Bilateral choroidal effusion following vaccination against SARS-CoV-2 virus

An 80-year-old systemically stable female presented with sudden blurring of vision post the first dose of Covishield™, a non-replicating viral vector vaccine. On examination, she was found to have bilateral serous choroidal effusions. A thorough systemic and ocular workup was performed to rule out other causes of choroidal effusion. The effusions resolved with tapering doses of oral and systemic steroids. To the best of our knowledge, at the time of submission, this is the first case of choroidal effusion being reported after the coronavirus disease 2019 (COVID-19) vaccine.

EXPRESIÓN DE LA PROTEÍNA CORE DEL VIRUS DE LA HEPATITIS C EN CÉLULAS HepG2 USANDO EL VIRUS DEL BOSQUE DE SEMLIKI / Hepatitis C virus core protein expression in HepG2 cells using Semliki Forest Virus

RESUMEN El Virus de la Hepatitis C (VHC) codifica la proteína Core. Que, además de ser la subunidad de la cápside, participa en diferentes mecanismos de patogénesis de la infección por VHC. Dado que el sistema de replicación in vitro del VHC presenta limitaciones, el uso de vectores virales podría ser una herramienta útil para estudiar las propiedades de la proteína Core. Con el fin de validar el vector con el Virus del Bosque de Semliki (SFV) para el estudio de Core en células HepG2, se evaluó la expresión de la proteína verde fluorescente (GFP) y la proteína Core utilizando este vector viral. Las expresiones de GFP y Core se detectaron en células HepG2 transducidas con rSFV de 24 a 96 horas postransducción. La expresión de la proteína Core fue inferior a la expresión de GFP en las células HepG2. Teniendo en cuenta que la proteína Core del VHC puede regular la actividad del gen p53, se evaluó el nivel transcripcional de este gen. Se observó una disminución en el nivel de mARN de p53 en las células luego de la transducción, comparado con las células control. Aunque las células transducidas con rSFV-Core presentaron el menor nivel de mARN de p53, la diferencia no fue significativa comparada con las células transducidas con rSFV-GFP. Los resultados confirman que rSFV permite la expresión transitoria de proteínas heterólogas en líneas celulares de hepatoma humano. Se necesitan estudios adicionales para determinar si la expresión disminuida de Core puede deberse a degradación de la proteína viral.

ABSTRACT The Hepatitis C Virus (HCV) encodes the structural protein Core, which in addition to being the capsid subunit, participates in different mechanisms of HCV infection pathogenesis. Since HCV in vitro replication system has limitations, the use of viral vectors could be a useful tool to study the Core protein properties. To validate the Semliki Forest Virus (SFV) strategy in transduced HepG2 cells to study the HCV Core protein, the expression of green fluorescent protein (GFP) and Core protein expressions were detected 24 to 96 hours post-transduction in HepG2 cells transduced with rSFV. Core protein expression was lower than GFP expression in HepG2 cells. Since HCV Core protein can regulate the activity of the p53 gene, the transcriptional level of this gene was evaluated. A decrease in the level of p53 mRNA was observed in the cells after transduction, compared to the control cells. Although the cells transduced with rSFV-Core had the lowest level of p53 mRNA, the difference was not significant compared to cells transduced with rSFV-GFP. The results confirm that rSFV allows the transient expression of heterologous proteins in human hepatoma cell lines. Additional studies are needed to determine whether the decreased expression of Core may be due to the degradation of the viral protein.

Research progress of poly (β-amino ester)s-based gene vector / 药学学报

Poly (β-amino ester)s (PβAEs) contain tertiary amine backbones and biodegradable ester bonds, making them highly biocompatible and pH-responsive. Meanwhile, originated from combinatorial libraries, PβAEs are simple to synthesize, easy to obtain raw materials and can be easily adapted to meet the different performance needs of gene carriers by adjusting the monomer type, monomer ratio and reaction time. Therefore, PβAEs are promising material for non-viral gene carriers. This paper provides a comprehensive overview of the properties and synthesis of PβAEs gene carriers and summarizes the progress of research on the gene delivery of each type of PβAEs.

