EIF4A3 is stabilized by the long noncoding RNA BC200 to regulate gene expression during Epstein-Barr virus infection.

Epstein‒Barr virus (EBV) regulates the expression of host genes involved in functional pathways for viral infection and pathogenicity. Long noncoding RNAs (lncRNAs) have been found to be important regulators of cellular biology. However, how EBV affects host biological processes via lncRNAs remains elusive. Eukaryotic initiation factor 4A3 (EIF4A3) was recently identified as an essential controller of cell fate with an unknown role in EBV infection. Here, the expression of lncRNA brain cytoplasmic 200 (BC200) was shown to be significantly upregulated in EBV-infected cell lines. RNA immunoprecipitation and RNA pulldown assays confirmed that BC200 bound to EIF4A3. Moreover, BC200 promoted EIF4A3 expression at the protein level but not at the mRNA level. Mechanistically, BC200 stabilized the EIF4A3 protein by impeding the K48-linked polyubiquitination of the K195 and K198 residues of EIF4A3. In addition, RNA-seq analysis of EBV-positive cells with knockdown of either BC200 or EIF4A3 revealed that a broad range of cellular genes were differentially regulated, particularly those related to virus infection and immune response pathways. This study is the first to reveal the key residues involved in EIF4A3 polyubiquitination and elucidate the novel regulatory role of EBV in host gene expression via the BC200/EIF4A3 axis. These results have implications for the pathogenesis and treatment of EBV-related diseases.

IFIT3 inhibits Epstein-Barr virus reactivation via upregulating innate immunity.

Epstein-Barr virus (EBV), a member of the γ-herpesvirus family, can establish latent infection in B lymphocytes and certain epithelial cells after primary infection. Under certain circumstances, EBV can enter into lytic replication. However, the regulation of EBV latent-lytic infection remains largely unclear. The important immune molecule, interferon-induced protein with tetratricopeptide repeats 3 (IFIT3), was upregulated in EBV latently infected cells. When the lytic replication of EBV was induced, the expression of IFIT3 was further increased. In turn, IFIT3 overexpression dramatically inhibited the lytic replication of EBV, while IFIT3 knockdown facilitated EBV lytic replication. Moreover, upon the lytic induction, the ectopic IFIT3 expression promoted the activation of the interferon (IFN) pathway, including the production of IFN-stimulated genes (ISGs), IFNB1, and the phosphorylation of IFN-regulatory factor 3 (IRF3). In contrast, the depletion of IFIT3 led to decreased ISGs and IFNB1 expression. Mechanically, IFIT3 inhibited EBV lytic replication through IFN signaling. This study revealed that the host innate immune-related factor IFIT3 played an important role in regulating EBV latent-lytic homeostasis. The results implied that EBV has evolved well to utilize host factors to maintain latent infection.

The emerging roles of IFIT3 in antiviral innate immunity and cellular biology.

The interferon-inducible protein with tetrapeptide repeats 3 (IFIT3) is one of the most important members in both the IFIT family and interferon-stimulated genes family. IFIT3 has typical features of the IFIT family in terms of gene and protein structures, and is able to be activated through the classical PRRs-IFN-JAK/STAT pathway. A variety of viruses can induce the expression of IFIT3, which in turn inhibits the replication of viruses, with the underlying mechanism showing its crucial role in antiviral innate immunity. Emerging studies have also identified that IFIT3 is involved in cellular biology changes, including cell proliferation, apoptosis, differentiation, and cancer development. In this review, we summarize the characteristics of IFIT3 with respect to molecular structure and regulatory pathways, highlighting the role of IFIT3 in antiviral innate immunity, as well as its diverse biological roles. We also discuss the potential of IFIT3 as a biomarker in disease diagnosis and therapy.

The implications of cell-free DNAs derived from tumor viruses as biomarkers of associated cancers.

Cancer is still ranked as a leading cause of death according to estimates from the World Health Organization (WHO) and the strong link between tumor viruses and human cancers have been proved for almost six decades. Cell-free DNA (cfDNA) has drawn enormous attention for its dynamic, instant, and noninvasive advantages as one popular type of cancer biomarker. cfDNAs are mainly released from apoptotic cells and exosomes released from cancer cells, including those infected with viruses. Although cfDNAs are present at low concentrations in peripheral blood, they can reflect tumor load with high sensitivity. Considering the relevance of the tumor viruses to the associated cancers, cfDNAs derived from viruses may serve as good biomarkers for the early screening, diagnosis, and treatment monitoring. In this review, we summarize the methods and newly developed analytic techniques for the detection of cfDNAs from different body fluids, and discuss the implications of cfDNAs derived from different tumor viruses in the detection and treatment monitoring of virus-associated cancers. A better understanding of cfDNAs derived from tumor viruses may help formulate novel antitumoral strategies to decrease the burden of cancers that attributed to viruses.

