Virus Genes DOI 10.1007/s11262-015-1177-x
Efficient replication of recombinant Enterovirus B types, carrying different P1 genes in the coxsackievirus B5 replicative backbone Nina Jonsson • Anna Sa¨vneby • Maria Gullberg • Kim Evertsson • Karin Klingel • A. Michael Lindberg
Received: 11 November 2014 / Accepted: 27 January 2015 Ó Springer Science+Business Media New York 2015
Abstract Recombination is an important feature in the evolution of the Enterovirus genus. Phylogenetic studies of enteroviruses have revealed that the capsid genomic region (P1) is type specific, while the parts of the genome coding for the non-structural proteins (P2–P3) are species specific. Hence, the genome may be regarded as consisting of two modules that evolve independently. In this study, it was investigated whether the non-structural coding part of the genome in one type could support replication of a virus with a P1 region from another type of the same species. A cassette vector (pCas) containing a full-length cDNA copy of coxsackievirus B5 (CVB5) was used as a replicative backbone. The P1 region of pCas was replaced with the corresponding part from coxsackievirus B3 Nancy (CVB3N), coxsackievirus B6 Schmitt (CVB6S), and echovirus 7 Wallace (E7W), all members of the Enterovirus B species. The replication efficiency after transfection with clone-derived in vitro transcribed RNA was studied and compared with that of pCas. All the recombinant viruses replicated with
similar efficiencies and showed threshold cycle (Ct) values, tissue culture infectivity dose 50 %, and plaque-forming unit titers comparable to viruses generated from the pCas construct. In addition to this, a clone without the P1 region was also constructed, and Western Blot and immunofluorescence staining analysis showed that the viral genome could be translated and replicated despite the lack of the structural protein-coding region. To conclude, the replicative backbone of the CVB5 cassette vector supports replication of intraspecies constructs with P1 regions derived from other members of the Enterovirus B species. In addition to this, the replicative backbone can be both translated and replicated without the presence of a P1 region. Keywords Enterovirus Cassette vector Replicative backbone Recombination Structural and non-structural genomic regions
Introduction Edited by Juergen A Richt. Nina Jonsson and Anna Sa¨vneby contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s11262-015-1177-x) contains supplementary material, which is available to authorized users. N. Jonsson A. Sa¨vneby M. Gullberg K. Evertsson A. M. Lindberg (&) Department of Chemistry and Biomedical Sciences, Linnaeus University, Kalmar, Sweden e-mail:
[email protected] K. Klingel Department of Molecular Pathology, University of Tu¨bingen, Tu¨bingen, Germany
Enterovirus genome replication involves interactions between the viral untranslated regions (UTRs), and viral nonstructural proteins and host proteins. Consequently, these interactions play a crucial role for efficient RNA replication, and therefore the outcome of the infection [1, 2]. The Enterovirus genus is a member of the Picornaviridae family and is classified into the species Enterovirus (EV) A–H and J, and Rhinovirus A–C [3]. Enteroviruses are small nonenveloped icosahedral viruses with a single positive-sense RNA genome of approximately 7500 nucleotides in length. The single open reading frame, flanked by UTRs, is translated into a polyprotein, which is processed by viral proteinases, generating mature viral proteins. The capsid proteins, VP1-4, are encoded within the P1 region, and the non-structural viral
123
Virus Genes
proteins required for virus replication are encoded in the P2 and P3 genomic regions of the virus genome [1]. Enteroviruses, like other RNA viruses, evolve rapidly with short replication times, large population sizes, high mutation rates due to the lack of proofreading by the RNAdependent RNA polymerase, and frequent recombination events [4–6]. Recombination of two strains of enteroviruses replicating within one cell is a well-known phenomenon. In the viable viral offspring, the recombination events have most often occurred at the boundaries outside of the P1 region, probably due to the structural requirements of the virion capsid or restrictions in terms of receptor binding [4, 6–8]. Incongruity between phylogenies of 50 UTR, structural and non-structural proteins have provided evidence for recombination in a wide range of enterovirus species [8]. While all enteroviruses have characteristic type-specific capsid protein sequences, the non-structural proteins are species specific, but not type specific [8–10]. Hence, enteroviruses could be considered to consist of two genomic regions, e.g., structural and non-structural coding regions, which may evolve independently. We have previously described the construction of an infectious cDNA clone, a cassette vector (pCas), derived from CVB5 strain Dalldorf (CVB5D) where the P1 region was replaced with a putative ancestor of all currently circulating CVB5 [11]. pCas may be regarded as a replicative backbone consisting of 50 UTR, P2, P3, and 30 UTR, while the P1 region can be replaced with a P1 region from another enterovirus (Fig. 1a). In this study, the P1 region of pCas was exchanged for the P1 regions of three other Enterovirus B species, creating three constructs used to study whether the replicative backbone could support replication of intraspecies recombinant viruses.
