Chapter 4 : Genetics and Reverse Genetics of Nidoviruses

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The genomes of nidoviruses are infectious, and virus replication is initiated as the genome is delivered to the cytoplasm and the replicase is translated by the host cell ribosomes. Nidovirus reverse-genetics systems are needed to better understand aspects of their complex replication strategy, pathogenesis, and mechanisms of host range expansion, and for the generation of safe and effective antiviral therapies. As the majority of existing research into the mechanisms of nidovirus host range expansion has been completed in coronavirus models, this chapter is devoted to coronaviruses. Although nidoviruses have the opportunity to expand their host ranges, they must be able to exploit such opportunities by rapidly adapting to fit their new host. Nidoviruses can explore the range of viable genetic variation through two mechanisms, mutation and recombination. Reverse-genetics systems allow viral genomes to be directly manipulated and linked to a given phenotype. A more recently described approach cloned the full-length genome into poxvirus vectors. All three of these systems, targeted RNA recombination, full-length infectious cDNA expressed in stable amplification systems, and infectious clones amplified as multicomponent cDNAs, are currently used in research and have relative strengths. The biology of nidoviruses makes them significant threats as existing, emerging, and reemerging pathogens.

Citation: Deming D, Baric R. 2008. Genetics and Reverse Genetics of Nidoviruses, p 47-64. In Perlman S, Gallagher T, Snijder E (ed), Nidoviruses. ASM Press, Washington, DC. doi: 10.1128/9781555815790.ch4

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Porcine reproductive and respiratory syndrome virus
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Figure 1

In vitro models for studying the mechanisms of coronavirus host range expansion. (A) Persistent-infection model. DBT cells were persistently infected with MHV-A59. After 51 passages, the mutant MHV-V51 was isolated and shown to have expanded its tropism from murine cells to include human and hamster cultures. (B) Mixed-cell model. Mixed cultures of DBT and BHK cells were coinfected with the A59 and JHM strains of MHV. Over successive passages, the ratio of the permissive DBT cells was diminished relative to the nonpermissive BHK cells until the culture consisted only of BHK cells. The MHV-H2 isolate was shown to have adapted to the changing selective pressures and evolved an extended host range tropism.

Citation: Deming D, Baric R. 2008. Genetics and Reverse Genetics of Nidoviruses, p 47-64. In Perlman S, Gallagher T, Snijder E (ed), Nidoviruses. ASM Press, Washington, DC. doi: 10.1128/9781555815790.ch4
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Figure 2

Schematic illustrating the DNA and RNA launch strategies for generating recombinant nidoviruses. DNA launch requires that the full-length cDNA copy of the viral genome be delivered to the nucleus of the cell by transfection. Once there, the host cell’s transcriptional machinery drives infectious transcripts from a CMV promoter engineered at the 5’ end of the nidovirus genome cDNA. The viral RNA is exported from the nucleus to the cytoplasm, where replication occurs. RNA launches begin with an in vitro transcription of infectious synthetic RNA using T7 or SP6 RNA polymerase. The RNA is electroporated into the cytoplasm of the cell, where infection begins.

Citation: Deming D, Baric R. 2008. Genetics and Reverse Genetics of Nidoviruses, p 47-64. In Perlman S, Gallagher T, Snijder E (ed), Nidoviruses. ASM Press, Washington, DC. doi: 10.1128/9781555815790.ch4
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Figure 3

Initial version of the targeted recombination reverse-genetics system for coronaviruses. Alb4, which contains a mutation in the N protein gene (circle), produced a limited number of small plaques at the nonpermissive temperature. Following transfection of subgenomic RNA7 and infection of Alb4, RNA recombinants are generated that result in wild-type plaque phenotypes. This process requires the integration of the wild-type N protein gene (square) and is evidenced by large plaques that can easily be distinguished from Alb4. HE, hemagglutinin-esterase; E, envelope protein; M, membrane protein; AAA, poly-A tail.

Citation: Deming D, Baric R. 2008. Genetics and Reverse Genetics of Nidoviruses, p 47-64. In Perlman S, Gallagher T, Snijder E (ed), Nidoviruses. ASM Press, Washington, DC. doi: 10.1128/9781555815790.ch4
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Figure 4

Improved targeted recombination system using cell specificity for selection of successful recombinants. A chimeric virus of MHV, fMHV, expressing the S protein gene of FIPV infects feline cells transfected with RNA from a donor molecule, pMH54, bearing a mutated MHV N protein gene (circle). Successfully recombined virus is screened by growth on murine cells, which requires the incorporation of the MHV S protein gene from the donor molecule. HE, hemagglutinin-esterase; E, envelope protein; M, membrane protein; AAA, poly-A tail.

Citation: Deming D, Baric R. 2008. Genetics and Reverse Genetics of Nidoviruses, p 47-64. In Perlman S, Gallagher T, Snijder E (ed), Nidoviruses. ASM Press, Washington, DC. doi: 10.1128/9781555815790.ch4
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Figure 5

Rearrangement of the recipient virus’s (fMHV.v2) structural genes to prevent double recombination. Using the targeted recombination system, it is possible to get a double recombinant which results in the exclusion of the desired mutation (in the N protein gene [circle]) while integrating the MHV S protein gene needed for replicating on murine cells. By rearranging the position of the MHV N and membrane protein (M protein) genes on the recipient chimeric virus, the opportunity for double recombination is reduced since a second event is likely to exclude at least part of one of the major structural genes encoding a protein critical for replication. HE, hemagglutinin-esterase; E, envelope protein; AAA, poly-A tail.