Non-viral vector for gene therapy in inherited retinal degeneration / 中华实验眼科杂志

Inherited retinal degeneration (IRD), a group of diseases often causing irreversible blindness, with multiple pathogenesis, still lacks effective treatments currently.Development of effective therapeutics is a primary research goal.Despite rapid advances in gene therapy during the past decades, the most challenging aspect of gene therapy in clinical applications for IRD is to deliver the curative molecules to achieve optimal expression levels in target cells safely.Apart from high gene transfection efficiency, there are still many limitations, such as immunogenicity, biosafety issue, etc.in the application of viral vectors, which drive the development of gene therapy based on non-viral vectors.As one of the hot research topics in non-viral vectors, encouraging progress has been made in DNA nanoparticles for IRD treatment.The polymer/DNA complex nanoparticle is compacted and encapsulated DNA via peptides, lipids, or polysaccharides.Besides, the non-viral delivery system shows cost, preparation, packaging capacity, and safety advantages, providing a promising non-viral platform for safe and effective treatment of IRD, such as retinitis pigmentosa, Stargardt disease, X-linked juvenile retinoschisis, Leber congenital amaurosis, and so on.In this article, advances in transfection efficiency, targeting ability and safety of non-viral gene therapy and its application in IRD were reviewed.

Vacunas para SARS-CoV-2: diferentes estrategias de los desarrollos en curso / Vaccines for SARS-CoV-2: different strategies of ongoing developments

El objetivo de este artículo es proporcionar una guía que sirva para la interpretación y seguimiento de los esfuerzos que se están desarrollando en todo el mundo con el objetivo de obtener una vacuna que pueda generar inmunidad contra el nuevo coronavirus SARS-CoV-2 de 2019, el agente causante de la enfermedad por coronavirus denominada COVID-19. Cinco meses después de haber sido detectada la enfermedad, ya hay 102 vacunas en distintos estadios de desarrollo, registradas por la Organización Mundial de la Salud (OMS), correspondientes a 8 plataformas vacunales con diferentes estrategias, y todos los días aparecen nuevas. Esto representará un enorme desafío de organismos internacionales, para la evaluación, comparación y selección de aquellas que cumplan con los criterios regulatorios indispensables de seguridad y eficacia y que, por otro lado, puedan ser producidas en cantidades suficientes para abastecer la demanda mundial. (AU)

The objective of this article is to provide a guide to help the interpretation and monitoring the efforts that are being carried out worldwide to obtain a vaccine that will be able to generate immunity against the new 2019 SARS-CoV-2 coronavirus, the viral agent causes the disease named COVID-19. Five months after the disease was detected, there are already 102 vaccines at different stages of development, registered by World Health Organization (WHO), corresponding to 8 vaccination platforms base on different strategies, and every day new ones appear. This will represent a huge challenge for international organizations, to evaluate, compare and selects those that will meet the essential regulatory criteria of safety and efficacy and that, would be able to be produced in enough quantities to supply the worldwide demand. Key words: SARS-Cov-2 vaccine, vaccine platform, COVID-19 strategy, attenuated virus, viral vector, viral proteins, viral DNA, viral RNA, nucleic acids, viral like particles, WHO. (AU)

Recent progress of non-viral vector polyethylenimine in the application of gene delivery / 药学学报

In recent years, non-viral gene vectors have attracted great attention for efficient gene delivery due to the advantages, including low toxicity, low immunogenicity and simple preparation. Polyethylenimine (PEI) is one of the typical non-viral gene carriers that have been widely utilized for gene delivery owing to its superior capabilities in gene compression and buffering capacity. This article discusses the processes of gene delivery and the barriers of PEI-based carrier during the gene delivery, such as low biocompatibility, cytotoxicity, lack of specific targeting and insufficient gene release, etc. Therefore, we summarize the multiple approaches for the modifications of PEI in terms of improved biocompatibility, degradability, specific targeting and buffering capacity. Furthermore, we also review on the recent impressive progresses of smart stimuli-responsive PEI carriers, including endogenous stimuli (pH, reactive oxygen species, glutathione, biomolecular, etc), exogenous stimuli (light, temperature, magnetic field, etc) and dual-responsive strategies, which might provide guidance for the development of more efficient and safer non-viral gene vectors.

