Recovery of a Neurovirulent Human Coronavirus OC43
http://www.100md.com
病菌学杂志 2006年第7期
Laboratory of Neuroimmunovirology, INRS-Institut Armand-Frappier, 531 Boulevard des Prairies, Laval, Quebec H7V 1B7, Canada
Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma, Darwin 3, 28049 Madrid, Spain
ABSTRACT
This study describes the assembly of a full-length cDNA clone of human coronavirus (HCoV)-OC43 in a bacterial artificial chromosome (BAC). The BAC containing the full-length infectious cDNA (pBAC-OC43FL) was assembled using a two-part strategy. The first step consisted in the introduction of each end of the viral genome into the BAC with accessory sequences allowing proper transcription. The second step consisted in the insertion of the whole HCoV-OC43 cDNA genome into the BAC. To produce recombinant viral particles, pBAC-OC43FL was transfected into BHK-21 cells. Recombinant virus displayed the same phenotypic properties as the wild-type virus, including infectious virus titers produced in cell culture and neurovirulence in mice.
TEXT
Human coronaviruses (HCoVs) are positive-stranded enveloped RNA viruses that are mainly associated with respiratory infections and are responsible for up to one-third of common colds (10) and possibly neurologic disease (3, 8). Like SARS-CoV, which was identified as the causative agent of severe acute respiratory syndrome (SARS) (11), HCoV-OC43 (ATCC VR-759 from the mid 1960s; GenBank accession number AY585228) (17) belongs to the second genetic group of the Coronaviridae (7, 14).
Several coronavirus infectious clones have been assembled, although this has proven difficult given the length of the viral genome (9), as well as the presence of unstable viral cDNA regions in bacteria (2, 6, 21). Uncommon strategies were therefore necessary to circumvent technical limitations engendered by the viral genome. Among the strategies available for the construction (2, 4, 5, 18, 19, 20, 21), the bacterial artificial chromosome (BAC) approach was selected given the availability of the technology, as well as the number of possibilities it offers for downstream applications (1, 2, 15, 16, 22). This approach uses a two-step amplification system that couples viral RNA expression in the nucleus from the cytomegalovirus (CMV) immediate-early promoter, with a second amplification step in the cytoplasm driven by the viral polymerase.
Construction of the full-length HCoV-OC43 cDNA clone. Prior to introducing the full-length viral cDNA into the pBeloBAC11 (13), pBAC-OC43-5'-3' was first assembled. This plasmid includes the first 720 nucleotides (nt) of the genome under the control of the CMV promoter, and the last 1,651 nt of the viral RNA, followed by a 25-bp synthetic poly(A) tail, the hepatitis delta virus ribozyme (HDV) and the bovine growth hormone termination and polyadenylation sequences (BGH) to lead to an accurate 3' end. In order to join the CMV promoter with the viral 5' end and the viral 3' end with the HDV-BGH sequences in a contiguous and very precise way, overlapping PCRs were performed.
Primers used to perform the overlapping PCRs are listed in Table 1. The vector pBAC-TGEV-5'-3', which contains the accessory sequences CMV, HDV, and BGH, was used as a template to amplify these latter sequences. After two rounds of amplification, an overlapping PCR was performed for each end of the viral genome (Fig. 1A). Both overlapping PCR amplicons were directly introduced into the pBeloBAC11 plasmid in order to create the pBAC-OC43-5'-3' (Fig. 1B). After each cloning step, the new BAC vector was sequenced to assess that no undesired mutations were introduced into the new construct.
Before performing the assembly of the full-length cDNA clone, 5' rapid amplification of cDNA ends was carried out with RNA extracted from BHK-21 cells transfected with pBAC-OC43-5'-3' in order to check that the transcription machinery was functional (data not shown). The HDV cleavage efficiency was also determined by using a reverse transcription-PCR approach and, as expected, it was estimated at approximately 60% (data not shown) (12).
The HCoV-OC43 infectious cDNA clone was assembled using a five-step cloning approach, starting with the pBAC-OC43-5'-3' as a backbone. Given the low availability of unique restriction sites in the HCoV-OC43 genome, three additional restriction sites (MluI, AsiSI, and SacII) were introduced into the sequence of the cDNA clone. This modification required the introduction of six silent point mutations (T6479C, T6482G, C12143T, T12146C, A24068G, and T24071G). These sequence changes were used as genetic markers to identify the recombinant virus (2, 19). For cDNA assembly, we took advantage of the presence of the unique SfoI restriction site in both the BAC vector and the HCoV-OC43 genome (Fig. 1B and 2A).
