Ljungan Virus Replication in Cell Culture
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(2) Organisation. Document name. DOCTORAL DISSERTATION University of Kalmar School of Pure and Applied Natural Sciences SE-391 82 Kalmar, Sweden. Date of issue. 2007-03-30 Sponsoring organisation. Author. University of Kalmar, Sweden. Jens-Ola Ekström Title and subtitle. Ljungan Virus Replication in Cell Culture Abstract. Ljungan virus (LV) is a recently identified picornavirus of the genus Parechovirus. LV has been isolated from voles trapped in Sweden and also in the United States. LV infected small rodents may suffer from diabetes type 1 and type 2 like symptoms, myocarditis and encephalitis. LV has been proposed as a human pathogen, with indications of causing diabetes type 1, myocarditis and intrauterine fetal deaths. In this thesis, cell culture adapted LV strains were utilised for development and adaptation of several basic methodological protocols to study the LV biology, e.g. real time PCR, highly specific antibodies and a reverse genetics system. These methods allowed detailed studies of this virus and how it interacts with the host cell. The genomic 5’-end was identified and modelling showed unique secondary structure folding of this region. The LV encodes an aphthovirus-like 2A protein with a DvExNPGP motif. This motif was found to mediate primary cleavage of the LV polyprotein in vitro and is proposed to constitute the carboxy terminus of the structural protein VP1 in LV. Rabbit polyclonal antibodies generated against recombinant structural proteins were used to verify that the LV virion is composed of the structural proteins VP0, VP1 and VP3. Cell culture studies showed that LV replicates to low titer with an absent or delayed cell lysis. LV is proposed to be able to spread by a, for picornaviruses, not previously demonstrated direct cell-to-cell transmission. All results taken together suggest a maintenance strategy of LV including low amounts of the LV genome and persistently infected hosts. Stability studies showed that the LV virion not only maintain activity in acidic and alkaline environments but also exhibit resistance to the commonly used disinfectant Virkon®. The results presented in this thesis show that LV has several unique properties, not previously observed for a picornavirus. Key words. Ljungan virus, picornavirus, parechovirus, 5’-RACE, 5’-end, infectious cDNA clone, real time PCR, virus purification, virion stability, disinfection, aphthovirus-like 2A. Classification system and/or index terms (if any) Supplementary bibliographical information. Language English. ISSN and key title 1650-2779. ISBN 978-91-89584-70-9. Recipient’s notes. Number of pages 156. Price. Security classification Distribution by: Jens-Ola Ekström, School of Pure and Applied Natural Sciences, University of Kalmar, Kalmar, Sweden. I, the undersigned, being the copyright owner of the abstract of the above-mentioned dissertation, hereby grant to all reference sources to publish and disseminate the abstract of the above-mentioned dissertation. Signature. Date. March 16, 2007.
(3) ȱȱȱȱ ȱȱãȱȱǰȱ ȱȱȱ¢ȱ§ǰȱ §ȱȱȈǷȈȱǻ
(4) ȱȱȱǼȱȱ ȱȈȱǯǯǯȈȱ ȱ ȱȱ.
(5) ȱ ¢ȱȚȱ
(6) ȬȱãǰȱŘŖŖŝȱ ȱ¢DZȱȱǰȱ¢ȱȱ ǰȱ ȱ ȱ DZȱŗŜśŖȬŘŝŝşȱ DZȱşŝŞȬşŗȬŞşśŞŚȬŝŖȬşȱ.
(7) ȱȱȱ ȱ§ȱǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯȱ ȱȱ ǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯ ȱ ǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯ ȱ ŗǯȱȱǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯŗȱ ŗǯŗǯȱȱȱȱȱǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯŗȱ ŗǯŘǯȱȱȱȱȱǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯŚȱ ŗǯřǯȱȱȱȱ¢ǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯşȱ Řǯȱȱ ǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯŗřȱ Řǯŗǯȱ ȱȱȱȱȱ ǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯŗřȱ ŘǯŘǯȱ ȱ ǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯŗśȱ Řǯřǯȱ ȱȱȱȱȱȱȱ ǯǯǯǯǯǯǯǯǯǯǯŗŜȱ ŘǯŚǯȱȱȱ¢ȱȱ ǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯŗŞȱ ŘǯśǯȱȱȱśȂȬȱȱ ǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯŘŚȱ ŘǯŜǯȱȱȱȱȱ ǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯŘşȱ Řǯŝǯȱȱ ȱ ǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯřŗȱ ŘǯŞǯȱȱ ǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯřśȱ ŘǯşǯȱȱȱŘŗȱȱǯ ǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯŚŖȱ řǯȱȱǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯŚŚȱ ǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯŚŜȱ ȱ ǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯǯŚŝȱ. ȱ.
(8) IV.
(9) ȱ§ȱȱ ȱ ȱ ªȱ ȱ ȱ ȱ ȱ ŗşşŖȬǯȱ ȱ ȱ ȱȱ ȱȱȱŗŜȱȱȱȱȱȱȱ §ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱȱȱȱȱ§ȱȱ§ǯȱȱȱªȱȱ ȱȱȱ§ȱȱȱ¢ȱȱȱȱȱ §ȱ ȱ ȱ ¢ȱ ŗȱ ȱ §ǯȱ ȱ ȱ ȱȱȱãȱȱȱãȱȱȱȱȱȱȱ ǯȱ ȱ §ȱ ȱ §ȱ ȱ ȱ ȱ ȱ ȱ ªȱ ¢ȱŗȱ ȱ ¢ȱŘȱ ǰȱ §ǰȱ ȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ȱ ǯȱ ȱ §ȱ ȱ ȱ ȱȱȱȱ§ǯȱȱȱãȱȱ §ȱ ȱ ããȱ §ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ŗȱ ȱ ȱ ȱ ȱ ȱ ªȱ ȱ ȱ ȱ ȱ ãȱ ȱ ªȱ ȱ ȱ ȱ ãǯȱȱ ȱ ãȱ ǯȱ ȱ ȱ ȱ ȱ §§ȱȱ§ȱȱ¡ȱǰȱȬȱȱãǰȱ ȱȱã¢ȱȱȱȱȬǯȱȱ§ȱ ȱ ȱ ȱ §ȱ §ȱ ȱ ȱ ã§ȱ ȱ §ǯȱ ȱ ȱ ȱ ȱ §ȱ ȱ ȱ ¢ǰȱ ȱ§ȱ §ȱ ȱ㢢ǰȱ ȱȱ ȱȱ ȱ ȱȱȱȱȱȱ§ȱãȱªǯȱȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ªȱ ȱ ȱ §ȱ ȱ ȱ ªȱ ȱ ǯȱ ¢ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ŗŖȱ ¢ȱ ȱ ȱ ȱ ȱ ȱǯȱȱ§ȱȱȱȱȱãȱȱªãȱȱ¢ȱ ǰȱ ȱ ȱ §ãȱ ȱ ȱ ȱ §ȱ ȱ ŗŖŖȬŗŖŖŖȱ ȱ ȱ ȱ ¡ȱ ȱ ȱ ǯȱ ȱ §ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱȱȱȱǯȱȱ§ȱȱ§ȱȱȱȱ ȱ ȱ ãȱ ªȱ ȱ ǯȱ ȱ ¢ȱ ȱ.