Research status and application progress of CRISPR/Cas9 delivery system / 中国药科大学学报

@#Based on the three delivery forms of CRISPR/Cas9 system at the levels of DNA, RNA and protein, this paper mainly approaches the development and new strategies of CRISPR/Cas9 delivery systems, as well as their application in the biomedical field and the clinical treatment of gene-related diseases. By summarizing and elaborating the CRISPR/Cas9 system delivery and gene therapy strategy, new ideas are provided for the discovery of innovative drugs and the development of gene therapy.

Efficient drug and gene delivery to liver fibrosis: rationale, recent advances, and perspectives

Liver fibrosis results from chronic damages together with an accumulation of extracellular matrix, and no specific medical therapy is approved for that until now. Due to liver metabolic capacity for drugs, the fragility of drugs, and the presence of insurmountable physiological obstacles in the way of targeting, the development of efficient drug delivery systems for anti-fibrotics seems vital. We have explored articles with a different perspective on liver fibrosis over the two decades, then collected and summarized the information by providing corresponding  and  cases. We have discussed the mechanism of hepatic fibrogenesis with different ways of fibrosis induction in animals. Furthermore, the critical chemical and herbal anti-fibrotics, biological molecules such as micro-RNAs, siRNAs, and growth factors, which can affect cell division and differentiation, are mentioned. Likewise, drug and gene delivery and therapeutic systems on  and  models are summarized in the data tables. This review article enlightens recent advances in emerging drugs and nanocarriers and represents perspectives on targeting strategies employed in liver fibrosis treatment.

Concept, mechanism and problem of gene therapy for osteoarthritis / 中国组织工程研究

BACKGROUND: Specific gene expression at specific injury sites enhances cell regeneration. OBJECTIVE: To summarize the concept of gene therapy, to review the strategies of gene therapy and the mechanism of gene therapy for osteoarthritis, and to introduce the research direction of gene therapy. METHODS: Extensive consulted the literature on osteoarthritis gene therapy and related content in foreign databases such as PubMed and Science Direct. The key words were “Gene therapy, Cartilage, Cartilage repair, Osteoarthritis, Expression vectors, miRNA”. Above data were analyzed and summarized. RESULTS AND CONCLUSION: Gene therapy for osteoarthritis can be applied to all aspects of the pathogenesis of osteoarthritis and affect the metabolism of related substances. The target gene is delivered to the target cells through viral or non-viral vectors. At present, the focus of gene therapy is to accelerate cell repair and reduce inflammatory response. At the same time, multiple choices of gene vectors and target genes provide better individualized treatment options. The specific mechanism of gene therapy for osteoarthritis in clinic is not clear, and there is the possibility of gene mutation, which needs to be further studied to verify the safety.

Ausencia de expresión de la proteína Core del virus de la hepatitis C en células dendríticas derivadas de monocitos humanos utilizando virus del Bosque de Semliki recombinante / Lack of expression of hepatitis c virus core protein in human monocyte-derived dendritic cells using recombinant semliki forest virus

ABSTRACT Hepatitis C Virus belongs to the Flaviviridae family. One proposed mechanism of HCV persistence in the ability to infect hematopoietic cells, including Dendritic cells (DCs). HCV infection of DCs could impair their functions that represent one of the mechanisms, thus hampering viral clearance by the host immune system. Among HCV-encoded proteins, the highly conserved Core protein has been suggested to be responsible for the immunomodulatory properties of this Hepacivirus. Recombinant viral vectors expressing the HCV Core protein and allowing its transduction and therefore the expression of the protein into DCs could be useful tools for the analysis of the properties of the Core protein. Vaccinia Virus and retrovirus have been used to transduce human DCs. Likewise, gene transfer into DCs using Semliki Forest Virus has been reported. This study aimed to express the HCV Core protein in human monocyte-derived DCs using an SFV vector, in which the subgenomic RNA encoding the structural proteins was replaced by the HCV Core sequence and then analyze the effects of its expression on DCs functions.

