun-cated envelope protein of WNV (rWNV-80E) and developed a WNV subunit vaccine formulation with a com-bination of aluminum hydroxide (alum) and a synthetic oligonucleotide CpG as adjuvants. C57BL/6 mice were immunized twice intramuscularly at 28-day intervals with 5 mu g purified rWNV-80E adjuvanted with Alum/ CpG. WNV E-specific IgG was detected by enzyme-linked immunosorbent assay and neutralizing antibodies (nAbs) was detected using single-round infectious particles of WNV. Furthermore, T cell immunity was detected by enzyme-linked immunospot assay and intracellular cytokine staining assay. Notably, rWNV-80E was highly immunogenic and elicited potent humoral and cell immunity, as evidenced by significant levels of IFN-gamma and TNF-alpha secretion in the T cells of mice. In summary, the Alum/CpG-adjuvanted rWNV-80E subunit vaccine elicited potent and balanced B-and T-cell immunity in mice, and therefore it is a promising candidate vaccine that warrants further investigation for use in human or veterinary applications.(c) 2023 Chinese Medical Association Publishing House. Published by Elsevier BV. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

目的 对全国32个省市级疾控机构新冠监测网络实验室新型冠状病毒(新冠病毒)变异株核酸检测能力进行室间质评,同时对常规所用新冠病毒核酸检测试剂性能进行评价.方法 制备新冠病毒武汉株及4个关切变异株、人冠状病毒OC43(HCoV-OC43)和H3亚型流感病毒(H3N2)核酸样本,发放给全国31个省、自治区及直辖市和新疆生产建设兵团疾病预防控制中心新冠监测网络实验室进行室间质评.每个实验室的考核样本盘包括两种不同Ct值(～35、～30)的新冠病毒武汉株、关切变异株(α和γ或β和δ)、HCoV-OC43、流感病毒核酸样本共10支.根据反馈结果计算检测符合率并对检测结果进行深入分析.同时,收集各考核实验室常用的新冠病毒核酸检测试剂盒进行检测性能验证.结果 全国省市级疾控机构新冠监测网络实验室室间质评总体达标率为100％(32/32),总体优秀率72％(23/32);初测中,新冠病毒、HCoV-OC43核酸样本检测总符合率为99.7％(319/320)、89％(57/64).对32个考核实验室送检的新冠病毒核酸检测试剂盒进行性能验证,66％(33/50)的试剂盒检测限与说明书相符,92％(11/12)的受检试剂盒特异性达标.结论 32个省市疾控机构实验室新冠病毒核酸检测能力全部达标,均能检测新冠病毒武汉株及阿尔法、贝塔、伽马和德尔塔变异株;送检的部分核酸检测试剂检测限与说明书不符,检测性能有待改进.

Objective:To construct a bivalent DNA vaccine against SARS-CoV-2 and influenza A virus H3N2 and to evaluate its immunogenicity in mice.Methods:The coding sequences for spike 1 (S1) protein of SARS-CoV-2 Beta variant and hemagglutinin (HA) of influenza A virus Cambodia (H3N2) strain were codon-optimized and synthesized. The two coding genes were ligated by the self-cleaving 2A peptide using over-lapping PCR to construct S1-2A-HA fragment, which was inserted into pVRC vector to construct the bivalent DNA vaccine, named as pVRC-S1-2A-HA. Indirect immunofluorescence assay (IFA) and Western blot were performed to detect the expression of S1 and HA proteins. BALB/c mice were immunized with pVRC-S1-2A-HA by intramuscular injection and electroporation. The humoral immune responses induced in mice were detected by indirect ELISA, pseudovirus neutralization assay and hemagglutination inhibition assay. Cellular immune responses were detected by IFN-γ ELISPOT, intracellular cytokine staining (ICS) and cytometric bead array (CBA).Results:The bivalent DNA vaccine pVRC-S1-2A-HA could express S1 and HA proteins in vitro. Specific cellular immune responses against S1 protein and specific IgG antibody against HA protein were significantly induced in mice with single-dose immunization. The antigen-specific immunity was significantly enhanced after booster immunization. The geometric mean titer (GMT) of specific IgG antibody increased to 3 251 for S1 protein and 45 407 for HA protein after two-dose immunization. Moreover, the S1-specific T cells increased to 1 238 SFC/10 6 cells. ICS results indicated that the booster vaccination induced CD4 + T and CD8 + T cells to produce IL-2, IFN-γ and TNF-α in mice. The secretion of various cytokines including IL-2, IL- 4, IL-6, IL-10 and IFN-γ in mouse splenocytes was induced after single-dose immunization. Conclusions:A bivalent DNA vaccine against SARS-CoV-2 and influenza A virus H3N2 was constructed and could induce S1- and HA-specific humoral and cellular immune responses in mice, suggesting the great potential of it for further development and application.