(10) ȱ ȱ ȱ ȱ ªȱ ȱ ȱ ȱ ȱ ȱȱȱªȱ§ȱȱȱȱ§ǯȱȱªȱ§ȱ ȱȱãȱȱȱȱȱªȱȱ ǯȱȱ ȱ§§ȱȱ§ȱȱȱȱȱãȱȱ ȱ țǯȱ ȱ ȱ §ȱ ȱ ªȱ ȱ ãȱ ȱ ȱ ǰȱ ȱ ȱ ȱ ªǯȱ ȱ ȱ ªȱ țȱ ȱ ȱ ȱ ª§ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ §ȱ ȱ ȱ ǯȱ. ȱȱ§ȱȱȱȱȱ§ȱȱȱȱ ȱ ȱ ȱ §ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ãȱ ªȱ ªȱ ȱ ǯȱ ȱ ȱ §ȱ ȱ ȱ ȱ ȱ ȱ ȱ ãǰȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱȱ㢢ǯȱȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ǯȱ ȱ ȱ §ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ãȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ §ǰȱ ȱ ããȱ ȱ ȱ ȱ ȱ §ȱ ȱ ȱ ȱ ȱ ǯȱ ȱ ȱ ªȱ ȱ ȱ §ȱ ȱ ȱ ȱ ȱ ȱ ªȱ ãȱ §ǯȱ. VI.
(11) ȱȱȱ ȱȱȱȱȱȱ ȱȱȱȱȱ ȱ ȱȱȱ¢ȱȱȱDZȱ ǯȱ. ǯȱȱ
(12) ǰȱ
(13) Ȭȱãǰȱȱǯȱǰȱ ȱǰȱ ȱ ¢¢§ǰȱ ȱ ȱ ȱ ǯȱ ȱ ǯȱ ǻŘŖŖŚǼǯȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ¢ȱ ǯȱ ȱ ȱ ¢ȱ ȱ ȱ řŗŝǻŚǼDZŗŖŘřȬŗŖŘşǯȱ. ǯȱ
(14) Ȭȱãǰȱ¢ȱǰȱȱǰȱǯȱȱ
(15) ǰȱ ȱ ǰȱ ȱ ǰȱ Ȭǯȱ ȱ ȱ ǯȱ ȱ ǯȱǻŘŖŖŝǼǯȱȱȱȱȱȱȱDZȱȱ ȱȱȬȱȱǯȱǯȱ ǯȱ
(16) Ȭȱ ãǰȱ ¢ȱ ǰȱ Ȭǯȱ ȱ ȱ ǯȱȱ ǯȱ ǻŘŖŖŝǼǯȱ ¢ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ DZȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ȱ ǰȱ ȱ ȱ Ȭ¢ȱ ȱ ȱ ȱ țȱȱǯȱǯȱ ǯȱ
(17) Ȭȱ ãǰȱ ¢ȱ ǰȱ Ȭǯȱ ȱ ȱ ǯȱ ȱ ǯȱǻŘŖŖŝǼǯȱȱȱȱȱȱȱȱŗȱȱ ȱȱǯȱǯȱ ǯȱ ¢ȱ ǰȱ
(18) Ȭȱ ãǰȱ ȱ ǰȱ ȱ ǰȱ ȱ ǰȱ
(19) Ȭ#ȱ ǰȱ Ȭǯȱ ȱ ȱ ǯȱȱ ǯȱ ǻŘŖŖŝǼǯȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱȱȱȱǯȱǯȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǯȱȱ ȱ ȱȱȱȱȱȱDZȱȱ ȱ. ȱ ǰȱ
(20) Ȭȱ ãǰȱ ȱ ǰȱ ǯȱ ȱ ǯȱ ǻŘŖŖśǼǯȱ ¢¢ȱ ȱ ȱ ¡ȱ Řȱ ȱ Ȭ ȱ¢ȱǯȱȱȱŗŗřǻŘǼDZŗŖŝȬŗŗśǯ ȱ.
(21) ȱ ŞŝȬŖŗŘȱ ŞŝȬŖŗŘ ȱ Ȭȱ ȱ ȱ ȱ ȱ Ȭŗȱ ȱ ȱ ȱ ȱ ȱ ȱ. ȱ. ȱ ȱ Ȭȱ ȱ ȱ Ȭȱ ȱ ȱ ȱ ǯǯȱ ȱ ȱ ȱ Ȭ ȱȱ ȱ ȱ ȱ ȱ. VIII. ȱȱ¢ȱȱŞŝȬŖŗŘȱ ȱȱȱȱŞŝȬŖŗŘȱ ¡¢ȱȱ ¢ȱȱ ȱȱ ȂȱȱȂȱȱ ȱȱȱ ȱȱȱ¢ȱŗȱ £¢Ȭȱȱ¢ȱ ȱ ¢ȱȱ ȱ ȬȬȱȱȱ ȱ¢ȱ¢ȱȱ ȱȬȱ ȱȱȱ ȱȱ ȱȱ¢ȱȱ ȱȱ ȱȱ ¢ȱȱȱ ȱȱ ȱȱȱ ȱ ȱȱȱ ȱȱ ȱȱȱȱȱ ȱȱ ȱ¢ȱȱ ȱ¢ȱȬ¢¢ȱȱ ȱ Ȭȱ Ȃȱȱ¢ȱȱ ȱȱ.