RESUMEN El virus de la Hepatitis C (VHC) pertenece a la familia Flaviviridae. Uno de los mecanismos propuestos de la persistencia del VHC es la capacidad de infectar células hematopoyéticas, incluidas las células dendríticas (DCs). La infección por VHC de DCs podría alterar sus funciones y corresponde a uno de los mecanismos que impiden el aclaramiento de la infección por VHC por el sistema inmunitario del hospedero. Entre las proteínas codificadas por el VHC, se ha sugerido que la proteína Core, altamente conservada, es responsable de las propiedades inmunomoduladoras de este Hepacivirus. Los vectores virales recombinantes que expresan la proteína Core y permiten su transducción a DCs podrían ser herramientas útiles para el análisis de las propiedades de esta proteína. El virus Vaccinia y el retrovirus se han utilizado para la transducción de DCs humanas. Del mismo modo, la transducción de DCs usando el virus del bosque de Semliki ha sido reportada. El objetivo de este estudio fue expresar la proteína Core de VHC en DCs derivadas de monocitos humanos utilizando un vector de SFV, en el que el ARN subgenómico que codifica las proteínas estructurales fue reemplazado por la secuencia Core del VHC y evaluar los efectos de su expresión en las funciones de DCs.

Respuesta inmune y terapia gó©nica con vectores virales adenoasociados / Immune response and gene therapy with adenoassociated viral vectors

En los ó;ºltimos aó;±os la terapia gó;©nica se ha posicionado como una opció;n real y segura en el desarrollo de alternativas terapó;©uticas para la cura y la prevenció;n de diferentes enfermedades. Consiste en la inserció;n de material genó;©tico en un tejido o có;©lula defectuosa, mediante el uso de un vector. Existen varias consideraciones para seleccionar el vector más apropiado, incluyendo el potencial de unió;n y entrada a la có;©lula diana, la capacidad de transferencia del material genó;©tico al nó;ºcleo, la habilidad de expresió;n del inserto y la ausencia de toxicidad. En el panorama actual, los vectores virales más utilizados son los derivados de los virus adenoasociados (AAV). Características como su bioseguridad, baja toxicidad y tropismo selectivo, han posibilitado su evaluació;n como opció;n terapó;©utica en un amplio nó;ºmero de enfermedades monogó;©nicas o complejas. A pesar de sus ventajas, los vectores AAV presentan inconvenientes, siendo el más importante la respuesta inmune del paciente al vector, especialmente la respuesta mediada por anticuerpos neutralizantes (NAb). Los NAb disminuyen la transducció;n del vector e impiden la expresió;n del gen que transporta, limitando su aplicació;n clínica. Por lo tanto, identificar y cuantificar la presencia y actividad de los NAbs, es el primer paso en cualquier protocolo de terapia gó;©nica con vectores AAV. La presencia de NAb depende principalmente de la exposició;n al virus en la naturaleza y varía drásticamente segó;ºn edad, localizació;n geográfica y estado de salud de la persona evaluada.

In recent years, gene therapy has been positioned as a real and safe option in the development of therapeutic alternatives for the cure and prevention of different diseases. It consists in the insertion of genetic material in a defective tissue or cell, through the use of a vector. There are several considerations for selecting the most appropriate vector, including the potential for binding and entry to the target cell, the ability of the genetic material to transfer to the nucleus, the ability to express the insert, and the absence of toxicity. In the current scenario, the most commonly used viral vectors are those derived from adeno-associated viruses (AAV). Characteristics such as biosafety, low toxicity and selective tropism have enabled its evaluation as a therapeutic option in many monogenic or complex diseases. Despite their advantages, AAV vectors have drawbacks, the most important being the patient’s immune response to the vector, especially the response mediated by neutralizing antibodies (NAb). NAbs decrease the transduction of the vector and prevent the expression of the gene it transports, limiting its clinical application. Therefore, identifying and quantifying the presence and activity of NAbs is the first step in any gene therapy protocol with AAV vectors. The presence of NAbs depends mainly on exposure to the virus in nature and varies drastically according to age, geographic location and health status of the person evaluated.