Competing risk of the specific mortality among Asian-American patients with prostate cancer: a surveillance, epidemiology, and end results analysis.

BACKGROUND: Adopted the competing-risk model to investigate the relevant factors affecting the prostate cancer (PCa)-specific mortality among Asian-American PCa patients based on the Surveillance, Epidemiology, and End Results (SEER) database. METHODS: The information of 26,293 Asian-American patients diagnosed with PCa between 2004 and 2015 were extracted from the SEER 18 database. Subjects were divided into three groups: died of PCa, died of other causes, survival based on the outcomes at the end of 155 months' follow-up. Multivariate analysis was performed by the Fine-gray proportional model. Meanwhile, subgroup analyses were conducted risk stratification by race and age. RESULTS: Age ≥ 65 years [Hazard ratio (HR) = 1.509, 95% confidence interval (CI) 1.299-1.754], race (HR = 1.220, 95% CI 1.028-1.448), marital status (unmarried, single or widowed, HR = 1.264, 95% CI 1.098-1.454), tumor grade II (HR = 3.520, 95% CI 2.915-4.250), the American Joint Committee on Cancer (AJCC) stage (T3: HR = 1.597, 95% CI 1.286-1.984; T4: HR = 2.446, 95% CI 1.796-3.331; N1: HR = 1.504, 95% CI 1.176-1.924; M1: HR = 9.875, 95% CI 8.204-11.887) at diagnosis, radiotherapy (HR = 1.892, 95% CI 1.365-2.623), regional nodes positive (HR = 2.498, 95% CI 1.906-3.274) increased risk of PCa-specific mortality for Asian-American PCa patients, while surgical (HR = 0.716, 95% CI 0.586-0.874) reduced the risk. CONCLUSION: The study findings showed that age, race, marital status, tumor grade (II), AJCC stages (T3, T4, N1, M1) at diagnosis, radiotherapy, regional nodes positive and surgery was associated with the specific mortality of PCa patients among Asian-Americans.

[Construction and Fluorescence Analysis of the Recombinant Listeria ivanovii Strain Expressing Green Fluorescent Protein].

OBJECTIVE: Constructing the recombinant Listeria ivanovii strain expressing green fluorescent protein to provide an important tool for study of Listeria ivanovii. METHODS: The promoter of Listeria monocytogenes Listeriolysin O (phly) and the green fluorescent protein (GFP) gene were fused by SOEing PCR,and then ligated the fusion gene into plasmid pCW to result in recombinant plasmid pCW-phly-GFP. Recombinant plasmid was electroporated into Listeria ivanovii,and fluorescence microscope was used to analyze the expression of GFP. To observe the stability of recombinant plasmid and the stable expression of GFP in Listeria ivanovii,bacteria were cultured in the BHI broth with or without erythromycin for several generations. The stability of recombinant plasmid pCW-phly-GFP and fluorescent protein in each generation of bacteriawas studied by extracting plasmids and observing fluorescence. RESULTS: The exactness of recombinant plasmid pCW-phly-GFP was confirmed with restrictive endonuclease assay and sequence analysis. Under the fluorescence microscope,the green fluorescence was obvious in Listeria ivanovii carried with pCW-phly-GFP. The recombinant plasmid pCW-phly-GFP was stable in Listeria ivanovii and the GFP kept expressing in a high level under the pressure of erythromycin. CONCLUSION: The prokaryotic expression plasmid pCW-phly-GFP containing GFP gene was successfully constructed. Listeria ivanovii carried with the plasmid efficiently expressed GFP. This research provides an important tool for further study of Listeria ivanovii as a vaccine carrier.

[Prediction and Analysis of Epitopes in clpP2 of Mycobacterium tuberculosis].