Citation: Deming D, Baric R. 2008. Genetics and Reverse Genetics of Nidoviruses, p 47-64. In Perlman S, Gallagher T, Snijder E (ed), Nidoviruses. ASM Press, Washington, DC. doi: 10.1128/9781555815790.ch4
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Figure 6

Diagram of the SARS-CoV genome and the division of its cDNA into multiple subclones. The SARS-CoV genome is amplified using a six-component system, with each fragment maintained separately in its own plasmid. Reassembly makes use of BglI, which cleaves at highly variable sequences. Since this variability includes those nucleotides involved in the resulting overhang, the entire coronavirus genome fragments can be excised from their bacterial amplification plasmids by BglI digestion and fragment purification and then seamlessly religated in the correct order and orientation. The T7 promoter sequence at the 5’ end of the genomic cDNA is used to drive transcripts in vitro for an RNA launch of recombinant SARS-CoV. E, envelope protein; M, membrane protein.

Citation: Deming D, Baric R. 2008. Genetics and Reverse Genetics of Nidoviruses, p 47-64. In Perlman S, Gallagher T, Snijder E (ed), Nidoviruses. ASM Press, Washington, DC. doi: 10.1128/9781555815790.ch4
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Figure 7

Diagram outlining the strategy for the stable propagation and amplification of full-length coronavirus cDNA in a vaccinia virus vector. The coronavirus cDNA is incorporated into a recombinant vaccinia virus genome which is then transfected into a cell infected with a helper poxvirus. The recombinant genome containing the coronavirus genome is then packaged in a vaccinia virus virion, which is itself infectious and can be amplified in subsequent rounds of infection. The coronavirus cDNA can be excised from the purified genome of the recombinant vaccinia virus and used as a template for DNA or RNA launch.

Citation: Deming D, Baric R. 2008. Genetics and Reverse Genetics of Nidoviruses, p 47-64. In Perlman S, Gallagher T, Snijder E (ed), Nidoviruses. ASM Press, Washington, DC. doi: 10.1128/9781555815790.ch4
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Figure 8

Phylogenetic analysis of human, bat, and civet/raccoon dog virus S protein sequences. Shown is an unrooted Bayesian phylogenetic gene tree of 24 SARS-CoVs divided into four groups. Group 1 includes viruses isolated from animals in southern China in 2003. Group 2 is a cluster of viruses isolated from animals and humans (asterisks) in 2003. Group 3 includes viruses from all three phases (early, middle, and late) of the human SARS epidemic of 2002-2003. Group 4 represents a cluster of viruses isolated from bats in 2005-2006. A multiple-sequence alignment of the S protein gene of each virus was created using ClustalX 1.83 with default settings. Bayesian inference was conducted with Mr. Bayes, with Markov chain Monte Carlo sampling of four chains for 500,000 generations, and a consensus tree was generated using the 50% majority rule with a burn-in of 1,000. Branch confidence values are shown as posterior probabilities. The three human isolates that fall within the animal cluster (GZ0402, GD03, and GZ0401) may represent infections where a human acquired the virus from animals. The dashed line between group 3 and group 4 is used to represent a much longer line in the tree (~10 times longer); thus, the distance of the line is not representative of the distance between bat and human SARS-CoVs.

Citation: Deming D, Baric R. 2008. Genetics and Reverse Genetics of Nidoviruses, p 47-64. In Perlman S, Gallagher T, Snijder E (ed), Nidoviruses. ASM Press, Washington, DC. doi: 10.1128/9781555815790.ch4
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Figure 9

The novel challenge SARS-CoV strain icGDO3-S. (A) A novel challenge strain of SARS-CoV was generated by replacing the Urbani S protein gene with a synthetic S protein gene of GDO3. Amino acid changes unique to GDO3 relative to Urbani are indicated, with the GDO3 S protein amino acid listed on the left of the colon and the corresponding Urbani amino acid on the right. The amino acid changes are shown in relation to the receptor-binding domain (RBD) and known neutralizing epitopes. Two mutations which arose during tissue culture passage of the chimeric icGDO3-S are shown in bold italics. (B). Urbani or icGDO3-S was treated with the indicated dilution of anti-Urbani S protein sera and the number of resulting plaques was compared to the average number of plaques formed after treatment with control antibody and expressed as a percentage. Relative to Urbani, icGDO3-S was more resistant to neutralization.

