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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces direct cytopathic effects, complicating the establishment of low-cytotoxicity cell culture models for studying its replication. We initially developed a DNA vector-based replicon system utilizing the CMV promoter to generate a recombinant viral genome bearing reporter genes. However, this system frequently resulted in drug resistance and cytotoxicity, impeding model establishment. Herein, we present a novel cell culture model with SARS-CoV-2 replication induced by Cre/LoxP-mediated DNA recombination. An engineered SARS-CoV-2 transcription unit was subcloned into a bacterial artificial chromosome (BAC) vector. To enhance biosafety, the viral spike protein gene was deleted, and the nucleocapsid gene was replaced with a reporter gene. An exogenous sequence was inserted within NSP1 as a modulatory cassette that is removable after Cre/LoxP-mediated DNA recombination and subsequent RNA splicing. Using the PiggyBac transposon strategy, the transcription unit was integrated into host cell chromatin, yielding a stable cell line capable of inducing recombinant SARS-CoV-2 RNA replication. The model exhibited sensitivity to the potential antivirals forsythoside A and verteporfin. An innovative inducible SARS-CoV-2 replicon cell model was introduced to further explore the replication and pathogenesis of the virus and facilitate screening and assessment of anti-SARS-CoV-2 therapeutics.

The prenatal BACs-on-Beads™ (BoBs) assay was introduced for rapid detection of abnormalities of chromosomes 13, 18, 21, X, and Y and specific nine significant microdeletion syndromes. The ability of prenatal BoBs to detect mosaicism ranged from 20 to 40%. However, there have been no prenatal studies of sex chromosome mosaicism in prenatal BoBs. Therefore, the present study was performed with an aim to uncover the detection level of sex chromosome mosaicism that application of prenatal BoBs assay, and then to assess the sensitivity of prenatal BoBs assay, thereby improving the prenatal diagnostic accuracy. A total of 31 samples of amniotic fluid (AF) and umbilical cord blood (UCB) for prenatal diagnosis were collected, and the results were confirmed through karyotyping, single nucleotide polymorphism microarray (SNP-array) and copy number variation sequencing (CNV-seq). 23 cases of sex chromosome mosaicism were prompted abnormal by prenatal BoBs, the minimum detection level of mosaicism was about 6% as detected by karyotype. The overall sensitivity of prenatal BoBs in the detection of sex chromosome mosaicism was 74.2% (23/31). This study evaluated the effectiveness of prenatal BoBs for detecting sex chromosome mosaicism in prenatal diagnosis, and the results will provide valuable information for genetic counseling.

Here, we report the generation and characterization of a novel Huntington\'s disease (HD) mouse model BAC226Q by using a bacterial artificial chromosome (BAC) system, expressing full-length human HTT with ~226 CAG-CAA repeats and containing endogenous human HTT promoter and regulatory elements. BAC226Q recapitulated a full-spectrum of age-dependent and progressive HD-like phenotypes without unwanted and erroneous phenotypes. BAC226Q mice developed normally, and gradually exhibited HD-like psychiatric and cognitive phenotypes at 2 months. From 3 to 4 months, BAC226Q mice showed robust progressive motor deficits. At 11 months, BAC226Q mice showed significant reduced life span, gradual weight loss and exhibited neuropathology including significant brain atrophy specific to striatum and cortex, striatal neuronal death, widespread huntingtin inclusions, and reactive pathology. Therefore, the novel BAC226Q mouse accurately recapitulating robust, age-dependent, progressive HD-like phenotypes will be a valuable tool for studying disease mechanisms, identifying biomarkers, and testing gene-targeting therapeutic approaches for HD.

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhoea in suckling piglets and has the potential for cross-species transmission. No effective PDCoV vaccines or antiviral drugs are currently available. Here, we successfully generated an infectious clone of PDCoV strain CHN-HN-2014 using a combination of bacterial artificial chromosome (BAC)-based reverse genetics system with a one-step homologous recombination. The recued virus (rCHN-HN-2014) possesses similar growth characteristics to the parental virus in vitro. Based on the established infectious clone and CRISPR/Cas9 technology, a PDCoV reporter virus expressing nanoluciferase (Nluc) was constructed by replacing the NS6 gene. Using two drugs, lycorine and resveratrol, we found that the Nluc reporter virus exhibited high sensibility and easy quantification to rapid antiviral screening. We further used the Nluc reporter virus to test the susceptibility of different cell lines to PDCoV and found that cell lines derived from various host species, including human, swine, cattle and monkey enables PDCoV replication, broadening our understanding of the PDCoV cell tropism range. Taken together, our reporter viruses are available to high throughput screening for antiviral drugs and uncover the infectivity of PDCoV in various cells, which will accelerate our understanding of PDCoV.