OBJECTIVES: To predict and analyze the antigenic epitopes in Mycobacterium tuberculosis protein caseinolytic protease P2 (clpP2), and explore its possibility to be applied as a new tuberculosis (TB) vaccine and drug development target. METHODS: Secondary structure of clpP2 based on nucleic sequence was predicted by DNA Star software. The homologous sequence conformation were analyzed by Swiss-Model online software. T cells antigenic epitopes were predicted through VaxiPred, and B cell epitopes were predicted by combining use of several different prediction programs, such as ABCpred, COBEPro and BepiPredPred. The immune characteristics of clpP2 were analyzed by DNA Star, SignalP, TMHMM online software and were searched through NCBI database. RESULTS: clpP2protein was diverse in structure, composing with a great deal of CTL and Th cell epitopes. clpP2 was also predicted to comprise rich potential liner and discontinuous B-cell epitopes. These epitopes were accessible on the protein surface, located in flexible and hydrophilic regions. CONCLUSION: clpP2 is prompted to induce immune responses and developes a novel target in surveillance, treatment and vaccine.

[Genetic Recombiniation and Protein Expression Detection of Listeria-based tuberculosis Vaccine Candidates.]

OBJECTIVES: Genetic construction of tuberculosis vaccine candidates based on Listeria(L.) monocytogenes,L.ivanovii,and evaluation their protein expression,in order to provide a novel method for research on tuberculosis controlling. METHODS: Two kinds of gene cassettes carrying tuberculosis antigen encoding gene Rv3875 or Rv0129c were inserted into targeting vector harboring L.monocytogenes,L.ivanovii homologous sequences via genetic connection methods and plasmid transformation technology in vitro.Targeting plasmids were electroporated into L.monocytogenes,L.ivanovii,and the recombinant strains were experienced serial passage at 42 °C and 30 °C.Subsequently,the tuberculosis antigen gene cassettes in targeting plasmids were integrated into L.monocytogenes and L.ivanovii attenuated strain (knocking out of virulence gene actA and plcB) and L.ivanovii wild type strain by homologous recombination and gene targeting technology.The recombinant strains were screened by blue-white spot and antibiotic resistance test;the intracellular and extracellular proteins of the recombinant strains were tested by Western blot. RESULTS: Five recombination strains carried antigen gene cassette were constructed,and the recombinant genome were confirmed by PCR and sequencing.No erythromycin resistance gene was found in 5 strains,which was coincident to expection.Recombination strains Li-Rv0129c,Li-ΔactAplcB-Rv0129c and Li-ΔactAplcB-Rv3875 expressed Mycobacterium tuberculosis antigenic protein,Ag85C or ESAT-6,as expected.But L.monocytogenes strains did not express proper antigenic protein. CONCLUSIONS: Three novel L.ivanovii-based tuberculosis vaccine candicates,carrying Mycobacterium tuberculosis Rv0129c antigen gene cassette (coding for Ag85C) or Rv3875 gene cassette (coding for ESAT-6),and expressing relevant antigenic proteins have been successfully selected.

The Antitumor Effects of α-Linolenic Acid.

α-linolenic acid (ALA), which is a member of the n-3 polyunsaturated fatty acid (n-3 PUFA) family, has often been ignored due to a lack of information. ALA has gradually attracted increased attention due to its nutritional and medicinal advantages. Studies have shown that ALA exerts beneficial effects on a variety of diseases, including cancer. In this review, we summarize the antitumor effects of ALA in the context of cell biology, including the inhibition of proliferation, the induction of apoptosis, the inhibition of metastasis and angiogenesis, and antioxidant effects. In addition, studies have shown that ALA can be used as a drug carrier or exert positive clinical effects when combined with drugs. Therefore, the use of ALA in clinical treatments is very promising and valuable.

LncRNA BC200 promotes the development of EBV-associated nasopharyngeal carcinoma by competitively binding to miR-6834-5p to upregulate TYMS expression.