评价一种SARS-CoV-2 Beta变异株和甲型流感病毒H3N2新型重组双价疫苗在小鼠模型中的免疫保护效果.本研究构建了表达SARS-CoV-2南非变异株(B.1.351)棘突蛋白1(S1)和H3N2柬埔寨分离株(A/Cambodia/e0826360/2020)血凝素(HA)的重组双价非复制Ad5载体疫苗,命名为HAdV5-S1-2A-HA,经单针肌肉注射免疫BALB/c雌鼠后,采用ELISA、血凝抑制实验、假病毒中和实验与Elispot实验进行体液与细胞免疫学检测,免后3～6周采用H3N2-X31病毒、H1N1-PR8与SARS-CoV-2(B.1.351)进行攻毒保护实验.HAdV5-S1-2A-HA免疫两周后,低剂量组(1×108vp/只)免疫小鼠后可检出HA特异体液免疫与S1特异的细胞免疫应答;而高剂量组(5X 109vp/只)诱导小鼠产生了较强的双抗原(S1,HA)特异的体液和细胞免疫应答,并能完全保护小鼠对H3N2-X31攻击,降低SARS-CoV-2(B.1.351)感染后小鼠肺部病毒载量,延迟H1N1-PR8病毒感染后小鼠死亡发生.本研究制备的重组双价疫苗HAdV5-S1-2A-HA在小鼠体内诱导抗原特异的体液与细胞免疫应答.同时,它还可以在小鼠中诱导对SARS-CoV-2和H3N2感染的免疫保护,具有较好的研发与应用前景.

Objective:To study the cell morphological changes and related molecular mechanisms of non-replicating vaccinia virus NTV infection with human cells and to provide a scientific basis for the further optimization, transformation and application of NTV vectors.Methods:HeLa cells were infected with vaccinia virus Tiantan strain VTT and non-replicating vaccinia virus NTV, and the morphological changes of cells were observed. Then cells were harvested, and rRNA break levels were detected by electrophoresis and the molecular signals associated with apoptosis were detected by Western blotting, and the pathways and mechanisms of NTV-induced early apoptosis were preliminarily determined.Results:In this study, in terms of cell morphology, it was observed that cells infected with NTV were able to have cell rounding, wrinkles and other lesions at an earlier time compared with VTT. DAPI staining showed that NTV-infected nuclei exhibited high chromatin aggregation, marginalization, and disintegration over time. The rRNA fracture level test result indicate that rRNA has been broken and degraded after 16 hours of NTV infection. The Western blotting test result showed that the molecular signals of PARP, caspase-3 and caspase-9 that were stronger than in normal cells could be detected in NTV-infected HeLa cells, but there was no significant increase in caspase-8, while the result of VTT were the opposite of those in NTV.Conclusions:NTV can induce apoptosis in the early stage and provide a theoretical basis for the modification of vaccinia vectors.

Introduction: The first imported case of monkeypox (MPX) from the mainland of China was reported in September 2022. Herein, the study reports the isolation and characterization of MPX virus (MPXV) in this case.Methods: Clinical specimens including skin blister fluid, oropharyngeal and nasopharyngeal swabs, and blood were collected and inoculated onto Vero cells. The isolated virus was identified as MPXV using quantitative polymerase chain reaction (qPCR), cytopathic effects (CPEs), immunofluorescence assay (IFA) and transmission electron microscopy (TEM). Plaque assays were employed to quantify infectious plaque-forming units (PFUs). The plaque reduction neutralization test (PRNT) was developed to determine the neutralizing antibody (nAb) against MPXV. Results: MPXV replication was confirmed with qPCR. Typical CPEs were observed 48 h post -incubation. The isolated virus was named MPXV-B.1-China-C-Tan-CQ01. IFA showed that MPXV reacted with serum of MPX case. Orthopoxvirus morphology was observed using TEM. The virus titer increased to >106 PFU/mL after three passages. The serum PRNT 50% neutralization titer (NT50) was 35 for the MPX patient 6 days after symptom onset.Discussion: The study successfully isolated the first MPXV strain in the mainland of China, MPXV-B.1-China-C-Tan-CQ01. Infectious titration and PRNT methods have been developed. The study provides key resources and technical platforms for further research as well as anti-viral drug and vaccine development against MPX.

Introduction:To identify Novaferon (Nova), a novel recombinant protein of interferon (IFN)-α, antiviral activity against ancestral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Omicron variant in vitro.Methods:Vero cells were infected with SARS-CoV-2 and Omicron variant in a biosafety level-3 laboratory. And viral replications were accessed using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR).Results:Results demonstrated that Nova has effective inhibition against ancestral SARS-CoV-2 and Omicron variant in vitro.Discussion:The in vivo effects of Nova need to be further tested in animal models. And large-scale randomized double-blind clinical trials are needed to reveal its potentially clinical application.