Citation: Deming D, Baric R. 2008. Genetics and Reverse Genetics of Nidoviruses, p 47-64. In Perlman S, Gallagher T, Snijder E (ed), Nidoviruses. ASM Press, Washington, DC. doi: 10.1128/9781555815790.ch4
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1. Almazan, F.,, M. L. Dediego,, C. Galan,, D. Escors,, E. Alvarez,, J. Ortego,, I. Sola,, S. Zuniga,, S. Alonso,, J. L. Moreno,, A. Nogales,, C. Capiscol, and, L. Enjuanes. 2006. Construction of a severe acute respiratory syndrome coronavirus infectious cDNA clone and a replicon to study coronavirus RNA synthesis. J. Virol. 80:10900-10906.
2. Almazan, F.,, J. M. Gonzalez,, Z. Penzes,, A. Izeta,, E. Calvo,, J. Plana-Duran, and, L. Enjuanes. 2000. Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proc. Natl. Acad. Sci. USA 97:5516-5521.
3. Baric, R. S.,, K. Fu,, W. Chen, and, B. Yount. 1995. High recombination and mutation rates in mouse hepatitis virus suggest that coronaviruses may be potentially important emerging viruses. Adv. Exp. Med. Biol. 380:571-576.
4. Baric, R. S.,, K. Fu,, M. C. Schaad, and, S. A. Stohlman. 1990. Establishing a genetic recombination map for murine corona-virus strain A59 complementation groups. Virology 177:646-656.
5. Baric, R. S.,, E. Sullivan,, L. Hensley,, B. Yount, and, W. Chen. 1999. Persistent infection promotes cross-species transmissibility of mouse hepatitis virus. J. Virol. 73:638-649.
6. Baric, R. S.,, B. Yount,, L. Hensley,, S. A. Peel, and, W. Chen. 1997. Episodic evolution mediates interspecies transfer of a murine coronavirus. J. Virol. 71:1946-1955.
7. Biebricher, C. K., and, M. Eigen. 2006. What is a quasispecies? Curr. Top. Microbiol. Immunol. 299:1-31.
8. Bridgen, A.,, M. Duarte,, K. Tobler,, H. Laude, and, M. Ackermann. 1993. Sequence determination of the nucleocapsid protein gene of the porcine epidemic diarrhoea virus confirms that this virus is a coronavirus related to human coronavirus 229E and porcine transmissible gastroenteritis virus. J. Gen. Virol. 74(Pt. 9):1795-1804.
9. Britton, P.,, S. Evans,, B. Dove,, M. Davies,, R. Casais, and, D. Cavanagh. 2005. Generation of a recombinant avian corona-virus infectious bronchitis virus using transient dominant selection. J. Virol. Methods 123:203-211.
10. Casais, R.,, B. Dove,, D. Cavanagh, and, P. Britton. 2003. Recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism. J. Virol. 77:9084-9089.
11. Casais, R.,, V. Thiel,, S. G. Siddell,, D. Cavanagh, and, P. Britton. 2001. Reverse genetics system for the avian coronavirus infectious bronchitis virus. J. Virol. 75:12359-12369.
12. Cavanagh, D. 2005. Coronaviruses in poultry and other birds. Avian Pathol. 34:439-448.
13. Cavanagh, D.,, P. Davis,, J. Cook, and, D. Li. 1990. Molecular basis of the variation exhibited by avian infectious bronchitis coronavirus (IBV). Adv. Exp. Med. Biol. 276:369-372.
14. Centers for Disease Control and Prevention. 2003. Prevalence of IgG antibody to SARS-associated coronavirus in animal traders—Guangdong Province, China, 2003. Morb. Mortal. Wkly. Rep. 52:986-987.
15. Chen, W., and, R. S. Baric. 1996. Molecular anatomy of mouse hepatitis virus persistence: coevolution of increased host cell resistance and virus virulence. J. Virol. 70:3947-3960.
16. Chen, Z.,, L. Zhang,, C. Qin,, L. Ba,, C. E. Yi,, F. Zhang,, Q. Wei,, T. He,, W. Yu,, J. Yu,, H. Gao,, X. Tu,, A. Gettie,, M. Farzan,, K. Y. Yuen, and, D. D. Ho. 2005. Recombinant modified vaccinia virus Ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies primarily targeting the receptor binding region. J. Virol. 79:2678-2688.
17. The Chinese SARS Molecular Epidemiology Consortium. 2004. Molecular evolution of the SARS coronavirus during the course of the SARS epidemic in China. Science 303:1666-1669.
18. Choi, Y. J.,, S. I. Yun,, S. Y. Kang, and, Y. M. Lee. 2006. Identification of 5’ and 3’ cis-acting elements of the porcine reproductive and respiratory syndrome virus: acquisition of novel 5’ AU-rich sequences restored replication of a 5’-proximal 7-nucleotide deletion mutant. J. Virol. 80:723-736.
19. Chou, T. H.,, S. Wang,, P. V. Sakhatskyy,, I. Mboudoudjeck,, J. M. Lawrence,, S. Huang,, S. Coley,, B. Yang,, J. Li,, Q. Zhu, and, S. Lu. 2005. Epitope mapping and biological function analysis of antibodies produced by immunization of mice with an inactivated Chinese isolate of severe acute respiratory syndrome-associated coronavirus (SARS-CoV). Virology 334:134-143.
20. Coley, S. E.,, E. Lavi,, S. G. Sawicki,, L. Fu,, B. Schelle,, N. Karl,, S. G. Siddell, and, V. Thiel. 2005. Recombinant mouse hepatitis virus strain A59 from cloned, full-length cDNA replicates to high titers in vitro and is fully pathogenic in vivo. J. Virol. 79:3097-3106.
21. Compton, S. R.,, C. B. Stephensen,, S. W. Snyder,, D. G. Weismiller, and, K. V. Holmes. 1992. Coronavirus species specificity: murine coronavirus binds to a mouse-specific epitope on its carcinoembryonic antigen-related receptor glycoprotein. J. Virol. 66:7420-7428.