genome, cloned into a pCR-Script Direct SK (?) vector (Stratagene), was used to construct a cassette vector. In this infectious full-length cDNA clone of CVB5D (pCVB5Dwt), a ClaI site was introduced at nucleotide position 3340 to generate a cassette vector (pCas). This modification resulted in one amino acid substitution in the 2A protein (valine to leucine at amino acid position 17). This substitution was regarded as a conservative change, as leucine is present in this position of the 2A protein genomic sequence in other enteroviruses, including echoviruses E9, E21, and E30. A synonymous mutation was also introduced to eliminate a natural SalI site. The P1 genomic regions of CVB3N, CVB6S, and E7W were amplified, using primers (supplementary material) containing the necessary restriction enzyme sites (Fig. 1b). Infected cell cultures were freeze thawed three times, after which the viral RNA was extracted (QIAmp viral RNA minikit, Qiagen), reverse transcribed (Superscript III, Invitrogen), and PCR amplified (PicoMaxx, Stratagene). PCR amplicons were visualized in agarose gels and purified using QIAquick gel extraction kit (Qiagen). The PCR products were cloned into pCas using the naturally present SalI and the introduced ClaI (Fig. 1a). The constructs were propagated in E. coli DH5a cells and purified (Midiprep kit, Promega). Nucleotide sequences of all constructs were verified by sequencing with an ABI Prism 3130 automated sequencer (Applied Biosystems). Sequences were analyzed using the Sequencher version 4.6 software package (Gene Codes Corporation). A cassette vector without the P1 region, pCasP1del, was constructed by digestion with SalI and HindIII. Digested plasmid was treated with the E. coli Klenow polymerase (Fermentas), re-ligated, transformed, and verified as described above. Infectivity assays and generation of viruses
Materials and methods Cells and viruses Green monkey kidney (GMK) cells were maintained in Dulbecco´s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 10 % newborn calf serum (NCS) (Sigma-Aldrich), at 37 °C, 5.0 % CO2. Type strains of CVB5 Dalldorf (CVB5D, provided by R.L. Crowell, Philadelphia, USA) [11–13], CVB3 Nancy (CVB3N) [14, 15], CVB6 Schmitt (ATCC VR-155) [16], and E7 Wallace (E7W, ATCC VR-37) [16], were propagated in GMK cells. Construction of recombinant cDNA constructs The cassette vector used to generate the constructs has been described previously [11]. Briefly, the complete CVB5D
123
The cassette vector construct clones were linearized using the NotI site at the 3´end of the virus genome. RNA was generated by in vitro transcription (MegaScriptÒ T7 Kit, Ambion) from the linearized constructs, and the RNA quality was determined using a non-denaturing agarose gel. The infectivity of the RNAs derived from cDNA clones was tested by plaque-forming unit (PFU) assay using LipofectamineÒ 2000 (Invitrogen) for transfection of GMK cells seeded in six-well plates with tenfold dilution of RNA. The inoculum was removed after 30 min of incubation at 37 °C and DMEM supplemented with 0.8 % (w/v) gum tragacanth (Sigma-Aldrich), 1 % NCS, 2 mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Sigma-Aldrich). The number of PFU/lg of transfected RNA was calculated (mean of two experiments). GMK cells were transfected with the RNA in sub-confluent 6-well plates, all in triplicate. Transfected cells were maintained at 37 °C until complete cytopathic effect (CPE)
Virus Genes
Fig. 1 Schematic diagram of cassette vector constructs and primers. a Genome organization of CVB5D with restriction enzyme sites indicated by arrows. The ClaI* site was introduced to construct a cassette vector with the ability to exchange the entire P1 region. Infectious cDNA clones, with P1 regions originating from other EV-B viruses, were constructed as indicated in the figure. A cDNA clone in
which the structural protein genes were deleted was also constructed using SalI and HindIII (pCasP1del). b Primers used to amplify the P1 region of the viruses introduced in the cassette vector. The restriction enzyme sites are positioned to give the same reading frame and size as in the original cassette vector (pCas)