(22) ŗǯȱȱȱ ŗǯŗǯȱȱȱȱȱȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǯȱȱȱȱȱŗşȱ¢ǰȱȱ ȱȱȱȱȱǰȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ǻ ȱ ȱ ȱ ȱ ǰȱ ŘŖŖŝǼǯȱ ȱ ŗŞşŞǰȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǰȱ ȱ ȱ ȬȬ ȱ ǯȱ ȱ ŗşŖŖȱ ȱ ŗşřŖȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ £ǰȱ ȱ ȱ ȱ ȱ ȱ £ǰȱȱȱȱȱ¢ȱȱȱȱȱ ǻȱȱǰȱŘŖŖŝǼǯȱȱǰȱȱǰȱ ȱȱȱ ȱȱȱŗşŖŞȱǻ ȱȱǰȱŘŖŖŝǼǯȱ ȱȱȱ ¢ȱ ȱȱ ȱȱȱǯȱȱŗşŚŞȱȱȱ¢ȱ ȱ ȱ ȱ ǰȱ ȱ ȱ ȱ ǯȱ ȱ ŗşŚşȱ ȱ ȱ ȱȱȱȱȬȱȱȱȱ ȱ ¢ȱȱǯȱ ȱŗşśŘȱȱȱȱ ȱǰȱȱȱȱȱȱȱȱ ǻ ȱ ȱ ȱ ȱ ǰȱ ŘŖŖŝǼǯȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱȱȱǯȱ ȱȱȱȱȱ¡¢ȱŘŝȬřŖȱǯȱȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¡¢ȱ ŞȱŖŖŖȱ ȱǻǼȱȱ¢ȱȱȱǯȱȱȱȱȱ ȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ǻ ȱȱǰȱŘŖŖŝǼǯȱ ȱȱȱ ȱȱȱ¢ǰȱ ǯǯȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ǯȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ȱȱǻǼǯȱȱȱȱȱ¡ȱ¢ȱ ȱ ¢ȱ ȱ ¢ȱ ȱ ¢ȱ ǯȱ ȱ śȂȬȱ ȱ. ȱ. ȱ. ȱ. ¢ȱ. ȱ. ǻ Ǽǰȱ. ȱ. Ȭȱȱȱȱȱǯȱȱȱ¢ȱ. ŗȱ.
(23) ȱ ¢ȱ ȱ ȱ ȱ ȱ ¢ȱ ¢ȱ ȱ ȱ ǻȱ ŗȱ ȱ ȱ ȱ ǰȱ ŘŖŖŝǼǯȱ ȱ ȱ ȱȱ ȱȱȱŘǰȱřǰȱřȱȱřȱȱȱȱȱ ȱ ǻȱ ȱ £ǰȱ ŗşşśǰȱ 㢢ȱ ȱ ǯǰȱ ŗşşŜǼǯȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǻȬǼȱ ȱ ȱ ǯȱ ȱȬȱȱŘȱȱȱȱȱȱȱȱ ȱ ¢ȱȱȱȱȱȱȱǰȱȱ ȱ¡ȱ ȱȱȱȱȱȱȱ ȱȱȱȱ ȱ ȱ ǯȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱȱ¢ȱȱ ȱȱȱȱȱȱŗǯŘȱȱŗǯřǯȱ. Structural proteins 5’-UTR. L VP4 VP2. VP3. VP1. Non-structural proteins 2A. 2B. 2C. 3A. 3B 3C. 3D. 3’-UTR. Figure 1. Schematic overview of the picornavirus genome.. ȱ ȱ ȱ ¢ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ǰȱ¢ȱȱȱȱȱDZȱȱǻȱ Ǽǰȱ ȱ ǻȱ ȱ ȱ ȱ ȱ Ǽǰȱ ȱ ǻȱ ȬȬȱ ȱ Ǽǰȱ ǰȱ ȱ ǻȱ ȱ ȱ Ǽǰȱ ǰȱ ǰȱ ȱ ȱ ȱ ǻȱ ȱ Řȱ ȱ ¢ȱ ȱ ǯǰȱ ŘŖŖśǼǯȱ ȱ ȱ ȱ ȱ ȱ ȱ¢ȱǰȱǯǯȱȱȱȱȱȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǰȱ ȱ ǻ ȱȱǯǰȱŘŖŖŜǼǯȱȱȱȱȱǰȱȱȱȱǻǼǰȱ ȱȱȱ ȱȱȱȱȱDzȱȱȱȱ ȱ ȱ ȱ ǻ Ǽǯȱ ¢ȱ ¢ȱ ȱ ȱ ¢ȱȱȱȱȱȱȱȱȱ ǰȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ŗȱ ǻ ȬŗǼǰȱ ȱ ȱ ȱȱȱȱȱȱǻȱŘǰȱ
(24) ȱȱǯǰȱ. 2.
(25) ŘŖŖŘǰȱ ǰȱŘŖŖŚǼǯȱȱȱȱ Ȭŗȱ ȱ¢ȱȱǻ ȱ ȱǯǰȱŘŖŖŜǰȱȱȱǯǰȱŘŖŖŝǼȱȱȱ¡ȱȱȱȱȱȱ ¢ȱȱȱǯȱȱ ȱ. HPeV1H 100. 100. HPeV2C HPeV2W 100. Parechovirus Ljungan virus. LV145SL 100 LV174F 100. DHV-1. Human parechovirus. DRL-62. LV87-012 M1146 HAV. 100. Hepatovirus. AEV HRV2. 100. Enterovirus. PV1S. 93. A-2 99. Kobuvirus. AichiV. Erbovirus. ERBV 78. EMCV. 100. 93. Cardiovirus. TMEV. 99. FMDV. 100. Aphthovirus. ERAV PorcENTV1. Teschovirus. SBV InFV 0.2. Figure 2. Phylogenetic relationship of picornaviruses. Relationships were inferred by employing the maximum-likelihood method (JTT substitution model) for amino acid sequence of the 3Dpol protein. The 3Dpol tree was rooted using corresponding sequences of two picorna-like insect viruses, infectious flacherie (InFV) and sacbrood viruses (SBV). Bootstrap values above 70% are indicated for 1 000 pseudo replicates. Courtesy of Min-Chul Kim, National Veterinary Research and Quarantine Service, Republic of Korea and A. Michael Lindberg, University of Kalmar, Sweden.. řȱ.