The genome of HCoV-OC43 was therefore amplified in five distinct overlapping regions, named A to E, which spanned the entire viral genome, except for the 5' and 3' ends (Fig. 2A). Each region was generated in such a way that it contained a given cloning site at one end and a SfoI site at the other end. Primers used for the generation of region A to E are described in Table 1. Each amplicon was first inserted into the pSMART HCKan vector in order to generate plasmids pSMART-A to -E (Fig. 2A) and positive clones with the appropriate sequence were obtained.
Viral DNA regions contained in clones pSMART-A to -E were used to fill the pBAC-OC43-5'-3' and to generate the pBAC-OC43FL (Fig. 2B). Regions A to E were respectively introduced into the BAC vector using the restriction sites they were sharing with the construct (Fig. 2A). Region E, which contained the disrupted unstable segment of ORF1a, was introduced at the last cloning step to minimize the toxicity problem usually observed with this region of the genome (6, 21). Junction sites created for each new construct were sequenced to make sure no unexpected mutations were introduced into the genome.
Recovery, replication, and neurovirulence of recombinant HCoV-OC43. One of the major advantage provided by the BAC system is that the recovery of recombinant viral particles is very efficient and simple. This was especially true for pBAC-OC43FL given that the cell line BHK-21 provided a good transfection rate and was also susceptible to infection by HCoV-OC43. For rescue of recombinant virus, 5 μg of pBAC-OC43FL were directly transfected into BHK-21 cells using Lipofectamine 2000 (Invitrogen). Virus titers obtained for the recombinant virus reached 106.5 50% tissue culture infective doses (TCID50)/ml, which are comparable to those obtained after infection with the HCoV-OC43 laboratory strain. To make sure rescued viruses were indeed recombinant particles, reverse transcription-PCR were performed and the presence of the newly introduced restriction sites, which are exclusive to the recombinant virus, was assessed (data not shown).
To test whether the recombinant virus displayed the same growth kinetics as the parental strain in HRT-18 cells, replication was assessed at different times postinfection in a multicycle growth curve. Wild-type and recombinant virus shared very similar overall patterns of replication, reaching virus titers of approximately 106.0 TCID50/ml after 4 days of infection, although the replication of the recombinant virus was slightly delayed during the first 2 days (Fig. 3A). As expected and like the wild-type HCoV-OC43 strain, recombinant virus did not form any plaques (data not shown). Taken together, these data confirm that the recombinant virus shares phenotypic properties with the wild type HCoV-OC43 strain in cell culture.
To determine whether the HCoV-OC43 strain, as well as the recombinant virus, displayed the same phenotype in vivo, their neurovirulence was assessed in mice. C57BL/6 and BALB/c mice were infected intracerebrally with either virus (8). In C57BL/6 mice, a survival rate of 30% was achieved for both viruses, whereas 70 and 80% of BALB/c mice survived the infection with the recombinant virus and the parent strain, respectively (Fig. 3B). Both recombinant and wild-type virus displayed very similar levels of virulence following injection into the brain. Furthermore, both viruses showed very similar patterns of replication in mouse brain (data not shown).
Overall, the virus recovered from the infectious cDNA clone exhibited the same genotypic and phenotypic properties as the HCoV-OC43 from ATCC, both in vitro and in vivo.
The cDNA infectious clones represent very interesting tools for the study of potential vaccine candidates, like genetically attenuated viruses, but also allow multiple possibilities for the characterization of any viral functions and mechanisms by reverse genetics. The pBAC-OC43FL will be invaluable in the characterization of the adaptation of the HCoV-OC43 virus in a neural environment and will provide an opportunity to better understand the biology of the HCoV-OC43, as well as other related coronaviruses, such as SARS-CoV.
ACKNOWLEDGMENTS
We are grateful to Francine Lambert for technical assistance with cell and virus culture. We thank R. C. Levesque (Universite Laval, Quebec, Canada) for providing vector pBeloBAC11 and Luc Gagnon (Ecopia Biosciences, Inc., Montreal, Quebec, Canada) for his valuable advice regarding cloning techniques in the BAC vector.
This work was supported by grant MT-9203 from Canadian Institutes for Health Research (Institute of Infection and Immunity) to P.J.T., who is the holder of a Tier-1 Canada Research Chair, and by a grant from the Department of Education and Science of Spain (BIO2004-00636) to L.E. J.R.S.-J. acknowledges a studentship from the Fonds Quebecois de Recherche sur la Nature et les Technologies.
Supplemental material for this article may be found at http://jvi.asm.org/.
REFERENCES
Almazán, F., C. Galán, and L. Enjuanes. 2004. The nucleoprotein is required for efficient coronavirus genome replication. J. Virol. 78:12683-12688.
Almazán, F., J. M. González, Z. Penzes, A. Izeta, E. Calvo, J. Plana-Durán, and L. Enjuanes. 2000. Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proc. Natl. Acad. Sci. USA 97:5516-5521.