Nasopharyngeal carcinoma (NPC) is closely related to Epstein-Barr virus (EBV) infection. Long noncoding RNAs (lncRNAs) play important roles in cancers. However, the molecular mechanism underlying the roles of lncRNAs in EBV-associated NPC remains largely unclear. In this study, we confirmed that the expression of the lncRNA brain cytoplasmic 200 (BC200) was significantly increased in EBV-infected NPC cells and tissues. BC200 facilitated the growth and migration of NPC cells, suggesting that it participated in NPC progression by functioning as an oncogene. Mechanistically, BC200 was found to act as a ceRNA by sponging and inhibiting miR-6834-5p. Thymidylate synthetase (TYMS), whose high expression was reported to be an independent indicator of poor prognosis in NPC via an unknown mechanism, was identified as a target gene of miR-6834-5p in the present study. BC200 upregulated TYMS expression in a manner that depends on miR-6834-5p. TYMS was abnormally upregulated in EBV-positive NPC cells and tissues, and its ectopic expression contributed to the proliferation and migration of NPC cells. This study highlights the role of lncRNA BC200, which is upregulated by EBV, in promoting the development of NPC, suggesting that BC200-mediated ceRNA network may be valuable biomarkers for the diagnosis and treatment of EBV-associated NPC.

Insight into LncRNA- and CircRNA-Mediated CeRNAs: Regulatory Network and Implications in Nasopharyngeal Carcinoma-A Narrative Literature Review.

Nasopharyngeal carcinoma (NPC) is a kind of head-and-neck malignant tumor, and distant metastasis treatment resistance is the leading cause of patient death. In-depth understanding of NPC progression and treatment failure remains to be explored. Long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) are noncoding RNAs that play key regulatory role in shaping tumor cell activities. Recent studies have revealed that lncRNA and circRNA function as competitive endogenous RNAs (ceRNAs) by regulating the posttranscriptional expression of genes as miRNA baits. The imbalanced ceRNA networks derived from lncRNA/circRNA-miRNA-mRNA interaction are widely found to contribute to NPC development. Herein, we summarize typical examples of lncRNA/circRNA-associated ceRNAs in recent years, which involved the potential molecular mechanisms in the regulation of proliferation, apoptosis, treatment resistance and metastasis of NPC, and discuss their potential clinical significance in the prognosis and treatment of NPC. Interpreting the involvement of ceRNAs networks will provide new insight into the pathogenesis and treatment strategies of NPC. However, ceRNA regulatory mechanism has some limitations currently. Screening the most effective ceRNA targets and the clinical application of ceRNA still has many challenges.

Plasma Exosomal Proteomic Pattern of Epstein-Barr Virus-Associated Hemophagocytic Lymphohistiocytosis.

Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a life-threatening syndrome, which is caused by EBV infection that is usually refractory to treatment and shows relapse. The development of new biomarkers for the early diagnosis and clinical treatment of EBV-HLH is urgently needed. Exosomes have been shown to mediate various biological processes and are ideal non-invasive biomarkers. Here, we present the differential plasma exosomal proteome of a patient with EBV-HLH before vs. during treatment and with that of his healthy twin brother. A tandem mass tag-labeled LC-MS technique was employed for proteomic detection. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that differential proteomic profiles were related to virus infection, coagulopathy, nervous system dysfunction, imbalance of immune response, and abnormal liver function. The candidate biomarkers were first identified in the patient's plasma exosomes at different treatment and follow-up time points. Then, 14 additional EBV-HLH exosome samples were used to verify six differentially expressed proteins. The upregulation of C-reactive protein, moesin, galectin three-binding protein, and heat shock cognate 71 kDa protein and the downregulation of plasminogen and fibronectin 1 could serve as potential biomarkers of EBV-HLH. This plasma exosomal proteomic analysis provides new insights into the diagnostic and therapeutic biomarkers of EBV-HLH.

Corrigendum: Plasma Exosomal Proteomic Pattern of Epstein-Barr Virus-Associated Hemophagocytic Lymphohistiocytosis.

[This corrects the article DOI: 10.3389/fmicb.2022.821311.].

Combination immunotherapy with two attenuated Listeria strains carrying shuffled HPV-16 E6E7 protein causes tumor regression in a mouse tumor model.