(26) ŗǯŘǯȱȱȱȱȱȱȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ǰȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱȱǯȱȱ Ȭȱ ȱ ȱ ȱ ǰȱ ǰȱ ǰȱ ȱ ȱ ȱǰȱȱȱ¢ȱȱȱǰȱȱȱ Ȭȱ ǻ ¢ȱ ȱ ǯǰȱ ŘŖŖśǰȱ ȱ ȱ ǯǰȱ ŘŖŖŜǼǯȱ ȱ ¢ȱ ¢ȱ ȱ Ȭȱ ȱȱ¢ȱ ȱȱȱȱȱȱ ȱ ȱ Ȭǰȱ ȱ Ȭȱ ȱ ȱ ¡ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ȱ Řȱ ȱ ǻ ȱ ȱ ǯǰȱŗşşŚǰȱ ȱȱǯǰȱŗşşśǰȱ ȱȱǯǰȱŗşşŞǼǯȱ¢ȱȱ ȱȱ ȱȱȱȱȱȱȬǯȱȱ Ȭȱȱ¢ȱȱȱǻ¢ȱȱǯǰȱŘŖŖŜǼǯȱ ȱ ȱ Ȭȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱǻȱȱǯǰȱŘŖŖřǼǯȱȱȱȱȱȱȱȱ Ȭȱȱȱřȱȱřȱȱǻ ȱȱǯǰȱŘŖŖśǼǯȱȱ ȱȱŖȬŚȱ ȱ ȱ ȱ Śȱ ȱ Řȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǰȱ ȱ Ŗȱ ǯȱ ȱ ȱ ȱ ȱ Śȱ ȱ Řȱ ȱȱȱ¢ȱȱȱȱȱǻ ȱȱ ǰȱ ŘŖŖŝǼǯȱ ȱ ȱ ȱ ȱ ȱ ǻȱ Ǽȱ ȱ ȱ ǰȱ ȱ Ȭŗǰȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǻ ¢ȱ ȱ ǯǰȱ ŗşşŚǰȱ ȱ ȱ ǯǰȱ ŗşşŞǰȱ
(27) ȱ ȱ ǯǰȱ ŘŖŖŘǰȱ ȱ ȱ ǯǰȱ ŘŖŖŜǼǯȱ ȱ ȬȱȱŖȦŚȱȱ ȱȱȱ¢¢ȱȱȱȱȱ ǰȱ ǰȱ ȱ ȱ ȱ ǰȱ ȱ ȱ ȱȱȱȱȱȱ¢ȱǻ ȱȱǯǰȱŗşŞŝǰȱȱȱ ǯǰȱ ŗşşŖǰȱ ȱ ȱ ǯǰȱ ŗşşŘǼǯȱ ȱ ǰȱ ȱ ȱ ȱ ȱ ȱ ǻ Ǽȱ ȱ ¢¢ȱ ȱ ŖȦŚǰȱ ȱ ¢ȱ ȱ ǰȱ ȱ ¢ȱ. 4.
(28) ǰȱȬȱȱǻȱȱǯǰȱŗşşřǰȱ ¢ȱȱ ǯǰȱŗşşŚǼǯȱ ȱ ŗǰȱ Řȱ ȱ řǰȱ ȱ Ŗȱ ȱ ǰȱ ȱ ȱ ȬŗǰȱȱȱȱȬȱȬȱȱȱȱǻȱ řǰȱ ȱȱǯǰȱŗşŞśǰȱǰȱŘŖŖŝǼǯȱȱȱȱȱȱȱȱ ȱ ȱ ȱ ȱ ǰȱ ȱ ǰȱ ȱ ȱ ȱ ǯȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǻȱ řȱ ȱ ȱ ȱ ǯǰȱ ŗşŞśǼǯȱ ȱ ȱ ȱȱȱȱȱȱȱȱȱȱȱȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǻ
(29) ȱ ȱ ǰȱ ŗşŜŞǰȱ ǰȱ ŗşŞŘǰȱ ȱ ȱ ǯǰȱ ŗşŞŜǰȱ ȱ ȱ ǯǰȱ ŗşşŖǰȱ ¢ȱ ȱ ǯǰȱ ŗşşŝǼǯȱ ȱ ȱ ȱ ȱ. ȱ ȱ Ȃȱ ȱ ¢ȱ ȱ ǻǼȱ ȱ ȱȱȱ¢ȱȱǻȱȱǰȱŗşşŝǰȱ ȱȱȱȱǰȱȱŘŖŖřǼǰȱȱȱȱȱȱȱȱȱȱȱȱ. (A). (B). VP1 Figure only permitted in the printed version. VP3 VP2. Science. Figure 3. Structure of the picornavirus. (A) Structural proteins VP1 through VP3 folds into an anti-parallel beta-fold motif, a folding motif present in structural proteins of several virus families. (B) 60 copies of each structural protein form the virion in an icosahedral shape. (A) is reprinted, with permission, from Hogle et al. (1985), Science 229:1358-1365. ©1985 AAAS.. śȱ.