was observed, or at a maximum of 5 days. Generated viruses were harvested from cells by three rounds of freeze thawing. Clone-derived viruses were further propagated for five subsequent serial passages in GMK cells. For each triplicate sample, 1 ml of lysate from the transfection or from the previous passage was added to sub-confluent GMK cells in a T25 flask and incubated at room temperature for 1 h. Inoculum was removed and fresh serumfree medium was added, after which the cells were incubated at 37 °C as the transfections described above. The identity of the generated viruses was verified by nucleotide sequencing of the P1 region (LIGHTrun, GATC Biotech).
time PCR was performed using an ABI Prism 7500 SDS machine (Applied Biosystems). Virus titers were determined for the fifth passage using TCID50 [17, 18], with ten-fold serial diluted virus. The degree of CPE was determined by visual inspection after 7 days. A PFU assay was performed on the fifth passage (P5) for determination of PFU/ml and to study the plaque morphology of the generated viruses, as described previously [19]. Briefly, ten-fold dilutions of P5 lysates were added to confluent GMK cells in sextuplicate and incubated at room temperature for 1 h. Inoculum was removed, cells were washed and medium supplemented with gum tragacanth was added as described above. Cells were stained 72 h post-infection with a crystal violet–ethanol solution for visualization of plaques. All quantifications were performed in triplicate.
Measurements of virus production Replication of constructs in infected cells at passage one, three, and five were determined using real-time PCR as described previously [16]. Briefly, RNA was extracted (QIAamp viral RNA mini kit, Qiagen) and reverse transcribed using random hexamers and TaqMan reverse transcriptase kit (Applied Biosystem) according to the manufacturer’s protocol. Reverse-transcribed cDNA was quantified using SYBR Green master mix (Applied Biosystem) according to the manufacturer’s description, with primers directed to the 50 UTR of the genome. Real-
Western Blot For analysis of the replication, cells were transfected in duplicate, as described above. Cell lysates were prepared at 12 h post-infection for pCasP1del and pCas, and analyzed for expression of the structural VP1 and the non-structural 3D protein. Briefly, cells were washed and incubated at room temperature in buffer containing 125 mM NaCl,
123
Virus Genes
20 mM Tris, pH 8.0, and 0.5 % NP40, until cells detached. Lysates were kept on ice for 5 min and cellular nuclei were pelleted by 10-min centrifugation at 13,000 rpm at 4 °C, followed by extraction of the supernatants and freezing at -20 °C. The protein samples were fractionated by 12 % SDS-PAGE and transferred to PVDF Plus membranes (Amersham). Briefly, membranes were washed and blocked with non-fat dry milk and 0.1 % Tween-20 in TBS overnight at 4 °C, and subsequently incubated with polyclonal antisera against VP1 of CVB5 (1:10,000, kindly provided by H. Norder, Swedish Institute for Communicable Disease Control, Stockholm, Sweden) or polyclonal rabbit antisera against the 3D region of CVB3 [20] (1:4,000) for 3 h at room temperature. Immunoreactive proteins were visualized with HRP-conjugated secondary antibodies (1:2,000 ab112767, abcam and 1:2,000 P0448, DAKO), incubated for 1 h at room temperature and visualized using a chemiluminescence system (ECL, Amersham). Immunofluorescence staining For immunofluorescence assays, monolayers of GMK cells, cultured on poly-L-lysine treated Lab-TEK II chamber glass slides (Nalge Nunc International), were transfected using 4 ll Lipofectamine 2000 (Life Technologies) and 0.4 lg RNA derived from in vitro transcription of pCas and pCasP1del per well. The mock control was treated the same way except that no RNA was added. After 8 h, the cells were fixed with 4 % formaldehyde for 10 min at room temperature, washed twice, and treated with 0.5 % TritonX100 in PBS for 15 min. The cells were washed again and blocked with 3 % BSA in PBS for 30 min. Following washing, the cells were stained with dsRNA-specific monoclonal mouse antibody 1:500 J2 (English & Scientific Consulting Kft.) over night at 4 °C. After washing, a secondary goat anti-mouse antibody labeled with Alexa FluorÒ 488 (Sigma-Aldrich) was added, and the slides were incubated in the dark at room temperature for 45 min. Finally, the slides were mounted with Vectashield (Immunkemi) containing 40 ,6-diamidino-2-phenylindole (DAPI). Images were captured with an epifluorescence microscope.