Transient expression of hemagglutinin antigen from canine influenza virus H3N2 in Nicotiana benthamiana and Lactuca sativa

PURPOSE: Canine influenza virus (CIV), H3N2, carries potentiality for zoonotic transmission and genetic assortment which raises a concern on possible epidemics, and human threats in future. To manage possible threats, the development of rapid and effective methods of CIV vaccine production is required. The plant provides economical, safe, and robust production platform. We investigated whether hemagglutinin (HA) antigen from Korea-originated CIV could be produced in Nicotiana benthamiana and lettuce, Lactuca sativa by a DNA viral vector system. MATERIALS AND METHODS: We used DNA sequences of the HA gene from Korean CIV strain influenza A/canine/Korea/S3001/2015 (H3N2) for cloning into a geminiviral expression vectors to express recombinant HA (rHA) antigen in the plant. Agrobacterium-mediated infiltration was performed to introduce HA-carrying vector into host plants cells. Laboratory-grown N. benthamiana, and grocery-purchased or hydroponically-grown lettuce plant leaves were used as host plants. RESULTS: CIV rHA antigen was successfully expressed in host plant species both N. benthamiana and L. sativa by geminiviral vector. Both complex-glycosylated and basal-glycosylated form of rHA were produced in lettuce, depending on presence of endoplasmic reticulum (ER) retention signal. In terms of rHA expression level, canine HA (H3N2) showed preference to the native signal peptide than ER retention signal peptide in the tested geminiviral vector system. CONCLUSION: Grocery-purchased lettuce leaves could serve as an instant host system for the transient expression of influenza antigen at the time of emergency. The geminiviral vector was able to induce expression of complex-glycosylated and basal-glycosylated rHA in lettuce and tobacco.

Development and evaluation considerations of viral vector production for CAR-T cell products / 中国肿瘤生物治疗杂志

@#Due to the spectacular therapy results in hematologic tumors, chimeric antigen receptor T (CAR-T) cell therapy has been the research hot-spot in the field of cell-immunotherapy. Viral vectors, as the critical raw material in CAR-T cell manufacturing, are closely related to the safety, efficacy and quality control of CAR-T cell products, in the aspects of the structure design of CAR gene, refinement of production process, quality control and setting of characterization and specification etc. Based on the research progress in lentiviral and γ-retroviral vectors development and the evaluation experiences, this paper discusses some common problems that need to be focused on in the preparation of viral vectors, expecting to provide references for the development and the authorization applications of domestic related products in the future.

Development of New Preventive and Therapeutic Vaccines for Tuberculosis

Tuberculosis (TB) is a contagious disease that has been responsible for the death of one billion people in the last 200 years. Until now, the only vaccine approved for the prevention of TB is Bacillus Calmette-Guérin (BCG), which is prepared by attenuating Mycobacterium bovis. However, one of the limitations of BCG is that its preventive effect against pulmonary TB varies from person to person. Therefore, there arises a need for a new TB vaccine to replace or supplement BCG. In this review, we have summarized the findings of current clinical trials on preventive and therapeutic TB vaccine candidates. In addition, we have discussed a novel vaccination approach using the cell-based vaccine presenting early secretory antigenic target-6 (ESAT-6), which is a potent immunogenic antigen. The role of ESAT-6 in hosts has also been described.