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which inflicts major economic losses on the global pig farming industry. Based on its similarity to highly pathogenic strains, the GDzj strain isolated in this study was predicted to be highly pathogenic. We therefore analyzed the pathogenicity of this strain experimentally in piglets. All piglets challenged with this virus experienced fever or high fever, loss of appetite, decreased food intake, daily weight loss, shortness of breath, and listlessness, and the necropsy results showed that they had experienced severe interstitial pneumonia. We then used the BAC system to construct a full-length cDNA infectious clone of GDzj, and the rescued virus displayed in vitro proliferation characteristics similar to those of the parental PRRSV strain. In summary, we successfully isolated a highly pathogenic PRRSV strain and constructed a full-length infectious cDNA clone from it, thereby providing an effective reverse genetics platform for further study of viral pathogenesis.

Human leukocyte antigen (HLA)-DP is much less studied than other HLA class II antigens, that is, HLA-DR and HLA-DQ, etc. However, the accumulating data have suggested the important roles of DP-restricted responses in the context of cancer, allergy, and infectious disease. Lack of animal models expressing these genes as authentic cis-haplotypes blocks our understanding for the role of HLA-DP haplotypes in immunity.
To explore the potential cis-acting control elements involved in the transcriptional regulation of the HLA-DPA1/DPB1 gene, we performed the expression analysis using bacterial artificial chromosome (BAC)-based transgenic humanized mice in the C57BL/6 background, which carried the entire HLA-DP401 gene locus. We further developed a mouse model of Staphylococcus aureus pneumonia in HLA-DP401 humanized transgenic mice, and performed the analysis on the expression pattern of HLA-DP401 and immunological responses in the model.
In this study, we screened and identified a BAC clone spanning the entire HLA-DP gene locus. DNA from this clone was analyzed for integrity by pulsed-field gel electrophoresis and then microinjected into fertilized mouse oocytes to produce transgenic founder animals. Nine sets of PCR primers for regional markers with an average distance of 15 kb between each primer were used to confirm the integrity of the transgene in the five transgenic lines carrying the HLA-DPA1/DPB1 gene. Transgene copy numbers were determined by real-time PCR analysis. HLA-DP401 gene expression was analyzed at the mRNA and protein level. Although infection with S aureus Newman did not alter the percentage of immune cells in the spleen and thymus from the HLA-DP401-H2-Aβ1 humanized mice. Increased expression of HLA-DP401 was observed in the thymus of the humanized mice infected by S aureus.
We generated several BAC transgenic mice, and analyzed the expression of HLA-DPA1/DPB1 in those mice. A model of Saureus-induced pneumonia in the HLA-DP401-H2-Aβ1-/- humanized mice was further developed, and S aureus infection upregulated the HLA-DP401 expression in thymus of those humanized mice. These findings demonstrate the potential of those HLA-DPA1/DPB1 transgenic humanized mice for developing animal models of infectious diseases and MHC-associated immunological diseases.

Multiple independent sequence variants of the hTERT locus have been associated with telomere length and cancer risks in genome-wide association studies. Here, we identified an intronic variable number tandem repeat, VNTR2-1, as an enhancer-like element, which activated hTERT transcription in a cell in a chromatin-dependent manner. VNTR2-1, consisting of 42-bp repeats with an array of enhancer boxes, cooperated with the proximal promoter in the regulation of hTERT transcription by basic helix-loop-helix transcription factors and maintained hTERT expression during embryonic stem-cell differentiation. Genomic deletion of VNTR2-1 in MelJuSo melanoma cells markedly reduced hTERT transcription, leading to telomere shortening, cellular senescence, and impairment of xenograft tumor growth. Interestingly, VNTR2-1 lengths varied widely in human populations; hTERT alleles with shorter VNTR2-1 were underrepresented in African American centenarians, indicating its role in human aging. Therefore, this polymorphic element is likely a missing link in the telomerase regulatory network and a molecular basis for genetic diversities of telomere homeostasis and age-related disease susceptibilities.