22. Cowley, J. A.,, C. M. Dimmock,, K. M. Spann, and, P. J. Walker. 2000. Gill-associated virus of Penaeus monodon prawns: an invertebrate virus with ORF1a and ORF1b genes related to arteri- and coronaviruses. J. Gen. Virol. 81:1473-1484.
23. Cowley, J. A.,, C. M. Dimmock,, C. Wongteerasupaya,, V. Boonsaeng,, S. Panyim, and, P. J. Walker. 1999. Yellow head virus from Thailand and gill-associated virus from Australia are closely related but distinct prawn viruses. Dis. Aquat. Org. 36:153-157.
24. Delmas, B.,, J. Gelfi,, R. L’Haridon,, L. K. Vogel,, H. Sjostrom,, O. Noren, and, H. Laude. 1992. Aminopeptidase N is a major receptor for the entero-pathogenic coronavirus TGEV. Nature 357:417-420.
25. Deming, D.,, T. Sheahan,, M. Heise,, B. Yount,, N. Davis,, A. Sims,, M. Suthar,, J. Harkema,, A. Whitmore,, R. Pickles,, A. West,, E. Donaldson,, K. Curtis,, R. Johnston, and, R. Baric. 2006. Vaccine efficacy in senescent mice challenged with recombinant SARS-CoV bearing epidemic and zoonotic spike variants. PLoS Med. 3:e525.
26. Drosten, C.,, S. Gunther,, W. Preiser,, S. van der Werf,, H. R. Brodt,, S. Becker,, H. Rabenau,, M. Panning,, L. Kolesnikova,, R. A. Fouchier,, A. Berger,, A. M. Burguiere,, J. Cinatl,, M. Eickmann,, N. Escriou,, K. Grywna,, S. Kramme,, J. C. Manuguerra,, S. Muller,, V. Rickerts,, M. Sturmer,, S. Vieth,, H. D. Klenk,, A. D. Osterhaus,, H. Schmitz, and, H. W. Doerr. 2003. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N. Engl. J. Med. 348:1967-1976.
27. Duan, J.,, X. Yan,, X. Guo,, W. Cao,, W. Han,, C. Qi,, J. Feng,, D. Yang,, G. Gao, and, G. Jin. 2005. A human SARS-CoV neutralizing antibody against epitope on S2 protein. Biochemi. Biophys. Res. Commun. 333:186-193.
28. Duarte, M., and, H. Laude. 1994. Sequence of the spike protein of the porcine epidemic diarrhoea virus. J. Gen. Virol. 75(Pt 5):1195-1200.
29. Dveksler, G. S.,, M. N. Pensiero,, C. B. Cardellichio,, R. K. Williams,, G. S. Jiang,, K. V. Holmes, and, C. W. Dieffenbach. 1991. Cloning of the mouse hepatitis virus (MHV) receptor: expression in human and hamster cell lines confers susceptibility to MHV. J. Virol. 65:6881-6891.
30. Erles, K.,, C. Toomey,, H. W. Brooks, and, J. Brownlie. 2003. Detection of a group 2 coronavirus in dogs with canine infectious respiratory disease. Virology 310:216-223.
31. Esper, F.,, C. Weibel,, D. Ferguson,, M. L. Landry, and, J. S. Kahn. 2005. Evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children. J. Infect. Dis. 191:492-498.
32. Fouchier, R. A.,, N. G. Hartwig,, T. M. Bestebroer,, B. Niemeyer,, J. C. de Jong,, J. H. Simon, and, A. D. Osterhaus. 2004. A previously undescribed coronavirus associated with respiratory disease in humans. Proc. Natl. Acad. Sci. USA 101:6212-6216.
33. Fu, K., and, R. S. Baric. 1992. Evidence for variable rates of recombination in the MHV genome. Virology 189:88-102.
34. Fu, K., and, R. S. Baric. 1994. Map locations of mouse hepatitis virus temperature-sensitive mutants: confirmation of variable rates of recombination. J. Virol. 68:7458-7466.
35. Gallagher, T. M.,, M. J. Buchmeier, and, S. Perlman. 1992. Cell receptor-independent infection by a neurotropic murine coronavirus. Virology 191:517-522.
36. Goebel, S. J.,, J. Taylor, and, P. S. Masters. 2004. The 3’ cis-acting genomic replication element of the severe acute respiratory syndrome coronavirus can function in the murine coronavirus genome. J. Virol. 78:7846-7851.
37. Gonzalez, J. M.,, Z. Penzes,, F. Almazan,, E. Calvo, and, L. Enjuanes. 2002. Stabilization of a full-length infectious cDNA clone of transmissible gastroenteritis coronavirus by insertion of an intron. J. Virol. 76:4655-4661.
38. Gorbalenya, A. E.,, L. Enjuanes,, J. Ziebuhr, and, E. J. Snijder. 2006. Nidovirales: evolving the largest RNA virus genome. Virus Res. 117:17-37.
39. Greenough, T. C.,, G. J. Babcock,, A. Roberts,, H. J. Hernandez,, W. D. Thomas, Jr.,, J. A. Coccia,, R. F. Graziano,, M. Srinivasan,, I. Lowy,, R. W. Finberg,, K. Subbarao,, L. Vogel,, M. Somasundaran,, K. Luzuriaga,, J. L. Sullivan, and, D. M. Ambrosino. 2005. Development and characterization of a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody that provides effective immunoprophylaxis in mice. J. Infect. Dis. 191:507-514.
40. Guan, Y.,, B. J. Zheng,, Y. Q. He,, X. L. Liu,, Z. X. Zhuang,, C. L. Cheung,, S. W. Luo,, P. H. Li,, L. J. Zhang,, Y. J. Guan,, K. M. Butt,, K. L. Wong,, K. W. Chan,, W. Lim,, K. F. Shortridge,, K. Y. Yuen,, J. S. Peiris, and, L. L. Poon. 2003. Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science 302:276-278.
41. Haijema, B. J.,, H. Volders, and, P. J. Rottier. 2003. Switching species tropism: an effective way to manipulate the feline coronavirus genome. J. Virol. 77:4528-4538.
42. Hamre, D. and, J. J. Procknow. 1966. A new virus isolated from the human respiratory tract. Proc. Soc. Exp. Biol. Med. 121:190-193.
43. Han, M. G.,, D. S. Cheon,, X. Zhang, and, L. J. Saif. 2006. Cross-protection in gnotobiotic calves between a human enteric coronavirus and a virulent bovine enteric coronavirus. J. Virol. 80:12350-12356.