Gene therapy and delivery strategies for ocular diseases / 药学学报

Gene therapy has obvious advantages in the treatment of ocular diseases due to the unique structure of the eye. In recent years, there are more and more therapeutic gene-based drugs for ophthalmic application in clinical trials. Most of the delivery vectors are adeno-associated virus and administered via intraocular injection, which has potential risks. Traditional remedies, such as topical instillationor systemic administration, have limited therapeutic effects on the diseases in the posterior segment of the eye, where the chemical drugs are hard to reach. This makes the research of new strategies for gene drug delivery extremely urgent. For better understanding of the latest hot topics of ocular gene therapy, this article is prepared to introduce application of gene therapy to the typical ocular diseases and the corresponding gene-based medicines. The absorption routes for gene delivery into eyes and existing barriers are summarized. Finally, the gene delivery strategies are highlighted. The clinical application of ocular gene therapy will be boosted by overcoming the absorbing barriers and reducing the potential pitfalls.

Chimeric rabies glycoprotein with a transmembrane domain and cytoplasmic tail from Newcastle disease virus fusion protein incorporates into the Newcastle disease virion at reduced levels

Rabies remains an important worldwide health problem. Newcastle disease virus (NDV) was developed as a vaccine vector in animals by using a reverse genetics approach. Previously, our group generated a recombinant NDV (LaSota strain) expressing the complete rabies virus G protein (RVG), named rL-RVG. In this study, we constructed the variant rL-RVGTM, which expresses a chimeric rabies virus G protein (RVGTM) containing the ectodomain of RVG and the transmembrane domain (TM) and a cytoplasmic tail (CT) from the NDV fusion glycoprotein to study the function of RVG's TM and CT. The RVGTM did not detectably incorporate into NDV virions, though it was abundantly expressed at the surface of infected BHK-21 cells. Both rL-RVG and rL-RVGTM induced similar levels of NDV virus-neutralizing antibody (VNA) after initial and secondary vaccination in mice, whereas rabies VNA induction by rL-RVGTM was markedly lower than that induced by rL-RVG. Though rL-RVG could spread from cell to cell like that in rabies virus, rL-RVGTM lost this ability and spread in a manner similar to the parental NDV. Our data suggest that the TM and CT of RVG are essential for its incorporation into NDV virions and for spreading of the recombinant virus from the initially infected cells to surrounding cells.

Advance in the development of modified vaccinia virus Ankara (MVA)vectors for novel influenza vaccines / 中国人兽共患病学报

Influenza,caused by influenza virus,is a respiratory infectious disease with a serious hazard to human health.Prevention of influenza through vaccine development is the most effective strategy.It is important to build a rapid response platform for research and production of influenza vaccine.As virus vectors,live vaccine provides a new prevention and treatment way for infectious disease.Modified vaccinia virus Ankara(MVA) is a replication-deficient viral vector that is safe and can encode one or more foreign antigens and induce humoral and cellular immune response.MVA holds great promise as a vaccine platform.In this review,we discuss the use of MVA for vaccine development against influenza virus.

Safety of the novel influenza viral vector Brucella abortus vaccine in pregnant heifers / Segurança de uma nova vacina vetorial empregando o vírus influenza para Brucella abortus em novilhas prenhes

ABSTRACT: The present study provides the first information about the safety of a new influenza viral vector vaccine expressing the Brucella ribosomal protein L7/L12 or Omp16 containing the adjuvant Montanide Gel01 in pregnant heifers. Immunization of pregnant heifers was conducted via the conjunctival (n=10) or subcutaneous (n=10) route using cross prime and booster vaccination schedules at an interval of 28 days. The vector vaccine was evaluated in comparison with positive control groups vaccinated with B. abortus S19 (n=10) or B. abortus RB51 (n=10) and a negative (PBS+Montanide Gel01; n=10) control group. Clinical studies, thermometry, assessment of local reactogenicity and observation of abortion showed that the vector vaccine via the conjunctival or subcutaneous route was completely safe for pregnant heifers compared to the commercial vaccines B. abortus S19 or B. abortus RB51. The only single adverse event was the formation of infiltration at the site of subcutaneous injection; this reaction was not observed for the conjunctival route.