CVB6S, and E7W from the EV-B species were cloned into the cassette vector, i.e., pCasCVB3N, pCasCVB6S, and pCasE7W. A vector where the majority of the P1 region was deleted was also constructed, in order to demonstrate the ability of the replicative backbone to replicate efficiently, when present within the infected cell without the region coding for the capsid proteins (Fig. 1a). Viability of recombinant virus RNA The replication capacity of the in vitro transcibed and subsequently transfected viral RNAs was tested by PFU assay. All transfected constructs generated replicating viruses thus demonstrating that the replicative backbone of CVB5D supports replication of chimeras containing the P1 region of other types of the same species. The titers of the replicating viruses after transfection were similar for all included constructs with a range between 3.29 and 3.70 log(PFU/ll) (Fig. 2). Replicating viruses were thus generated from in vitro transcribed RNA transfected into GMK cells, with all EV-B constructs giving complete CPE within 3 days posttransfection. To monitor the synthesis of viral RNA during replication, the amount of viral genomes was measured by reverse transcription real-time PCR (RT-PCR) (Fig. 3). This was done at the first passage, 48 h post-infection—the time point where the positive control pCas gave full CPE and at the third and the fifth passage at full CPE or a maximum of 5 days after infection. All constructs with recombinant P1 regions gave Ct values indicating efficient replication capacity with high concentrations of RNA copies at the first passage. TCID50 and PFU assay were performed on passage five to further investigate viral load and viability. The results for the TCID50 ranged from 5.27 to 6.64 log(TCID50/ml), and the PFU values were between 5.69 and 8,28 log(PFU/ ml) for the construct generated viruses. Although the
Results To study the viability of viruses generated by intraspecies recombination events between the P1 and P2-P3 regions, a previously described cassette vector [11] was used. This vector contains the CVB5D genome as a genetic backbone, in which the P1 region was slightly modified so that it can be replaced by the corresponding genomic regions from other viruses. To determine if the replicative backbone supported replication within a species, the P1 region of CVB3N,
123
Fig. 2 Infectivity and virus quantification assays. The ability of viruses, derived from transfection with RNA from the constructs, to generate plaques was determined. At the fifth passage, titers were established by PFU and TCID50. All values are presented as means of duplicate (transfection) or triplicate (passage 5)
Virus Genes
double-stranded RNA (=replicative intermediate) molecules are formed. Both the positive control, pCas, and the pCasP1del transfections showed signs of cells with doublestranded RNA, while no such cells could be found in the mock-transfected control cells (Fig. 4b).
Discussion
Fig. 3 RT-PCR quantification of clone-derived virus RNA. RNA was purified and used for RT-PCR analysis in the first passage (at 48 h, the time point where pCas gave complete CPE), and at the third and fifth passage at complete CPE or after incubation for a maximum of 5 days. Values are results of triplicates and error bars represent confidence intervals
generated viruses exhibited similar replication efficiencies, different plaque morphologies could be observed (Fig. 2). Sequencing of the P1 region after the fifth passage revealed that the progeny of pCas and pCasCVB6 had one non-silent mutation each, VP1Q132K and VP2R232S respectively, while pCasCVB3 and pCasE7 did not display any mutations.