44. Han, Y.,, H. Geng,, W. Feng,, X. Tang,, A. Ou,, Y. Lao,, Y. Xu,, H. Lin,, H. Liu, and, Y. Li. 2003. A follow-up study of 69 discharged SARS patients. J. Tradit. Chin. Med. 23:214-217.
45. He, Y.,, Q. Zhu,, S. Liu,, Y. Zhou,, B. Yang,, J. Li, and, S. Jiang. 2005. Identification of a critical neutralization determinant of severe acute respiratory syndrome (SARS)-associated coronavirus: importance for designing SARS vaccines. Virology 334:74-82.
46. Herrewegh, A. A.,, I. Smeenk,, M. C. Horzinek,, P. J. Rottier, and, R. J. de Groot. 1998. Feline coronavirus type II strains 79-1683 and 79-1146 originate from a double recombination between feline coronavirus type I and canine coronavirus. J. Virol. 72:4508-4514.
47. Holland, J. J. 2006. Transitions in understanding of RNA viruses: a historical perspective. Curr. Top. Microbiol. Immunol. 299:371-401.
48. Jia, W.,, K. Karaca,, C. R. Parrish, and, S. A. Naqi. 1995. A novel variant of avian infectious bronchitis virus resulting from recombination among three different strains. Arch. Virol. 140:259-271.
49. Kan, B.,, M. Wang,, H. Jing,, H. Xu,, X. Jiang,, M. Yan,, W. Liang,, H. Zheng,, K. Wan,, Q. Liu,, B. Cui,, Y. Xu,, E. Zhang,, H. Wang,, J. Ye,, G. Li,, M. Li,, Z. Cui,, X. Qi,, K. Chen,, L. Du,, K. Gao,, Y. T. Zhao,, X. Z. Zou,, Y. J. Feng,, Y. F. Gao,, R. Hai,, D. Yu,, Y. Guan, and, J. Xu. 2005. Molecular evolution analysis and geographic investigation of severe acute respiratory syndrome coronavirus-like virus in palm civets at an animal market and on farms. J. Virol. 79:11892-11900.
50. Keck, J. G.,, G. K. Matsushima,, S. Makino,, J. O. Fleming,, D. M. Vannier,, S. A. Stohlman, and, M. M. Lai. 1988. In vivo RNA-RNA recombination of coronavirus in mouse brain. J. Virol. 62:1810-1813.
51. Keng, C.-T.,, A. Zhang,, S. Shen,, K.-M. Lip,, B. C. Fielding,, T. H. P. Tan,, C.-F. Chou,, C. B. Loh,, S. Wang,, J. Fu,, X. Yang,, S. G. Lim,, W. Hong, and, Y.-J. Tan. 2005. Amino acids 1055 to 1192 in the S2 region of severe acute respiratory syndrome coronavirus S protein induce neutralizing antibodies: implications for the development of vaccines and antiviral agents. J. Virol. 79:3289-3296.
52. Kottier, S. A.,, D. Cavanagh, and, P. Britton. 1995. Experimental evidence of recombination in coronavirus infectious bronchitis virus. Virology 213:569-580.
53. Kroneman, A.,, L. A. Cornelissen,, M. C. Horzinek,, R. J. de Groot, and, H. F. Egberink. 1998. Identification and characterization of a porcine torovirus. J. Virol. 72:3507-3511.
54. Ksiazek, T. G.,, D. Erdman,, C. S. Goldsmith,, S. R. Zaki,, T. Peret,, S. Emery,, S. Tong,, C. Urbani,, J. A. Comer,, W. Lim,, P. E. Rollin,, S. F. Dowell,, A. E. Ling,, C. D. Humphrey,, W. J. Shieh,, J. Guarner,, C. D. Paddock,, P. Rota,, B. Fields,, J. DeRisi,, J. Y. Yang,, N. Cox,, J. M. Hughes,, J. W. LeDuc,, W. J. Bellini, and, L. J. Anderson. 2003. A novel coronavirus associated with severe acute respiratory syndrome. N. Engl. J. Med. 348:1953-1966.
55. Kuiken, T.,, R. Fouchier,, G. Rimmelzwaan, and, A. Osterhaus. 2003. Emerging viral infections in a rapidly changing world. Curr. Opin. Biotechnol. 14:641-646.
56. Kuo, L.,, G. J. Godeke,, M. J. Raamsman,, P. S. Masters, and, P. J. Rottier. 2000. Retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier. J. Virol. 74:1393-1406.
57. Kusters, J. G.,, E. J. Jager,, H. G. Niesters, and, B. A. van der Zeijst. 1990. Sequence evidence for RNA recombination in field isolates of avian coronavirus infectious bronchitis virus. Vaccine 8:605-608.
58. Lai, M. M.,, R. S. Baric,, S. Makino,, J. G. Keck,, J. Egbert,, J. L. Leibowitz, and, S. A. Stohlman. 1985. Recombination between nonsegmented RNA genomes of murine coronaviruses. J. Virol. 56:449-456.
59. Lassnig, C.,, A. Kolb,, B. Strobl,, L. Enjuanes, and, M. Muller. 2005. Studying human pathogens in animal models: fine tuning the humanized mouse. Transgenic Res. 14:803-806.
60. Lau, S. K.,, P. C. Woo,, K. S. Li,, Y. Huang,, H. W. Tsoi,, B. H. Wong,, S. S. Wong,, S. Y. Leung,, K. H. Chan, and, K. Y. Yuen. 2005. Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proc. Natl. Acad. Sci. USA 102:14040-14045.
61. Lee, C.,, J. G. Calvert,, S. K. Welch, and, D. Yoo. 2005. A DNA-launched reverse genetics system for porcine reproductive and respiratory syndrome virus reveals that homodimerization of the nucleocapsid protein is essential for virus infectivity. Virology 331:47-62.
62. Lee, C. W., and, M. W. Jackwood. 2000. Evidence of genetic diversity generated by recombination among avian coronavirus IBV. Arch. Virol. 145:2135-2148.
63. Lee, C. W., and, M. W. Jackwood. 2001. Spike gene analysis of the DE072 strain of infectious bronchitis virus: origin and evolution. Virus Genes 22:85-91.
64. Levis, R.,, C. B. Cardellichio,, C. A. Scanga,, S. R. Compton, and, K. V. Holmes. 1995. Multiple receptor-dependent steps determine the species specificity of HCV-229E infection. Adv. Exp. Med. Biol. 380:337-343.
