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Laboratory of Virology


Sandra Huerfano Meneses, Ph.D.


E-mail: huerfano@natur.cuni.cz
Phone: +420 325 873 914, +420 325 873 955


BIOCEV, Průmyslová 595, Vestec, 252 42, rooms 007, 008, 009, 011, 012, 013, 015
Viničná 5, Prague 2, 128 43 - Nové Město, rooms 004, 004A, 004B, 110

Team Members

        Methodology and Technical Support


        Scientific Collaborations

Proposed Topics of Ph.D., M.Sc. and B.Sc. Theses for New Students

Research Description:

(additional information can be found also here: http://web.natur.cuni.cz/molbio/virology/)

The Laboratory of Virology studies the biology of small non-enveloped DNA viruses. It offers a unique environment for both basic and applied research, with many opportunities for bachelor, undergraduate and graduate students. For many years the Laboratory has studied different molecular aspects of the mouse polyomavirus, type species of the Polyomaviridae family which includes a number of clinically important human pathogens such as BKPyV, JCPyV and Merkel carcinoma cell polyomavirus (MCPyV). The study of mouse polyomavirus which can be easily cultivated may provide the insight into the life cycle of its human counterparts that still lack effective production systems and broaden the knowledge of the cellular antiviral response. The important line of research in our Laboratory is directed at the production of polyomavirus-based nanoparticles (virus-like particles, VLPs) for the basic research as well as for therapeutic and diagnostic purposes. These VLPs have the potential to be developed not only as effective vaccines but also as efficient vehicle systems for the transfer of exogenous genetic information, peptides or other molecules into target cells. The Laboratory is also engaged in research leading to the preparation of a veterinary vaccine against porcine circovirus and bovine papillomaviruses.

The entry of polyomaviruses into host cells and trafficking from the cell membrane towards the cell nucleus has been recently intensively investigated because it can bring many interesting findings of cell endocytosis of extracellular material into cells. It was shown that polyomavirus virions get into the endo-lysosomal compartments and the endoplasmic reticulum, where they are partially disassembled. The mechanism by which the virus is released from these compartments and delivers its viral genome into the nucleus is, however, unclear. In this process minor capsid proteins can play a vital role. We constructed series of viruses mutated in distinct domains of minor proteins in order to analyze consequences for the delivery of their genomes into the cell nucleus. Using electron and confocal microscopy, inhibitors of nuclear import and siRNA technology we are trying to identify critical events associated with the translocation of the viral genome into the nucleus and its subsequent expression and replication. A detailed understanding of these processes is crucial for the successful utilization of polyomavirus-derived vectors for therapeutic and diagnostic purposes.

We are also interested in the characterization of possible cellular interaction partners of structural proteins of polyomaviruses. We examine the role of identified cellular proteins in the virus replication cycle, mainly their role in late phase of infection and virus assembly. A better understanding of the participation of cellular proteins in these processes would help to develop the efficient antiviral treatment of polyomavirus-associated pathologies.

Polyomavirus capsid proteins can spontaneously self-assemble into VLPs when produced in heterologous expression systems. The modification of the major capsid protein VP1 within virions can change the tropism of the virus and the same approach can be applied in principle to VLPs in order to change their binding specificity for target cells. We use several different approaches (genetic and chemical modifications of VLPs) to change the surface of VLPs and achieve the "re-targeting" of particles from the natural viral receptor to the specific cancer cell receptor. As an experimental cancer cell model we use androgen-resistant human prostate tumor cells that express a distinct surface cancer marker, prostate membrane antigen (PSMA). The project also includes the development of technology for the in vitro encapsidation of low molecular weight substances (e.g. MRI contrast agents for the diagnosis) and therapeutic nucleic acids into these modified particles, as well as methods that will lead to their effective release within the target cell. In terms of nanotechnologies VLPs have several unique bio-chemical properties (they are biodegradable and unlike other polymers have a well-defined shape and structure) and can therefore be used as a scaffold for the construction of nano-objects for biomedical applications. In cooperation with the Synthetic Nanochemistry research group of the Institute of Organic Chemistry and Biochemistry (IOCB), Czech Academy of Sciences (Petr Cígler), we use polyomavirus VLPs as templates for the production of nanoplasmonic materials. All these projects are performed in cooperation with other laboratories, particularly with the Biochemistry and Molecular Biology group of the IOCB (Jan Konvalinka), and the Coordination and Bioinorganic Chemistry group of the Department of Inorganic Chemistry of our faculty (Jan Kotek).

PCV2 is a small non-enveloped virus with an ssDNA genome that causes severe disease in pigs - porcine circoviral associated disease. The main antigenic determinant of the virus is a protein Cap which forms the viral capsid. However, the cultivation of PCV2 and heterologous production of Cap protein in the form of VLPs (the preferable structures for the induction of neutralizing antibodies) is very inefficient. We thus use capsid structures of mouse polyomavirus as carriers for Cap protein or its epitopes for vaccine production. In cooperation with the Dyntec company we have prepared a number of constructs with selected immunogenic Cap epitopes located on the surface or inside the VLPs derived from mouse polyomavirus. Alternatively we prepare a fusion between Cap protein and main mouse polyomavirus capsid protein VP1. Although this protein fusion prevents VP1 from assembly to complete VLPs, the formation of viral capsomers is not disrupted. Interestingly, our data show that all of these approaches (VLPs as well as capsomeric structures) lead to the induction of specific immune responses against the Cap protein and may be utilized for the preparation of an efficient and commercially-attractive vaccine against PCV2.

We are also preparing a vaccine against bovine papillomaviruses. The bovine papillomavirus (BPV) induces skin and mucosal lesions or cancers of the bladder in cattle. The disease can have serious economic consequences on milking cows and suckling calves because cows with papillomas on udder teats cannot be milked (infection may also be accompanied by inflammation of the udder) and infected calves with lesions in the oral cavity cannot suck. In addition, these viruses can be transmitted to horses. So far, the only available vaccine is based on the inactivated virus prepared from organ extracts of infected animals. Therefore we are preparing a preventive vaccine based on VLPs composed from BPV L1 and L2 capsid proteins with the use of the baculovirus expression system.

Human polyomaviruses BKPyV and MCPyV cause latent infection in most people. In immunocompromised patients, however, they can induce severe illness with a serious impact on the treatment. MCPyV was identified as an etiologic agent in Merkel cell carcinoma and BKPyV causes kidney transplant recipients nephropathy, accompanied by the risk of acute transplant rejection and (in recipients of hematopoietic stem cell transplant) also hemorrhagic cystitis. In infected persons, antibodies are directed mainly against the capsid protein VP1. On the basis of polymorphisms in the VP1 protein comprising the receptor binding region, four subtypes of BKPyV can be distinguished. It has been shown that people with high levels of antibodies against the subtype BKPyV-I are not able to neutralize an infection with the BKPyV-IV subtype. This may result in serious health consequences after transplantations when the BKPyV subtype between the recipient and the donor differs. Species-specific antigens based on recombinant VP1 were previously prepared and used only experimentally for limited serological studies. In cooperation with the VIDIA company, we are developing a multiplex diagnostic confirmation LIA (immuno-line) test based on the recombinant VLP technology to simultaneously detect subtype-specific antibodies against BKPyV and against MCPyV and JCPyV, for clinical use.

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