It has previously been demonstrated through comparative analyses that all enteroviruses have a type-specific characteristic capsid protein sequences and that regions coding for the non-structural proteins are species specific but not type specific [8–10]. The apparent independent evolution of these two parts of the genome is possibly due to welldefined roles of the different regions of the genome during the replication cycle [6, 8, 10]. Furthermore, high mutation rate and efficient recombination are two key attributes of the members of the Picornaviridae family, which will increase rapid adaptation within a host, and thus lead to change in tissue tropism [10]. Natural recombination of picornaviruses is widely reported and has been shown for most genera within the family [21]. We have previously shown that the structural proteins of an enterovirus genome can be replaced with the corresponding genomic region within the same type using the cassette vector described above [11]. In the previous report,
Replication efficiency of pCasP1del The pCasP1del construct was included to the study whether the transfected RNA containing only genes coding for nonstructural proteins is able to replicate without the region coding for the structural proteins. The replication of pCasP1del cannot be measured by the same methods as it has no way of forming virions without the P1 region. Analysis of the protein production at 12 h post-transfection revealed that the polymerase, 3D, could be detected for both pCas, the positive control and pCasP1del, while VP1 only was detected for pCas, containing a complete P1 region sequence (Fig. 4a). The presence of the polymerase showed that the in vitro transcribed RNA from the pCasP1del construct can be translated although, as expected, no virions were formed. Viral RNA replication was determined with immunofluorescence staining, performed on GMK cells transfected with in vitro transcribed RNA using an antibody against double-stranded RNA. Double-stranded RNA does not occur naturally in the cells. However, when enteroviruses replicate, a negative-sense RNA copy is formed and used as a template for new copies of the genome, and during this process
Fig. 4 a Expression of 3D polymerase and VP1 capsid protein. Protein production by pCasP1del was determined 12 h post-transfection using Western Blot, with pCas as a positive control. Blots are representative of duplicates. Top image shows produced viral 3D polymerase and the bottom image shows produced viral capsid protein VP1. b Immunofluorescence staining confirmation of viral replication. GMK cells were transfected with RNA in vitro transcribed from pCas and pCasP1del. Double-stranded RNA can be seen as green, while the cell nuclei are blue. Images are representatives of three triplicates. Scale bar 10 lm
123
Virus Genes
the phylogenetic relationship among circulating CVB5 isolates was investigated and based on the circulating CVB5 P1 sequences, a CVB5 virus with a putative ancestral virion origin was reconstructed. The ancestral P1 region was synthesized and cloned into the cassette vector, and the generated virus showed the same replication characteristics both as clinical isolates and the CVB5 typespecific strain (CVB5D) used in our studies [11]. In this work, the compatibility of the replicative backbone and P1 regions from different virus types within the species EV-B is explored (Fig. 1). Studies of the sequence variation within the different enterovirus species show a general pattern of high sequence identity in the 50 UTR, the genes coding for non-structural proteins, and the 30 UTR, while there is a significant genetic variation in regions of the structural proteins [4, 6, 10]. Recombination events may occur throughout the genome, but they are rarely found within the P1 region of recovered circulating viral progeny, which is probably due to restriction based on structural constraints and receptor usage. However, it has been shown in vitro that the 50 UTR, structural and nonstructural genomic regions of the EV-B species CVA9 and CVB3 could be interchanged, still yielding viable viruses [22]. Recombination within a species is frequently observed, while recombination between species is a rare event. For EV-B, interspecies recombination has only been recorded in the highly conserved non-structural genomic regions [23, 24]. The sequence divergence between enterovirus species is considered to be the main constraint in the generation of viable intraspecies recombinants in nature [8, 10, 25]. It has previously been shown for poliovirus (PV), of the EV-C species, that the structural proteins can be deleted [26] or replaced by sequences coding foreign proteins [27– 29], creating replicons that are able to replicate, and also to be encapsidated provided that capsid proteins are added in trans [30]. A vector without the majority of the P1 region, pCasP1del, was made to verify that the replicative backbone in this experimental setup is replication competent without including the genomic sequences coding for the structural genes of the P1 region. To determine if translation of the genome was possible without the P1 region, Western Blot was performed visualizing the expression of the 3D polymerase and the VP1 capsid protein of pCas and pCasP1del. The expression of 3D and VP1 was examined at 12 h post-transfection, showing that the 3D protein is produced in both constructs, while the VP1 protein is only detected in pCas (Fig. 4a). Using immunofluorescence staining the presence of double-stranded RNA could be determined, showing that the viral RNA can replicate without the presence of the P1 proteins (Fig. 4b). This shows that both translation and processing of proteins as well as genome replication are possible for the construct lacking the P1 region.