Cervical cancer continues to impose a heavy burden worldwide, and human papilloma virus (HPV) infection, especially persistent infection with type 16 (HPV-16), is known to be the primary etiological factor. Therapeutic vaccines are urgently needed because prophylactic vaccines are ineffective at clearing pre-existing HPV infection. Here, two recombinant Listeria strains (LMΔ-E6E7 & LIΔ-E6E7) with deletions of the actA and plcB genes, expressing the shuffled HPV-16 E6E7 protein were constructed. The strains were delivered into the spleen and liver by intravenous inoculation, induced antigen-specific cellular immunity and were eliminated completely from the internal organs several days later. Intravenously treating with single strain for three times, or with both strains alternately for three times significantly reduced the tumor size and prolonged the survival time of model mice. Combination immunotherapy with two strains seemed more effective than immunotherapy with single strain in that it enhanced the survival of the mice, and the LMΔ-E6E7-prime-LIΔ-E6E7-boost strategy showed significant stronger efficacy than single treatment with the LIΔ-E6E7 strain. The antitumor effect of this treatment might due to its ability to increase the proportion of CD8+ T cells and reduce the proportion of T regulatory cells (Tregs) in the intratumoral milieu. This is the first report regarding Listeria ivanovii-based therapeutic vaccine candidate against cervical cancer. Most importantly we are the first to confirm that combination therapy with two different recombinant Listeria strains has a more satisfactory antitumor effect than administration of a single strain. Thus, we propose a novel prime-boost treatment strategy.

Nutrients recycling and biomass production from Chlorella pyrenoidosa culture using anaerobic food processing wastewater in a pilot-scale tubular photobioreactor.

Microalgae cultivation in anaerobic food wastewater was a feasible way for high biomass production and nutrients recycling. In this study, Chlorella pyrenoidosa culture on anaerobic food wastewater was processed outdoors using a pilot-scale tubular photobioreactor. The microalgae showed rapid growth in different seasons, achieving high biomass production of 1.83-2.10 g L-1 and specific growth rate of 0.73-1.59 d-1. The biological contamination and dissolved oxygen were controlled at suitable levels for algal growth in the tubular photobioreactor. Lipids content in harvested biomass was 8.1-15.3% of dried weight, and the analysis in fatty acids revealed high quality with long carbon chain length and high saturation. Additionally, algal growth achieved effective pollutants purification from wastewater, removing 42.3-53.8% of CODCr, 82.6-88.7% of TN and 59.7-67.6% of TP. This study gave a successful application for scaled-up microalgae culture in anaerobic food processing wastewater for biodiesel production and wastewater purification.

Cellular Deubiquitylating Enzyme: A Regulatory Factor of Antiviral Innate Immunity.

Deubiquitylating enzymes (DUBs) are proteases that crack the ubiquitin code from ubiquitylated substrates to reverse the fate of substrate proteins. Recently, DUBs have been found to mediate various cellular biological functions, including antiviral innate immune response mediated by pattern-recognition receptors (PRRs) and NLR Family pyrin domain containing 3 (NLRP3) inflammasomes. So far, many DUBs have been identified to exert a distinct function in fine-tuning antiviral innate immunity and are utilized by viruses for immune evasion. Here, the recent advances in the regulation of antiviral responses by DUBs are reviewed. We also discussed the DUBs-mediated interaction between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and antiviral innate immunity. The understanding of the mechanisms on antiviral innate immunity regulated by DUBs may provide therapeutic opportunities for viral infection.

Intranasal vaccination with Listeria ivanovii as vector of Mycobacterium tuberculosis antigens promotes specific lung-localized cellular and humoral immune responses.

We have previously demonstrated that a recombinant Listeria ivanovii (LI) strain expressing the ESAT-6 or Ag85C protein of Mycobacterium tuberculosis (Mtb) as a tuberculosis (TB) vaccine candidates induced antigen-specific cellular immune responses after intravenous immunization of mice. However, whether such recombinant strains could induce desired immune responses in the lung, where TB infection occurs, is not clear. In this paper, C57BL/6 J mice were intranasally vaccinated with attenuated LIΔactAplcB-Rv3875 (Δ refers to gene deletion in the bacterial genome) or LIΔactAplcB-Rv0129c, the two vaccine candidates that utilize LI as an antigen delivery vector. Bacterial load in the target organs, histological changes in the infected organs, the percentage of specific cytokine-secreting T cells in the lung and spleen, IgG levels in the serum and secretory IgA (SIgA) levles in bronchoalveolar lavage (BAL) fluid were determined at specific days post inoculation (dpi). The results showed that both strains were mainly confined to the lung and were eliminated at 10 dpi. The histological damage caused by the infection in the lung was slight and recovered by day 5. Intranasal vaccination of the mice twice at an interval of 4 weeks notably elicited TB antigen-specific CD4+ and CD8+ T cell responses in the lung and SIgA secretion in the pulmonary mucosa, and significantly enhanced the percentage of double-functional CD8+ T cells (IFN-γ+ TNF-α+ CD8+). To our knowledge, this is the first report regarding the used of LI vector vaccines to induce promising lung-localized cellular and humoral immune responses by intranasal vaccination. These data suggest that LI could be a novel and promising live vector to construct an intranasal vaccine against respiratory diseases.