65. Li, W.,, Z. Shi,, M. Yu,, W. Ren,, C. Smith,, J. H. Epstein,, H. Wang,, G. Crameri,, Z. Hu,, H. Zhang,, J. Zhang,, J. McEachern,, H. Field,, P. Daszak,, B. T. Eaton,, S. Zhang, and, L. F. Wang. 2005. Bats are natural reservoirs of SARS-like coronaviruses. Science 310:676-679.
66. Li, W.,, C. Zhang,, J. Sui,, J. H. Kuhn,, M. J. Moore,, S. Luo,, S. K. Wong,, I. C. Huang,, K. Xu,, N. Vasilieva,, A. Murakami,, Y. He,, W. A. Marasco,, Y. Guan,, H. Choe, and, M. Farzan. 2005. Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2. EMBO J. 24:1634-1643.
67. Liu, L.,, S. Hagglund,, M. Hakhverdyan,, S. Alenius,, L. E. Larsen, and, S. Belak. 2006. Molecular epidemiology of bovine coronavirus on the basis of comparative analyses of the S gene. J. Clin. Microbiol. 44:957-960.
68. Louz, D.,, H. E. Bergmans,, B. P. Loos, and, R. C. Hoeben. 2005. Cross-species transfer of viruses: implications for the use of viral vectors in biomedical research, gene therapy and as live-virus vaccines. J. Gene Med. 7:1263-1274.
69. Majhdi, F.,, H. C. Minocha, and, S. Kapil. 1997. Isolation and characterization of a coronavirus from elk calves with diarrhea. J. Clin. Microbiol. 35:2937-2942.
70. Makino, S.,, J. G. Keck,, S. A. Stohlman, and, M. M. Lai. 1986. High-frequency RNA recombination of murine coronaviruses. J. Virol. 57:729-737.
71. Manrubia, S. C.,, C. Escarmis,, E. Domingo, and, E. Lazaro. 2005. High mutation rates, bottlenecks, and robustness of RNA viral quasispecies. Gene 347:273-282.
72. Masters, P. S. 2006. The molecular biology of coronaviruses. Adv. Virus. Res. 66:193-292.
73. Masters, P. S., and, P. J. Rottier. 2005. Coronavirus reverse genetics by targeted RNA recombination. Curr. Top. Micro-biol. Immunol. 287:133-159.
74. McIntosh, K.,, J. H. Dees,, W. B. Becker,, A. Z. Kapikian, and, R. M. Chanock. 1967. Recovery in tracheal organ cultures of novel viruses from patients with respiratory disease. Proc. Natl. Acad. Sci. USA 57:933-940.
75. Meulenberg, J. J.,, J. N. Bos-de Ruijter,, R. van de Graaf,, G. Wensvoort, and, R. J. Moormann. 1998. Infectious transcripts from cloned genome-length cDNA of porcine reproductive and respiratory syndrome virus. J. Virol. 72:380-387.
76. Molenkamp, R.,, S. Greve,, W. J. Spaan, and, E. J. Snijder. 2000. Efficient homologous RNA recombination and requirement for an open reading frame during replication of equine arteritis virus defective interfering RNAs. J. Virol. 74:9062-9070.
77. Neumann, E. J.,, J. B. Kliebenstein,, C. D. Johnson,, J. W. Mabry,, E. J. Bush,, A. H. Seitzinger,, A. L. Green, and, J. J. Zimmerman. 2005. Assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the United States. J. Am. Vet. Med. Assoc. 227:385-392.
78. Normile, D. 2004. Infectious diseases. Viral DNA match spurs China’s civet roundup. Science 303:292.
79. Ontiveros, E.,, L. Kuo,, P. S. Masters, and, S. Perlman. 2001. Inactivation of expression of gene 4 of mouse hepatitis virus strain JHM does not affect virulence in the murine CNS. Virology 289:230-238.
80. Peiris, J. S.,, C. M. Chu,, V. C. Cheng,, K. S. Chan,, I. F. Hung,, L. L. Poon,, K. I. Law,, B. S. Tang,, T. Y. Hon,, C. S. Chan,, K. H. Chan,, J. S. Ng,, B. J. Zheng,, W. L. Ng,, R. W. Lai,, Y. Guan, and, K. Y. Yuen. 2003. Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study. Lancet 361:1767-1772.
81. Pensaert, M. B., and, P. de Bouck. 1978. A new coronavirus-like particle associated with diarrhea in swine. Arch. Virol. 58:243-247.
82. Pewe, L.,, H. Zhou,, J. Netland,, C. Tangudu,, H. Olivares,, L. Shi,, D. Look,, T. Gallagher, and, S. Perlman. 2005. A severe acute respiratory syndrome-associated coronavirus-specific protein enhances virulence of an attenuated murine coronavirus. J. Virol. 79:11335-11342.
83. Poon, L. L.,, D. K. Chu,, K. H. Chan,, O. K. Wong,, T. M. Ellis,, Y. H. Leung,, S. K. Lau,, P. C. Woo,, K. Y. Suen,, K. Y. Yuen,, Y. Guan, and, J. S. Peiris. 2005. Identification of a novel coronavirus in bats. J. Virol. 79:2001-2009.
84. Poon, L. L.,, Y. Guan,, J. M. Nicholls,, K. Y. Yuen, and, J. S. Peiris. 2004. The aetiology, origins, and diagnosis of severe acute respiratory syndrome. Lancet Infect. Dis. 4:663-671.
85. Rest, J. S., and, D. P. Mindell. 2003. SARS associated corona-virus has a recombinant polymerase and coronaviruses have a history of host-shifting. Infect. Genet. Evol. 3:219-225.
86. Rottier, P. J.,, K. Nakamura,, P. Schellen,, H. Volders, and, B. J. Haijema. 2005. Acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein. J. Virol. 79:14122-14130.
87. Saif, L. J. 1996. Mucosal immunity: an overview and studies of enteric and respiratory coronavirus infections in a swine model of enteric disease. Vet. Immunol. Immunopathol. 54:163-169.
88. Schickli, J. H.,, L. B. Thackray,, S. G. Sawicki, and, K. V. Holmes. 2004. The N-terminal region of the murine coronavirus spike glycoprotein is associated with the extended host range of viruses from persistently infected murine cells. J. Virol. 78:9073-9083.