BACKGROUND: Pseudorabies virus (PRV) is a preferred vector for recombinant vaccine construction. Previously, we generated a TK&gE-deleted PRV (PRVΔTK&gE-AH02) based on a virulent PRV AH02LA strain. It was shown to be safe for 1-day-old piglets with maternal PRV antibodies and 4 ~ 5 week-old PRV antibody negative piglets and provide rapid and 100 % protection in weaned pigs against lethal challenge with the PRV variant strain. It suggests that PRVTK&gE-AH02 may be a promising live vaccine vector for construction of recombinant vaccine in pigs. However, insertion site, as a main factor, may affect foreign gene expression.
RESULTS: In this study, we constructed four recombinant PRV-S bacterial artificial chromosomes (BACs) carrying the same spike (S) expression cassette of a variant porcine epidemic diarrhea virus strain in different noncoding regions (UL11-10, UL35-36, UL46-27 or US2-1) from AH02LA BAC with TK, gE and gI deletion. The successful expression of S gene (UL11-10, UL35-36 and UL46-27) in recombinant viruses was confirmed by virus rescue, PCR, real-time PCR and indirect immunofluorescence. We observed higher S gene mRNA expression level in swine testicular cells infected with PRV-S(UL11-10)ΔTK/gE and PRV-S(UL35-36)ΔTK/gE compared to that of PRV-S(UL46-27)ΔTK/gE at 6 h post infection (P < 0.05). Moreover, at 12 h post infection, cells infected with PRV-S(UL11-10)ΔTK/gE exhibited higher S gene mRNA expression than those infected with PRV-S(UL35-36)ΔTK/gE (P = 0.097) and PRV-S(UL46-27)ΔTK/gE (P < 0.05). Recovered vectored mutant PRV-S (UL11-10, UL35-36 and UL46-27) exhibited similar growth kinetics to the parental virus (PRVΔTK&gE-AH02).
CONCLUSIONS: This study focuses on identification of suitable sites for insertion of foreign genes in PRV genome, which laids a foundation for future development of recombinant PRV vaccines.

To rapidly and accurately manipulate genome such as gene deletion, insertion and site mutation, the whole genome of a very virulent strain Md5 of Marek\'s disease virus (MDV) was inserted into bacterial artificial chromosome (BAC) through homogeneous recombination. The recombinant DNA was electroporated into DH10B competent cells and identified by PCR and restriction fragment length polymorphism analysis. An infectious clone of Md5BAC was obtained following transfection into chicken embryo fibroblast (CEF) cells. Furthermore, a lorf10 deletion mutant was constructed by two step Red-mediated homologous recombination. To confirm the specific role of gene deletion, the lorf10 was reinserted into the original site of MDV genome to make a revertant strain. All the constructs were rescued by transfection into CEF cells, respectively. The successful packaging of recombinant viruses was confirmed by indirect immunofluorescence assay. The results of growth kinetics assay and plaques area measurement showed that the lorf10 is dispensable for MDV propagation in vitro. Overall, this study successfully constructed an infectious BAC clone of MDV and demonstrated its application in genome manipulation; the knowledge gained from our study could be further applied to other hepesviruses.
为了快速且准确地对疱疹病毒基因组进行基因敲除、插入或者点突变等修饰，通过同源重组将马立克氏病病毒 (MDV) 超强毒株Md5基因组克隆到细菌人工染色体 (BAC)。将筛选的阳性重组体DNA电转进DH10B菌株，用PCR及限制性片段多态分析 (RFLP) 方法鉴定含Md5全基因组的BAC克隆。将阳性重组体DNA转染入鸡胚成纤维细胞 (CEF)，拯救出重组病毒，命名为Md5BAC。进一步利用Red酶介导的两步法基因重组技术构建MDVlorf10基因敲除毒株。为了验证被敲除基因功能的特异性，将lorf10插入原位点以构建基因复原毒株。将构建的重组毒株分别感染CEF细胞，用间接免疫荧光试验确认重组病毒均包装成功；病毒生长曲线结果表明，lorf10敲除不影响病毒的体外增殖。总之，这为其他疱疹病毒的基因组编辑提供了技术参考。.