为了评估连花清瘟颗粒等中药(Traditional Chinese Medicines,TCMs)体外的抗新型冠状病毒作用.采用Cell Counting Kit-8(CCK8)法检测药物对Vero细胞的毒性,确定药物的细胞毒性.再以Vero细胞为模型,设置药物组、瑞德西韦对照组、病毒对照组,用新型冠状病毒感染细胞,药物作用48h,提取核酸,用荧光定量PCR法检测新型冠状病毒RNA拷贝数,评估药物的抗病毒作用.细胞活性结果和病毒RNA拷贝数变化的结果显示,连花清瘟等8种药物中,连花清瘟抗新型冠状病毒活性较好,半数抑制浓度达到了 0.43 mg/mL,药物六神丸和藿香正气水的细胞毒性较大,其他5种药物的抗新型冠状病毒活性较低.本研究揭示了莲花清瘟等8种药物体外的抗新型冠状病毒效果,为进一步的研究奠定了基础.

Objective:To screen and identify H-2 d-restricted T cell epitopes in fusion (F) and attachment (G) glycoproteins of Nipah virus (NiV) in mice. Methods:The complete peptides (single peptide contains 15 amino acids, and 10 amino acids were repeated in the front and back peptides) derived from F and G antigens were mixed into peptide libraries. BALB/c mice were immunized with DNA vaccines expressing NiV F and G proteins alone and in combination. The full sequence peptide libraries of F and G antigens were mixed into peptide pools by matrix design, and spleen cells of immunized mice were collected and analyzed by IFN-γ ELISPOT assay to detect the dominant H-2 d-restricted epitope peptides. Results:Twelve dominant H-2 d-restricted peptides were screened from the F protein-specific peptide library and the 56th peptide produced the strongest reaction. Four dominant peptides were screened from the G protein-specific peptide library and the 72nd peptide produced the strongest reaction. Conclusions:In this study, 12 F antigen-specific and 4 G antigen-specific H-2 d restricted dominant T cell epitopes of NiV were screened and identified by IFN-γ ELISPOT, which could provide reference for immunological analysis of NiV and vaccine research.

Objective:To improve the consistency of test results through reducing inter-laboratory variation in SARS-CoV-2 antibody detection with WHO SARS-CoV-2 antibody candidate international standard (IS, sample G) and antibody reference panel (samples E, F, H, I, J).Methods:Ten WHO samples (A-J) including the candidate IS and reference panel were evaluated using different methods, such as microneutralization tests based on live SARS-CoV-2, pseudovirus neutralization assay and commercial ELISA kits. The test results were compared using statistical analysis.Results:Using IS (sample G) as a reference, the relative concentrations of other samples could be determined with less variation. ELISA and pseudovirus neutralization assay had consistent results with those obtained with the microneutralization test based on SARS-COV-2 strain HB02. Weakly positive samples could be detected only by a certain kit.Conclusions:The availability of an IS for antibodies would facilitate the standardization of SARS-CoV-2 antibody detection methods. The reference panel fitted all the assays based on the SARS-CoV-2 prototype Wuhan strain. Pseudovirus neutralization assay and ELISA could be used as alternatives to live SARS-CoV-2-based neutralization test to some extent.

Objective:Using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) technology to edit Vaccinia virus (VACV) thymidine kinase (TK) Region for targeted recombination to establish an efficient vaccinia virus insertion recombination technology.Methods:We designed and synthesized guide RNAs(gRNA)targeting the TK region and then cloned individual gRNA into the PX458 vector that removes nuclear localization signals, and modified the original TK region recombinant plasmid. The gRNA and Cas9 co-expression plasmids were transfected into 293T cells separately to mediate the homologous recombination of vaccinia virus (VACV) and TK region recombination plasmid, and then the rate of viral recombination was evaluated by the appearance of blue and white spots.Results:The recombination efficiency mediated by the gRNA sequence designed and synthesized in this study is more than 1%, which is more than 10 times higher than the classical homologous recombination method .Conclusions:This study used CRISPR/Cas9 technology to establish a highly efficient recombinant vaccinia virus system, which provides technical support for pre-clinical research in vaccines or multivalent research of emerging infectious diseases, as well as tumor treatment.

NTV is a highly attenuated virus that was created by genetically deleting 26 genes related to host range and virulence from TianTan strain. Since NTV is highly attenuated, it has been used widely as an optimizing viral vector. In this study, we explored the biological characteristics in vitro and the host restriction mechanism of NTV. Most cell lines do not support sufficient dissemination and replication of NTV, and in non-permissive cell line HeLa, the replication block of NTV occurred at the translation stage of viral late protein expression. Lack of PKR activity was not sufficient to rescue expression of viral late proteins and replication, even though the phosphorylation level of eIF2α increased in NTV-infected HeLa cells. Moreover, the translation inhibition of NTV in HeLa cells was dependent upon a SAMD9 signaling pathway, as demonstrated by silencing SAMD9 expression with siRNA and observing the colocalization of SAMD9 and AVGs. Reinserting C7L or K1L into NTV rescued the late viral protein expression and replication of NTV in HeLa cells. Among the genes deleted in NTV, C7L or/and K1L gene was mainly responsible for its replication defect. Protein C7 interacted with SAMD9, which antagonized the antiviral response of SAMD9 to ensure viral protein translation and replication of NTV in non-permissive cell lines. Our finding will serve as a baseline for modification of NTV in future application.