(30) ȱ¢ȱȱȱȱȱǯȱȱȱ ȱȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ǯȱ ȱȱȱȱȱȱȬȬȱȱȱǻǼȱ ȱȱȱȱȱȱȱȱȱȱ¢ȱ ǻ¢ȱȱǯǰȱŗşşśǼǯȱȱȱȱȱ¢ȱȱȱȱ ȱ ȱ ȱ ŗȱ ǯȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¡ȱ ȱ ȱ ¢ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ¢ȱȱ¢ȱȱȱȱȱȱǻ ȱȱ¢ȱȱǯǰȱ ŘŖŖřǼǯȱ Řȱ ȱŘȱȱȱȱȱȱȱȱȱ¢ȱ ȱ¢ȱȱȱȱǻ ȱȱ ¢ǰȱŘŖŖŖǼǯȱȱ Ȭ ȱ Řȱ ȱ ȱ ȱ ȱ Řǰȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ řȱ ȱ ǻ ¢ȱ ȱ ǰȱ ŗşşřǼǯȱ ȱ ȱ Řȱ ȱ ȱ ȱ ¢¢Ȭȱ ¢ȱ ǰȱ ȱȱ¢ȱȱȱȱȱ¢ȱ ȱ ȱ Ȭȱ ȱ ŗȱ ȱ ȱ Ȭȱ ȱ ȱ Řȱ ȱ ȱ ǻ ¢ȱ ȱ ǰȱ ŗşşřǰȱ ȱ ȱ ǯǰȱ ŗşşşǼǯȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ Ȭȱ ǻȱ ȱ ǯǰȱ ŗşşŗǰȱ ȱȱǯǰȱŗşşşǼǯȱ ȱȱ¢ȱȱŘȱȱȱȱ¡ ȱȱȱ¡ȱ ȱȱȁȬȂȱ¢ȱ¢ȱȱȁȱȬȱȂȱ ȱ ǰȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱȱȱ ǻ¢ȱȱǯǰȱŗşşşǼǯȱȱȱȱȱǰȱȱȱ. ǰȱ ȱ ȱ ȱ ȱ ǻ
(31) ȱ ȱ ǯǰȱ ŘŖŖŘǼǯȱ ȱ ȱ ȱȱȱȱȱȱȱȱŘǯşǯȱȱ¡ ȱȱȱ ȱ ȱȱ ȱȱ ȱȱȱȱDZȱ ȱȱȱ ȱȱǰȱǰȱǰȱȱ ǰȱȱȱȱ¢ȱȱȱǰȱȱ Ȭŗȱ ǻ¢ȱ ȱ ǯǰȱ ŗşşşǰȱ ȱ ȱ ¢ǰȱ ŘŖŖŖǰȱ
(32) ȱ ȱ ǯǰȱ ŘŖŖŘǰȱ £ȱȱǯǰȱŘŖŖŘǰȱ ȱȱǯǰȱŘŖŖŜǰȱ ȱȱǯǰȱŘŖŖŜǼǯȱȱ. 6.
(33) ȱ Ȭ¡Ȧȱ ȱ Řȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ȱ ǻȱ Ǽǰȱ ǰȱ ȱ ¢ȱ ȱ ¢ȱȱȱȱ ȱȱǻ ȱȱ ¢ǰȱ ŘŖŖŖǰȱ
(34) ȱȱǯǰȱŘŖŖŘǼǯȱȱŘȱȱȱȱȱ ȱȱ ȬŗŖŝȱȱ¢ǰȱ ȱȱȱȱȱȱȱȱ ȱ ǻȱ ȱ ǯǰȱ ŗşşŝǰȱ ȱ ȱ ǯǰȱ ŗşşŞǰȱ ȱ ȱ ¢ǰȱ ŘŖŖŖǼǯȱ ȱ Ȭŗȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǰȱ ¢ȱ ȱ ȱ řȂȬȱ ȱ ȱ ȱ ȱ ȱȱȱȱȱȱȱȱ ȱȱǻȱȱǯǰȱ ŘŖŖŚǼǯȱ ȱ ȱ Řȱ ȱ ȱ ȱ ȱ ȱ ȱ Řȱ ȱ ǻ ȱȱ ¢ǰȱŘŖŖŖǼǯȱȱȱȱȱ ȱŘȱȱȱȱȱ ȱ ȱ ȱ ȱ ȱ ȱ ŗȱ ǰȱ ȱ ȱ ȱ ¢ȱ ǻȱ ȱ ǯǰȱ ŗşşşǼǰȱ ȱ ȱ ȱ ¢ȱ ȱȱŗȬŘȱȱȱŗȱǻȱȱǰȱŗşşŖǰȱ ȱ ȱ ǰȱ ŗşşřǰȱ ȱ ȱ ǰȱ ŗşşřǼǯȱ ȱ ȱ ¢ǰȱ ȱ ȱ ŗȬŘȱ ȱ ȱ ȱ ȱ ȱ ŗȱ ȱ Řȱ ȱ ¢ȱ řȱ ȱ ȱ ȱ ǰȱ ¢ȱ ȱ ȱ ȱ ȱ ǻ£ȱȱǯǰȱŗşşśǰȱȱȱǯǰȱŗşşşǰȱ ȱȱǯǰȱŘŖŖřǼǯȱȱ ȱ Řȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ Ȭȱ ȱǻȱȱǯǰȱŘŖŖŖǼǯȱ ȱ Ȭŗȱȱȱ¢ȱȱŘȱȱȱȱȱǻȱȱ ǯǰȱŘŖŖŝǼǯȱ ȱȱ ȱȱȱ ȱ ŗȬȱǰȱȱȱ ȱȱȱȱȱȱȱȱǻȱȱǯǰȱŘŖŖŗǰȱȱ ȱǯǰȱŘŖŖŝǼǯȱ ȱȱ¢ȱȱŘȱȱȱȱ¢ȱȱǯȱȱ ȱ ȱ Řȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¡ ȱ ȱ ¢ǰȱ ȱ ȱ ȱ ¡¢ȱ ŗŖŖȱ ȱ ȱ ȱ ¡ȱ ȱȱȱ¡ ȱȱǯȱȱ¡ȱȱ ȱ ȱȱȱȱȱȱȱȱȱȱȱŘȱȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǰȱ ȱ. ŝȱ.