89. Schickli, J. H.,, B. D. Zelus,, D. E. Wentworth,, S. G. Sawicki, and, K. V. Holmes. 1997. The murine coronavirus mouse hepatitis virus strain A59 from persistently infected murine cells exhibits an extended host range. J. Virol. 71:9499-9507.
90. Smati, R.,, A. Silim,, C. Guertin,, M. Henrichon,, M. Marandi,, M. Arella, and, A. Merzouki. 2002. Molecular characterization of three new avian infectious bronchitis virus (IBV) strains isolated in Quebec. Virus Genes 25:85-93.
91. Song, H. D.,, C. C. Tu,, G. W. Zhang,, S. Y. Wang,, K. Zheng,, L. C. Lei,, Q. X. Chen,, Y. W. Gao,, H. Q. Zhou,, H. Xiang,, H. J. Zheng,, S. W. Chern,, F. Cheng,, C. M. Pan,, H. Xuan,, S. J. Chen,, H. M. Luo,, D. H. Zhou,, Y. F. Liu,, J. F. He,, P. Z. Qin,, L. H. Li,, Y. Q. Ren,, W. J. Liang,, Y. D. Yu,, L. Anderson,, M. Wang,, R. H. Xu,, X. W. Wu,, H. Y. Zheng,, J. D. Chen,, G. Liang,, Y. Gao,, M. Liao,, L. Fang,, L. Y. Jiang,, H. Li,, F. Chen,, B. Di,, L. J. He,, J. Y. Lin,, S. Tong,, X. Kong,, L. Du,, P. Hao,, H. Tang,, A. Bernini,, X. J. Yu,, O. Spiga,, Z. M. Guo,, H. Y. Pan,, W. Z. He,, J. C. Manuguerra,, A. Fontanet,, A. Danchin,, N. Niccolai,, Y. X. Li,, C. I. Wu, and, G. P. Zhao. 2005. Cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human. Proc. Natl. Acad. Sci. USA 102:2430-2435.
92. Stanhope, M. J.,, J. R. Brown, and, H. Amrine-Madsen. 2004. Evidence from the evolutionary analysis of nucleotide sequences for a recombinant history of SARS-CoV. Infect. Genet. Evol. 4:15-19.
93. Stavrinides, J., and, D. S. Guttman. 2004. Mosaic evolution of the severe acute respiratory syndrome coronavirus. J. Virol. 78:76-82.
94. St-Jean, J. R.,, M. Desforges,, F. Almazan,, H. Jacomy,, L. Enjuanes, and, P. J. Talbot. 2006. Recovery of a neuroviru-lent human coronavirus OC43 from an infectious cDNA clone. J. Virol. 80:3670-3674.
95. Sui, J.,, W. Li,, A. Murakami,, A. Tamin,, L. J. Matthews,, S. K. Wong,, M. J. Moore,, A. S. Tallarico,, M. Olurinde,, H. Choe,, L. J. Anderson,, W. J. Bellini,, M. Farzan, and, W. A. Marasco. 2004. Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association. Proc. Natl. Acad. Sci. USA 101:2536-2541.
96. Tangudu, C.,, H. Olivares,, J. Netland,, S. Perlman, and, T. Gallagher. 2006. 7. Severe acute respiratory syndrome coronavirus protein 6 accelerates murine coronavirus infections. J. Virol. 81:1220-1229.
97. Thackray, L. B., and, K. V. Holmes. 2004. Amino acid substitutions and an insertion in the spike glycoprotein extend the host range of the murine coronavirus MHV-A59. Virology 324:510-524.
98. Thackray, L. B.,, B. C. Turner, and, K. V. Holmes. 2005. Substitutions of conserved amino acids in the receptor-binding domain of the spike glycoprotein affect utilization of murine CEACAM1a by the murine coronavirus MHV-A59. Virology 334:98-110.
99. Thiel, V.,, J. Herold,, B. Schelle, and, S. G. Siddell. 2001. Infectious RNA transcribed in vitro from a cDNA copy of the human coronavirus genome cloned in vaccinia virus. J. Gen. Virol. 82:1273-1281.
100. Thiel, V., and, S. G. Siddell. 2005. Reverse genetics of corona-viruses using vaccinia virus vectors. Curr. Top. Microbiol. Immunol. 287:199-227.
101. Tresnan, D. B.,, R. Levis, and, K. V. Holmes. 1996. Feline aminopeptidase N serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup I. J. Virol. 70:8669-8674.
102. Tripp, R. A.,, L. M. Haynes,, D. Moore,, B. Anderson,, A. Tamin,, B. H. Harcourt,, L. P. Jones,, M. Yilla,, G. J. Babcock,, T. Greenough, et al. 2005. Monoclonal antibodies to SARS-associated coronavirus (SARS-CoV): identification of neutralizing and antibodies reactive to S, N, M and E viral proteins. J. Virol. Methods 128:21-28.
103. Truong, H. M.,, Z. Lu,, G. F. Kutish,, J. Galeota,, F. A. Osorio, and, A. K. Pattnaik. 2004. A highly pathogenic porcine reproductive and respiratory syndrome virus generated from an infectious cDNA clone retains the in vivo virulence and transmissibility properties of the parental virus. Virology 325:308-319.
104. Tsunemitsu, H.,, Z. R. el-Kanawati,, D. R. Smith,, H. H. Reed, and, L. J. Saif. 1995. Isolation of coronaviruses antigenically indistinguishable from bovine coronavirus from wild ruminants with diarrhea. J. Clin. Microbiol. 33:3264-3269.
105. Tu, C.,, G. Crameri,, X. Kong,, J. Chen,, Y. Sun,, M. Yu,, H. Xiang,, X. Xia,, S. Liu,, T. Ren,, Y. Yu,, B. T. Eaton,, H. Xuan, and, L. F. Wang. 2004. Antibodies to SARS coronavirus in civets. Emerg. Infect. Dis. 10:2244-2248.
106. van der Hoek, L.,, K. Pyrc,, M. F. Jebbink,, W. Vermeulen-Oost,, R. J. Berkhout,, K. C. Wolthers,, P. M. Wertheim-van Dillen,, J. Spaargaren, and, B. Berkhout. 2004. Identification of a new human coronavirus. Nat. Med. 10:368-373.
107. van Dinten, L. C.,, J. A. den Boon,, A. L. Wassenaar,, W. J. Spaan, and, E. J. Snijder. 1997. An infectious arterivirus cDNA clone: identification of a replicase point mutation that abolishes discontinuous mRNA transcription. Proc. Natl. Acad. Sci. USA 94:991-996.