123
The encapsidation signal for enteroviruses has yet to be identified. Previous studies of enteroviruses have indicated that this signal is not present in the capsid proteins or the UTRs, leaving only the non-structural proteins [2, 27, 31– 33]. It has been shown that trans-encapsidation of the PV genome in other virus capsid proteins does occur, but with considerably lower efficiencies than with homologous PV capsid proteins [30]. Recently, a novel mechanism in which a protein–protein interaction between viral proteins 2CATPase and VP3 facilitate enteroviral encapsidation was suggested [34, 35]. This study demonstrates a cassette vector in which parts of the genome can be replaced to study recombination, encapsidation, and replication of enteroviruses. The progeny of all viral constructs showed Ct values, TCID50, and PFU titers comparable to viruses generated from the pCas construct. Hence, the replicative backbone of CVB5D supports replication of different members of the EV-B species. Recently, naturally occurring interspecies recombination involving Enterovirus B has been reported [23]. This opens up the question as to whether the replicative backbone pCas can support replication of chimeras containing P1 regions of species other than members of Enterovirus B. The cassette vector could be used as a tool to study the fitness of interspecies recombinants and determination of what the limiting factors may be. In conclusion, the conservative non-structural coding region of the replicative backbone of CVB5D is able to generate efficiently replicating virus with entire P1 regions from other types of the same species. This cassette vector could be a useful tool when studying replication, recombination, and encapsidation. Furthermore, the replicative backbone can initiate replication of the viral genome without a complete P1 region, showing that the structural proteins are not necessary for the replication of the viral genome. Acknowledgments We thank Kjell Edman (Linnaeus University, Kalmar, Sweden) for valuable discussions and for reading the manuscript. We also thank Merja Roivainen (National Institute for Health and Welfare, Helsinki, Finland) and Richard Crowell for providing viruses and Helene Norder for providing CVB5 antisera. This study was supported by Grants from the Faculty of Health and Life Sciences, Linnaeus University, the Swedish Knowledge Foundation and Crafoord Foundation.