(35) ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǻȱ ȱ ǯǰȱ ŗşşŝǼǯȱȱ ŘȬřȱ ȱ ȱ Řȱ ȱ řȱ ȱ ȱ ȱ ȱ ȱ ȱȱȱǯȱȱȱȱȱȱȱȱ ȱȱȱȱ¡ȱȱȱȱȱȱ ȱ ȱ ȱ ¢ǰȱ ȱ ȱ ȱ ǯȱ ȱȱ¡ȱȱ¢ȱȱȱȱȱ ȱȱ ȱ ȱȱǻ£ȱȱǯǰȱ ŗşŞřǰȱȱȱǯǰȱŗşşŜǰȱ ǰȱ ŘŖŖŝǼǯȱ ȱȱ ǰȱ Ȧȱȱȱ ȱ¢ǰȱ ȱȱ¢ȱȱȱȱ¢ȱȱȱŘǰȱŘǰȱȱȱŘǰȱřȱ ȱ řȱ ȱ ȱ ȱ ȱ ǻȱ ȱ ǯǰȱ ŗşşŚǰȱ ȱ ȱ ǰȱ ŗşşśǰȱȱȱǯǰȱŗşşŜǰȱȱ ȱȱǯǰȱŗşşŝǼǯȱȱ ȱ Řȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ Ȭȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǻȱ ȱ ǰȱ ŗşşśǰȱ ȱ ȱ ǯǰȱ ŗşşŜǼǯȱ Řȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ȱŗȱȱ ȱȱȱ ȱȱȱȱ¡ȱǻ£ȱ ȱǯǰȱŗşşŖǰȱȱȱǰȱŗşşśǼǯȱŘȱȱȱǰȱȱȱ ȱǰȱ¡ȱȱ¢ȱȱȱȱȱ¢ȱȱȱȱ ȱ ¢ȱ ǻ ¢ȱ ȱ ǯǰȱ ŗşŞşǰȱ ȱ ȱ ǰȱ ŗşşŖǰȱ £ȱ ȱ ǰȱ ŗşşřǰȱ ȱ ȱ ǯǰȱ ŗşşŝǰȱ ȱ ȱ ǯǰȱ ŗşşŝǰȱ ȱȱǯǰȱŘŖŖŖǼǯȱřȱȱřȱ¢ȱǻȱȱǯǰȱŗşşŚǼȱȱřȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ȱ ǯȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǻ ȱ ȱ ǰȱ ŘŖŖŝǼǯȱ řǰȱ ȱ ȱ ȱ řǰȱ ȱ ȱ ȱ ȱ ȱ ǻřǼǯȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ¡ȱ ȱ ȱ ȱ ȱ Ŗȱ ǻȱ Řȱ ȱ ŚǼǰȱ ȱ ȱ ȱ ǰȱ ¢ȱ ȱ ȱ ȱ ¢ȱȱȱŘȱȱǻ ȱȱǰȱŘŖŖŝǼǯȱřȱȱȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǻȱ ȱ ǰȱ. 8.
(36) ŗşşŖǰȱ ȱ ȱ ǯǰȱ ŗşşŘǰȱ ȱ ȱ Ǽǯȱ řȱ ȱ ȱ ȱ ȱȱ¢ǰȱȱȱȱȱȱȱ ȱ ȱ ¢ȱ ǻ ȱ ȱ ǰȱ ŘŖŖŝǼǯȱ ȱ ȱ ȱ¡ȱ ȱ Ȭŗȱ ȱȱȱȱȱ Ȭȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǻ £ȱ ȱ ǯǰȱ ŘŖŖŘǰȱȱȱǯǰȱŘŖŖŜǰȱ ȱȱǯǰȱŘŖŖŝǼǯȱȱȱȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¡ǯȱ ȱ ŗǯřǯȱȱȱȱ¢ȱȱ ȱ ȱ ȱ ȱ ¢ȱ ȱ Ȭȱ ¢ǰȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱȱȱȱȱ¡ȱȱȱȱȱȱȱȱȱ ȱ ǻ ȱ ȱ ǰȱ ŘŖŖŝǼǯȱ ȱ ǰȱ ȱ ȱ ȱ ȱ Ȭǯȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ȱȱȱȱȱȱȱȱȱȱȱȱȱȱ ȱǯȱ ȱǰȱȱȱȱȱȬ¢ȱ¢ȱȱȱ ȱ ȱ ȱ ȱ ȱ ǰȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱȱ ȱȱȱ ȱȱȱ ȱ ȱȱȱǻǰȱ ŘŖŖŝǼǯȱ ȱ ȱ ǰȱ ȱ ȱ ȱ Ȭȱ ǰȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ǻǰȱ ŘŖŖŝǼǯȱ ǰȱȱȱȬȱȱǰȱȱȱȱ ¢ȱ ȱ ȱ ȱ DZȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǻȱ ȱ ȱ ȱ ǰȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ Ǽȱ ȱ ȱ ȱ ȱ ¢ǯȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ǰȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǯȱ ȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ. şȱ.