108. Vega, V. B.,, Y. Ruan,, J. Liu,, W. H. Lee,, C. L. Wei,, S. Y. Se-Thoe,, K. F. Tang,, T. Zhang,, P. R. Kolatkar,, E. E. Ooi,, A. E. Ling,, L. W. Stanton,, P. M. Long, and, E. T. Liu. 2004. Mutational dynamics of the SARS coronavirus in cell culture and human populations isolated in 2003. BMC Infect. Dis. 4:32.
109. Vennema, H. 1999. Genetic drift and genetic shift during feline coronavirus evolution. Vet. Microbiol. 69:139-141.
110. Vennema, H.,, A. Poland,, J. Foley, and, N. C. Pedersen. 1998. Feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses. Virology 243:150-157.
111. Vignuzzi, M.,, J. K. Stone,, J. J. Arnold,, C. E. Cameron, and, R. Andino. 2006. Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population. Nature 439:344-348.
112. Vijgen, L.,, E. Keyaerts,, P. Lemey,, P. Maes,, K. Van Reeth,, H. Nauwynck,, M. Pensaert, and, M. Van Ranst. 2006. Evolutionary history of the closely related group 2 coronaviruses: porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, and human coronavirus OC43. J. Virol. 80:7270-7274.
113. Wang, L.,, D. Junker, and, E. W. Collisson. 1993. Evidence of natural recombination within the S1 gene of infectious bronchitis virus. Virology 192:710-716.
114. Wang, S.,, T. H. Chou,, P. V. Sakhatskyy,, S. Huang,, J. M. Lawrence,, H. Cao,, X. Huang, and, S. Lu. 2005. Identification of two neutralizing regions on the severe acute respiratory syndrome coronavirus spike glycoprotein produced from the mammalian expression system. J. Virol. 79:1906-1910.
115. Weiss, M.,, F. Steck, and, M. C. Horzinek. 1983. Purification and partial characterization of a new enveloped RNA virus (Berne virus). J. Gen. Virol. 64(Pt 9):1849-1858.
116. Wentworth, D. E.,, D. B. Tresnan,, B. C. Turner,, I. R. Lerman,, B. Bullis,, E. M. Hemmila,, R. Levis,, L. H. Shapiro, and, K. V. Holmes. 2005. Cells of human aminopeptidase N (CD13) transgenic mice are infected by human coronavirus-229E in vitro, but not in vivo. Virology 335:185-197.
117. Woo, P. C.,, S. K. Lau,, C. M. Chu,, K. H. Chan,, H. W. Tsoi,, Y. Huang,, B. H. Wong,, R. W. Poon,, J. J. Cai,, W. K. Luk,, L. L. Poon,, S. S. Wong,, Y. Guan,, J. S. Peiris, and, K. Y. Yuen. 2005. Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia. J. Virol. 79:884-895.
118. Woo, P. C.,, S. K. Lau,, C. C. Yip,, Y. Huang,, H. W. Tsoi,, K. H. Chan, and, K. Y. Yuen. 2006. Comparative analysis of 22 coronavirus HKU1 genomes reveals a novel genotype and evidence of natural recombination in coronavirus HKU1. J. Virol. 80:7136-7145.
119. Woode, G. N.,, D. E. Reed,, P. L. Runnels,, M. A. Herrig, and, H. T. Hill. 1982. Studies with an unclassified virus isolated from diarrheic calves. Vet. Microbiol. 7:221-240.
120. Woolhouse, M. E., and, S. Gowtage-Sequeria. 2005. Host range and emerging and reemerging pathogens. Emerg. Infect. Dis. 11:1842-1847.
121. Yeager, C. L.,, R. A. Ashmun,, R. K. Williams,, C. B. Cardellichio,, L. H. Shapiro,, A. T. Look, and, K. V. Holmes. 1992. Human aminopeptidase N is a receptor for human coronavirus 229E. Nature 357:420-422.
122. Yeh, S. H.,, H. Y. Wang,, C. Y. Tsai,, C. L. Kao,, J. Y. Yang,, H. W. Liu,, I. J. Su,, S. F. Tsai,, D. S. Chen, and, P. J. Chen. 2004. Characterization of severe acute respiratory syndrome corona-virus genomes in Taiwan: molecular epidemiology and genome evolution. Proc. Natl. Acad. Sci. USA 101:2542-2547.
123. Youn, S.,, J. L. Leibowitz, and, E. W. Collisson. 2005. In vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication. Virology 332:206-215.
124. Yount, B.,, K. M. Curtis, and, R. S. Baric. 2000. Strategy for systematic assembly of large RNA and DNA genomes: transmissible gastroenteritis virus model. J. Virol. 74:10600-10611.
125. Yount, B.,, K. M. Curtis,, E. A. Fritz,, L. E. Hensley,, P. B. Jahrling,, E. Prentice,, M. R. Denison,, T. W. Geisbert, and, R. S. Baric. 2003. Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus. Proc. Natl. Acad. Sci. USA 100:12995-13000.
126. Yount, B.,, M. R. Denison,, S. R. Weiss, and, R. S. Baric. 2002. Systematic assembly of a full-length infectious cDNA of mouse hepatitis virus strain A59. J. Virol. 76:11065-11078.
127. Yount, B.,, R. S. Roberts,, L. Lindesmith, and, R. S. Baric. 2006. Rewiring the severe acute respiratory syndrome coronavirus (SARS-CoV) transcription circuit: engineering a recombination-resistant genome. Proc. Natl. Acad. Sci. USA 103:12546-12551.
128. Zhang, X. M.,, W. Herbst,, K. G. Kousoulas, and, J. Storz. 1994. Biological and genetic characterization of a hemagglutinating coronavirus isolated from a diarrhoeic child. J. Med. Virol. 44:152-161.
129. Zhang, X. W.,, Y. L. Yap, and, A. Danchin. 2005. Testing the hypothesis of a recombinant origin of the SARS-associated coronavirus. Arch. Virol. 150:1-20.
130. Zheng, B. J.,, K. H. Wong,, J. Zhou,, K. L. Wong,, B. W. Young,, L. W. Lu, and, S. S. Lee. 2004. SARS-related virus predating SARS outbreak, Hong Kong. Emerg. Infect. Dis. 10:176-178.

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