RESUMO: O presente estudo fornece as primeiras informações sobre a segurança de uma nova vacina usando o vetor viral influenza para expressar as proteínas de Brucella L7/L12 ou Omp16, contendo o adjuvante Montanide Gel01 em novilhas prenhes. A imunização de novilhas prenhes foi realizada através das vias conjuntiva (n=10) ou subcutânea (n=10), empregadas na primovacinação e na dose de reforço. O intervalo foi de 28 dias. A vacina empregando o vetor foi comparada com os grupos de controle positivo, vacinados com B. abortus B19 (n=10) ou B. abortus RB51 (n=10) e um grupo de controle negativo (PBS + Montanide Gel01; n=10). Os estudos clínicos, termometria, reação local e observação do aborto mostraram que a vacina empregando o vetor, aplicada pela via conjuntival ou subcutânea, foi completamente segura para novilhas prenhes, em comparação com as vacinas comerciais B. abortus B19 ou B. abortus RB51. O único efeito adverso foi a formação de infiltrado no local da administração subcutânea; essa reação não foi observada no grupo vacinado pela via conjuntival.

Advances in virus-based therapies for colorectal cancer / 中国肿瘤临床

Virus-based anti-tumor therapies are novel biological treatments. Viral vectors can infect tumors to kill cancers directly (on-colysis), act as cancer vaccines to activate the immune system, and deliver genes with anti-tumor activity to the cancer cells. Genetic engineering has been applied to viruses to achieve more specific and efficient cancer treatment. Simultaneously, a reasonable combi-nation of viral vectors and existing anti-tumor therapy can improve the therapeutic effect. Consequently, virus-based therapy is expect-ed to serve as an effective anti-tumor strategy. We reviewed recent studies on the anti-tumor viral therapy of colorectal carcinoma.

Construction of recombinant infectious clones of HCoV-OC43 expressing green fluorescent protein and viral rescue / 中华微生物学和免疫学杂志

Objective To investigate the feasibility of using recombinant infectious clones of hu-man coronavirus OC43 (HCoV-OC43) as a vector for the expression of exogenous genes and to analyze the insertion sites. Methods Based upon pBAC-OC43FL, a full-length cDNA infectious clone of HCoV-OC43, three recombinant expression plasmids ( pBAC-OC43-GFPΔNS2, pBAC-OC43-GFPΔNS12. 9 and pBAC-OC43-N-GFP) were respectively constructed by replacing NS2 and NS12. 9 genes with the reporter gene en-coding the green fluorescent protein ( GFP ) and inserting the reporter gene after the N gene by using the overlapping-PCR and in vitro ligation. Reverse genetics techniques were used for viral rescue. All of the res-cued virus strains were characterized by immunofluorescence assay ( IFA) and Western blot ( WB) assay af-ter transfecting BHK-21 cells with the recombinant viruses. Results Two recombinant viruses, OC43-GFPΔNS2 and OC43-GFPΔNS12. 9, could be successfully rescued by transfection the BHK-21 cells with pBAC-OC43-GFPΔNS2 and pBAC-OC43-GFPΔNS12. 9 plasmids. The expressed GFP was observed in BHK-21 cells transfected with pBAC-OC43-GFPΔNS2 or pBAC-OC43-GFPΔNS12. 9 plasmids, but not in the cells transfected with the pBAC-OC43-N-GFP plasmid. An efficient and stable expression of GFP was observed in the pBAC-OC43-GFPΔNS2 plasmid-transfected cells. The 10th generation of OC43-GFPΔNS2 virus was ob-tained after repeated freezing and thawing. The expression of GFP and N protein were detected in cells infec-ted with the OC43-GFPΔNS2 virus after 10 passages. Conclusion The NS2 gene of HCoV-OC43 could be used as a promising insertion site of the pBAC-OC43 FL infectious clone for the expression of exogenous genes. This study might provide a platform for further researches on the replication of HCoV-OC43 and the development of human coronavirus-based vectors.