(37) ȱȱȱȱȱȱǻȱȱǯǰȱŘŖŖŖǰȱȱȱ ǯǰȱŘŖŖŖǰȱȱȱǯǰȱŘŖŖŗǰȱ ȱȱǯǰȱŘŖŖŘǰȱȱȱǯǰȱŘŖŖŜǼǯȱȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǻȱ ŗśŜȱ ȱ ŗřśǼǰȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ȱ ǻǼǰȱ ȱ ¡ȱ ȱ ȱ ȱ ǻǼǰȱ ǻ ȱ ȱ ȱ ȱ ǰȱ ŘŖŖŘǼǯȱ ȱ ǰȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱȱȱȱȱȱȱȱȱȱȬȱǰȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǻȱ ȱ ǯǰȱ ŗşşŗǰȱ ȱ ȱ ǯǰȱ ŗşşŘǼǯȱ ȱ ȱ ȱ ȱ¡ȱȱȱȬȱȱŗȱȱȱȱȱŚȱ ȱ ǻ ȱ ȱ ǯǰȱ ŗşŞŝǰȱ ȱ ȱ ǰȱ ŗşşŖǼǯȱ Śȱ ȱ ȱ Ȭȱȱŗȱȱ¢ȱ¡ȱȱȱȱȱȱȱ ȱȱȱřŝȱ°ȱǻȱȱǯǰȱŗşşřǰȱȱȱǯǰȱŗşşŚǼȱȱȱȱ Ȭ¢ȱȱȱȱȱǻȱǰȱ ȱȱǯǰȱŗşŞśǼǯȱ ȱȱȱ ȱȱȱȱȱȱȱȱȱȱȱ ǰȱ ȱ ȱ ¡ȱ ¢ȱ ¢ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱȱǯȱȱȱŗȱȬȱȱȱ ȱ ȱ ȱ ȱ ¡ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ŗřśȱ ȱȱȱȱ¢ȱȱȱȱǻȱȱ ǰȱŗşşŖǰȱ ȱ ȱ ǯǰȱ ŘŖŖŗǼǯȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȬȱȱŗȱȦȱȱŚȱȱȱȱȱȱȱȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ȱǻȱȱ ǰȱŗşşŝǰȱȱȱǯǰȱŘŖŖŖǰȱȱȱ ǯǰȱ ŘŖŖŗǰȱ ȱ ȱ ǯǰȱ ŘŖŖŘǰȱ ȱ ȱ ǯǰȱ ŘŖŖřǰȱ ȱ ȱ ǯǰȱ ŘŖŖŚǰȱ ȱȱǯǰȱŘŖŖśǰȱȱȱǯǰȱŘŖŖŜǼǯȱȱ ȱ ǰȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱȱȱȱȱȱȱǻ¡ȱȱǰȱŗşŞŖǰȱȱ ȱǯǰȱŘŖŖŚǼǯȱȱȱȱȱȱȱȱȱ¢ȱ¢ȱȱ ȱ ȱȱȱ¢ȱȱȱȱ ȱȱ ȱ ȱ ȱ ȱ ǻ¡ȱ ȱ ǰȱ ŗşŞŖǼǯȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ. 10.
(38) ȱȱȱ¢ȱȱȱȱȱȱȱǻȇȱȱ ǯǰȱŘŖŖśǼǯȱȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ȱ ȱȱȱȱȱȱȱȱ¢ǯȱȱȱȱ ȱȱȱȱ¢ȱȱȱȱȱȱȱȱ ȱ ¢ǯȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ȱȱȱȱȱȱǰȱȱȱȱ ȱ¢ȱ¢ȱȱ¢ȱȱŘȱȱȱȬȱǻ¢ȱ ȱǯǰȱŗşŞŞǰȱ ȱȱǯǰȱŗşşŜǰȱ ȱȱǯǰȱŗşşŞǰȱȱȱǯǰȱŗşşşǰȱȱ ȱ ǯǰȱ ŘŖŖŖǼǯȱ ǰȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǻȱ ȱ ǯǰȱ ŗşŜśǰȱ
(39) ǰȱ ŗşŝŚǰȱ £ȱ ȱ ǯǰȱ ŗşŞřǰȱ ȱ ȱ ǯǰȱ ŘŖŖŖǰȱ ȱ ȱ £ǰȱ ŘŖŖŘǰȱ ȱ ȱ ǯǰȱ ŘŖŖŚǼǯȱȱȱȱȱȱȱȱȱȱǰȱȱ ȱ Ȭȱ ȱ ȱ ȱ ¡ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǻ ȱ ȱ ǰȱ ŘŖŖŝǼǯȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱǻȱȱǰȱȱȱǯǰȱŗşşŜǰȱȱ ȱȱǯǰȱ ŗşşŝǼǯȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ¢ȱ ȱ ǰȱ ȱ ¢ȱ ȱ ȱ ȱ ȱȱȱȱȱȱȱȱǻȱȱǯǰȱŗşşŜǰȱ¢ȱ ȱ ǯǰȱ ŘŖŖŖǼǯȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǻ ȱ ȱ ȱ ȱ ¢ǰȱ ŘŖŖŚǼȱ ȱ ȱ ¢ȱ ¢ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǯȱ ȱ ȱ ȱ ȱ ¡ǰȱ ȱ ȱ ǻƸȱǼȱ ȱ ȱ ȱ ȱ ȱ ȱ ǻȬȱǼȱ ǯȱ ǰȱ ȱ ǰȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǻȱŚȱȱ ȱȱǰȱŘŖŖŝǼǯȱȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ǰȱ ȱ ȱ ȱ ȱ ȱ ǰȱ ȱ ȱ ȱ ȱ ȱ ǰȱ ȱ ȱ ȱ ȱ ȱ ǰȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ȱǻǼǯȱȱǰȱȱȱǰȱ¢ȱȱȱȱȱ. ŗŗȱ.
(40) Receptor binding and genome delivery. (-) strand RNA synthesis. Translation Polyprotein processing. (+) strand RNA synthesis. Assembly, encapsidation and maturation. Release. ȱ Figure 4. Schematic overview of a picornavirus infection and replication. Viral RNA is delivered into the host cell cytoplasm and translated to a polyprotein. The polyprotein is processed to single viral proteins by virally encoded proteases. Nonstructural proteins are responsible for synthesis of replication intermediate (-) strand and genomic (+) strand RNAs. (+) strand RNAs and structural proteins assemble into new virions, which are released from the cell.. ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǻǯǯǼǯȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ǻ ȱȱǰȱŘŖŖŝǼǯȱȱȱ¢ȱȱȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ¢ȱ ǻȱ ȱ ǰȱ ŗşśŚǼǯȱ. ǰȱȱȱȱȱ ȱȱȱ¢ȱȱ¡ȱ Ȭ¢ȱȱǰȱȱ¢ȱȱȱǻȱȱǯǰȱ ŗşşŘǰȱȱȱǯǰȱŗşşřǰȱ ȱȱǰȱŗşşŝǰȱȱȱǯǰȱŗşşşǰȱ ¢ȱ ȱ ǯǰȱ ŘŖŖŖǰȱ ȱ ȱ ǯǰȱ ŘŖŖŖǰȱ ȱ ȱ ǯǰȱ ŘŖŖŗǰȱ ȱ ȱ ǯǰȱ ŘŖŖŘǼǯȱ
(41) ȱ ȱ ǯȱ ǻŘŖŖśǼȱ ¢ȱ ȱ ȱ ȱ ȱ ȱȱȱȱȱȬ¢ȱȱǯȱȱ. 12.