References 1. K.M. Bedard, B.L. Semler, Microbes Infect. 6, 702–713 (2004) 2. W. Xiang, K.S. Harris, L. Alexander, E. Wimmer, J. Virol. 69, 3658–3667 (1995) 3. International Committee on Taxonomy of Viruses (ICTV), Virus Taxonomy: 2013 Release. http://ictvonline.org/virusTaxonomy. asp. Accessed 13 May 2014
Virus Genes 4. P. Simmonds, J. Welch, J. Virol. 80, 483–493 (2006) 5. P. Simmonds, J. Virol. 80, 11124–11140 (2006) 6. M.S. Oberste, S. Penaranda, M.A. Pallansch, J. Virol. 78, 2948–2955 (2004) 7. L. Heath, E. van der Walt, A. Varsani, D.P. Martin, J. Virol. 80, 11827–11832 (2006) 8. A.N. Lukashev, V.A. Lashkevich, O.E. Ivanova, G.A. Koroleva, A.E. Hinkkanen, J. Ilonen, J. Virol. 77, 10423–10431 (2003) 9. M.S. Oberste, K. Maher, D.R. Kilpatrick, M.A. Pallansch, J. Virol. 73, 1941–1948 (1999) 10. J. Santti, T. Hyypia, L. Kinnunen, M. Salminen, J. Virol. 73, 8741–8749 (1999) 11. M. Gullberg, C. Tolf, N. Jonsson, M.N. Mulders, C. SavolainenKopra, T. Hovi, M. Van Ranst, P. Lemey, S. Hafenstein, A.M. Lindberg, J. Virol. 84, 9695–9708 (2010) 12. R.L. Crowell, J.T. Syverton, J. Exp. Med. 113, 419–435 (1961) 13. K.J. Reagan, B. Goldberg, R.L. Crowell, J. Virol. 49, 635–640 (1984) 14. R. Kandolf, P.H. Hofschneider, P. Natl. Acad. Sci. 82, 4818–4822 (1985) 15. A.M. Lindberg, P.O.K. Stalhandske, U. Pettersson, Virology 156, 50–63 (1987) 16. N. Jonsson, M. Gullberg, A.M. Lindberg, Microbiol. Immunol. 53, 149–154 (2009) 17. R. Dulbecco, M. Vogt, J. Exp. Med. 99, 167–182 (1954) 18. J.C. Hierholzer, R.A. Killington, in Virology methods manual, ed. by H.O. Kangro, B.W.J. Mahy, (Academic Press, San Diego, 1996), pp. 25–47 19. P. Dobos, J. Clin. Microbiol. 3, 373–375 (1976) 20. S. Werner, W.M. Klump, H. Schonke, P.H. Hofschneider, R. Kandolf, DNA 7, 307–316 (1988) 21. A.N. Lukashev, Rev. Med. Virol. 20, 327–337 (2010)
22. H. Harvala, H. Kalimo, L. Dahllund, J. Santti, P. Hughes, T. Hyypia, G. Stanway, J. Gen. Virol. 83, 1697–1706 (2002) 23. D.V. Nguyen, H. Harvala, E.M. Ngole, E. Delaporte, M.E. Woolhouse, M. Peeters, P. Simmonds, J. Virol. 88, 5967–5976 (2014) 24. Simmonds P, in The Picornaviruses, ed. by E. Ehrenfeld, E. Domingo, R.P. Roos (ASM Press, Washington, 2010), pp. 229–237 25. M.S. Oberste, K. Maher, M.A. Pallansch, J. Virol. 76, 1244–1251 (2002) 26. S. Kajigaya, H. Arakawa, S. Kuge, T. Koi, N. Imura, A. Nomoto, Virology 142, 307–316 (1985) 27. W. Barclay, Q. Li, G. Hutchinson, D. Moon, A. Richardson, N. Percy, J.W. Almond, D.J. Evans, J. Gen. Virol. 79(Pt 7), 1725–1734 (1998) 28. D.C. Porter, D.C. Ansardi, W.S. Choi, C.D. Morrow, J. Virol. 67, 3712–3719 (1993) 29. D.C. Porter, D.C. Ansardi, C.D. Morrow, J. Virol. 69, 1548–1555 (1995) 30. X.Y. Jia, M. Van Eden, M.G. Busch, E. Ehrenfeld, D.F. Summers, J. Virol. 72, 7972–7977 (1998) 31. V.H. Johnson, B.L. Semler, Virology 162, 47–57 (1988) 32. M. Gromeier, L. Alexander, E. Wimmer, Proc. Natl. Acad. Sci. 93, 2370–2375 (1996) 33. J.B. Rohll, D.H. Moon, D.J. Evans, J.W. Almond, J. Virol. 69, 7835–7844 (1995) 34. P. Jiang, Y. Liu, H.C. Ma, A.V. Paul, E. Wimmer, Microbiol. Mol. Biol. Rev. 78, 418–437 (2014) 35. Y. Liu, C.L. Wang, S. Mueller, A.V. Paul, E. Wimmer, P. Jiang, Plos Pathog. 6(8), e1001066 (2010)
123