Vaccination of Mice with Listeria ivanovii Expressing the Truncated M Protein of Porcine Reproductive and Respiratory Syndrome Virus Induces both Antigen-Specific CD4+ and CD8+ T Cell-Mediated Immunity.

Porcine reproductive and respiratory syndrome (PRRS), a serious disease of swine caused by the PRRS virus (PRRSV), had a severe economic impact worldwide. As commonly used PRRS vaccines, the attenuated or inactivated vaccines, provide unsatisfactory immune protection, a new PRRS vaccine is urgently needed. In this study, a part of the PRRSV ORF6 gene (from 253 to 519 bp) encoding the hydrophilic domain of PRRSV M protein was integrated into two Listeria strains via homologous recombination to generate two PRRS vaccine candidates, namely LI-M' and LM-ΔactAplcB-M'. Both candidate vaccines showed similar growth rate as their parent strains in culture media, but presented different bacterial loads in target organs. As the integrated heterogenous gene was not expressed, LM-ΔactAplcB-M' was excluded from the immunological test. In a mouse model, LI-M' provoked both CD4+ and CD8+ T cell-mediated immunity. In addition, LI-M' boosting dramatically enhanced CD8+ T cell-mediated immunity without affecting the response intensity of CD4+ T cell-mediated immunity. All of these data suggest that LI-M' is a promising PRRS vaccine candidate.

Coating With Chitooligosaccharides Enhances the Cytokine Induction of Listeria ivanovii-Based Vaccine Strain.

Listeria ivanovi (LI) is an available live bacterial vaccine vector. This work attempted to coat LI-based vaccine candidates (LI-Rv0129c) with chitooligosaccharides (COSs) as an adjuvant to enhance the cellular immune responses induced. COS-bacteria composite was achieved by mixing the bacteria suspension with equal volume of COS solution, and this process accompanied with the increase of bacteria superficial zeta potential and formation of special superficial configurations. COS coating improved the ratio swallowed by the macrophage-like RAW264.7 cells from 0.54% to 2.88% (p < 0.001). In vivo, the COS-coated LI-Rv0129c strain did elicit significantly higher specific CD4+ IFN-γ, CD4+ TNF-α or CD8+ IFN-γ secretion (0.91%, 1.00%, 0.30%, respectively) than naked LI-Rv0129c (0.32%, 0.38%, 0.07%, respectively) (p < 0.01). These results demonstrated that COS is a promising adjuvant to enhance the protective cellular immune responses induced by LI-based vaccine strains. Our work provided a notion for developing adjuvant for Listeria and other bacterial vector-based vaccines.

The Protein Expression Level of a Heterogeneous Gene Inserted in LIPI-1 of the Listeria ivanovii Genome Relies on Its Insertion Orientation.

Due to its capability to multiply in either phagocytic or nonphagocytic cells, and to subsequently elicit a robust cellular immune response, Listeria ivanovii (LI) is thought to be feasible for developing bacteria-based live attenuated vaccines. We previously generated several recombinant LI strains expressing Mycobacterium tuberculosis antigens. Since the expression level of heterogeneous protein was sometimes very low, we attempted to elucidate the principle of heterogeneous protein expression in such recombinant LI strains. In this study, we inserted the M. tuberculosis antigen gene Rv0129c into LI strains at the same site as the genome but with a different insertion orientation. RT-qPCR and Western blot showed that when the insertion orientation of the heterogeneous gene was opposite to the LIorfXYZ gene in the Listeria pathogenicity island 1 in the bacterial genome, the heterogeneous gene could be transcribed well but the protein expression level seemed limited, both in vitro and in vivo. When inserted at an orientation consistent with LIorfXYZ at the same site in the genome, the expected 43-kD protein was observed in vitro as well as in a mouse model. Bacterial virulence was found to have decreased after recombination. This work confirms that the protein expression level of the heterogenous gene in such genome-recombinant LI-based vaccines is related to its inserted orientation in the bacterial genome, and a foreign gene inserted at this position of LIPI-1 will abolish Listeria virulence without affecting its growth.