(42) Řǯȱȱȱ Řǯŗǯȱ ȱȱȱȱȱȱ ȱȱȱȱŗşŝşȬŗşşŘǰȱŗŜȱ ȱȱȱȱȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ¢ȱ ǻ·ȱ ȱ ǯǰȱ ŗşşŜǼǯȱ ȱ ȱ ȱ Ȭȱ ǰȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ¡ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǯȱ ȱ ȱ ǯȱ ǻŗşşŞǼȱ ȱ ȱ ȱ ȱ ȱȱ£ȱȱȱȱȱǻ¢ȱǰȱȱ ȱ śǼȱ ȱ ȱ ¢ȱ ȱ ȱ ¢ȱ ŗȱ ȱ ǯȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ǰȱ ȱ ¢ȱ ȱ ȱ ǻ¢ȱ ¢ǰȱ ȱ Ǽǰȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ǻȱ ȱ ǯǰȱ ŗşşśǼǯȱ ȱ ȱ ȱȱȱȱȱȱ¡ȱȱȱȱȱ ȱ ǻȱ ȱ ǯǰȱ ŗşşşǼǯȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǰȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ǰȱ ǯȱȱȱȱȱȱȱȱȱȱȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǻ
(43) ȱ ȱ ǯǰȱ ŘŖŖŘǼǯȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǻǯȱǰȱ ȱ ǰȱ ǯȱ ¢Ǽȱ ȱ ȱ ȱ ȱ ȱ ǻ
(44) ǰȱ ŗşŜśǰȱ ¢ȱ ȱ ǯǰȱ ŗşŝŖǰȱ ȱ ȱ ǯǰȱ ŗşŝŜǰȱ
(45) ȱ ȱ ǯǰȱ ŘŖŖřǰȱȱȱȱǯǰȱŘŖŖŝǼǯȱȱȱȱȱȱȱ ȱ. Figure 5. Bank vole, the most abundant mammal in Sweden. Reprinted with courtesy of B. Niklasson.. ŗřȱ.
(46) ȱ ȱ ȱ ȱ ǰȱ ŗŗŚŜǰȱ ȱ ȱ ȱ ŘŖŖřȱ ǻ
(47) ȱ ȱ ǯǰȱ ŘŖŖřǼǯȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱȱȱ ȱȱȱȱȱǯȱ ȱ ǰȱ ȱ ǰȱ ȱ ¢ȱ ȱ ȱ ¢ȱ ŗȱ Ȭȱ ¢ǰȱ ȱ ȱ ȱ ȱ ΆȬȱ ǻȱ ȱ ǯǰȱ ŘŖŖřǰȱȱȱǯǰȱŘŖŖřǼǰȱȱ¢ȱǻȱȱǯǰȱŘŖŖŜǼǯȱ ȱ ¡ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ¢ȱ ȱ ǻȱ ȱ ǯǰȱ ŘŖŖřǰȱ ȱ ȱ ǯǰȱ ŘŖŖřǰȱ ȱȱǯǰȱŘŖŖřǰȱȱȱǯǰȱŘŖŖŜǼǯȱ ȱ ȱ¢ȱȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ¢ȱ ȱȱȱȱȱȱȱȱȱǻȱȱ ǯǰȱŘŖŖŜǼǯȱ ȱȱ ȱȱȱȱȱȱȱȱȱǯȱ ȱ ȱ Ȭŗȱ ȱ ǰȱ ¢ȱ Řȱ Ȭȱ ¢ȱ ȱ ȱ ȱȱ¡ȱȱȱǻȱȱǯǰȱŘŖŖŜǼǯȱȱȱ¢ȱ ȱ ȱ ȱ ȱ ȱ ǰȱ ȱ ȱ ȱ ¢ȱ ¢ȱ ǻȱ ȱ ǯǰȱ ŘŖŖŜǼǯȱ ȱ Ȭŗȱ ȱ ȱ ȱ ȱ ¢ȱȱȱȱȱȱ ȱǻȱǰȱȱȱ ǯǰȱŘŖŖŜǼǯȱȱ ȱȱ¢ȱȱȱȱ¢ȱȱȱȱȱ ȱ ȱ ȱ ȱ ¡ȱ ǯȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǻȱ ȱ ǯǰȱ ŗşşŞǼǰȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ŗȱ ȱ ǻȱ ȱ ǯǰȱ ŘŖŖřǼǯȱ ǰȱ ȱ ȱ ȱ ȱ ȱ ȱ ¢ȱ ȱ ¢ȱ ¢ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǰȱ ȱ ȱ ȱ ȱ ŘŖȱ ȱ ȱ ȱȱǻȱȱǯǰȱŘŖŖŝǼǯȱ ȱ. 14.
(48) ŘǯŘǯȱ ȱȱ. Ȭŗȱ ȱ ȬŘȱ ȱ ȱ ȱ ȱ ŗşśŜȱ ȱ ȱ ȱ ȱ ȱ ȱ ǰȱ ȱ ȱ ǰȱ ȱ ȱ ȱ ȱ ȱ ȱȱŘŘȱȱŘřǰȱ¢ȱǻȱȱǰȱŗşŜŗǼǯȱ ȱ ȱ ȱ ¢ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ¡ȱȱȱ¢ȱȱȱȱȱ ȱȱȱȱ ȱ ȱ ȱ ȱ ǻȱ ȱ ǯǰȱ ŗşŜŗǰȱ ȱ ȱ ǰȱ ŗşŜŗǼǯȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ŘŘȱ ȱ Řřȱ ȱ ȱȱȱȱǻȱȱ ¢¢§ǰȱŗşşŖǰȱȱȱ ǯǰȱŗşşŖǼǯȱȱȱȱȱȱ ȱǰȱ ȱ ¢ȱ ǰȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ǰȱ ȱ ȱ ȱ ȱ ȱ ǻ ¢¢§ȱ ȱ ǯǰȱ ŗşşŘǰȱ ȱ ȱ £ǰȱ ŗşşśǰȱ £ȱ ȱ ǯǰȱ ŗşşŞǰȱ ȱ ȱ ǯǰȱ ŗşşŞǰȱ
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