Arbour, N., R. Day, J. Newcombe, and P. J. Talbot. 2000. Neuroinvasion by human respiratory coronaviruses. J. Virol. 74:8913-8921.
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.
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.
Gonzalez, J. M., Z. Penzes, F. Almazán, 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.
Gorbalenya, A. E., E. J. Snijder, and W. J. Spaan. 2004. Severe acute respiratory syndrome coronavirus phylogeny: toward consensus. J. Virol. 78:7863-7866.
Jacomy, H., and P. J. Talbot. 2003. Vacuolating encephalitis in mice infected by human coronavirus OC43. Virology 315:20-33.
Masters, P. S. 1999. Reverse genetics of the largest RNA viruses. Adv. Virus Res. 53:245-264.
Myint, S. H. 1994. Human coronaviruses: a brief review. Rev. Med. Virol. 4:35-46.
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.
Sharmeen, L., M. Y. P. Kuo, G. Dinter-Gottlieb, and J. Taylor. 1988. Antigenomic RNA of human hepatitis delta virus can undergo self-cleavage. J. Virol. 62:2674-2679.
Shizuya, H., B. Birren, U.-J. Kim, V. Mancino, T. Slepak, Y. Tachiiri, and M. Simon. 1992. Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc. Natl. Acad. Sci. USA 89:8794-8797.
Snijder, E. J., P. J. Bredenbeek, J. C. Dobbe, V. Thiel, J. Ziebuhr, L. L. Poon, Y. Guan, M. Rozanov, W. J. M. Spaan, and A. E. Gorbalenya. 2003. Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J. Mol. Biol. 331:991-1004.
Sola, I., S. Alonso, S. Zúiga, M. Balasch, J. Plana-Durán, and L. Enjuanes. 2003. Engineering the transmissible gastroenteritis virus genome as an expression vector inducing lactogenic immunity. J. Virol. 77:4357-4369.
Sola, I., J. L. Moreno, S. Zúiga, S. Alonso, and L. Enjuanes. 2005. Role of nucleotides immediately flanking the transcription-regulating sequence core in coronavirus subgenomic mRNA synthesis. J. Virol. 79:2506-2516.
St-Jean, J. R., H. Jacomy, M. Desforges, A. Vabret, F. Freymuth, and P. J. Talbot. 2004. Human respiratory coronavirus OC43: genetic stability and neuroinvasion. J. Virol. 78:8824-8834.
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.
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.
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.
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.
Zúiga, S., I. Sola, S. Alonso, and L. Enjuanes. 2004. Sequence motifs involved in the regulation of discontinuous coronavirus subgenomic RNA synthesis. J. Virol. 78:980-994.(Julien R. St-Jean, Marc D)
Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma, Darwin 3, 28049 Madrid, Spain
ABSTRACT
This study describes the assembly of a full-length cDNA clone of human coronavirus (HCoV)-OC43 in a bacterial artificial chromosome (BAC). The BAC containing the full-length infectious cDNA (pBAC-OC43FL) was assembled using a two-part strategy. The first step consisted in the introduction of each end of the viral genome into the BAC with accessory sequences allowing proper transcription. The second step consisted in the insertion of the whole HCoV-OC43 cDNA genome into the BAC. To produce recombinant viral particles, pBAC-OC43FL was transfected into BHK-21 cells. Recombinant virus displayed the same phenotypic properties as the wild-type virus, including infectious virus titers produced in cell culture and neurovirulence in mice.
TEXT
Human coronaviruses (HCoVs) are positive-stranded enveloped RNA viruses that are mainly associated with respiratory infections and are responsible for up to one-third of common colds (10) and possibly neurologic disease (3, 8). Like SARS-CoV, which was identified as the causative agent of severe acute respiratory syndrome (SARS) (11), HCoV-OC43 (ATCC VR-759 from the mid 1960s; GenBank accession number AY585228) (17) belongs to the second genetic group of the Coronaviridae (7, 14).
Several coronavirus infectious clones have been assembled, although this has proven difficult given the length of the viral genome (9), as well as the presence of unstable viral cDNA regions in bacteria (2, 6, 21). Uncommon strategies were therefore necessary to circumvent technical limitations engendered by the viral genome. Among the strategies available for the construction (2, 4, 5, 18, 19, 20, 21), the bacterial artificial chromosome (BAC) approach was selected given the availability of the technology, as well as the number of possibilities it offers for downstream applications (1, 2, 15, 16, 22). This approach uses a two-step amplification system that couples viral RNA expression in the nucleus from the cytomegalovirus (CMV) immediate-early promoter, with a second amplification step in the cytoplasm driven by the viral polymerase.
Construction of the full-length HCoV-OC43 cDNA clone. Prior to introducing the full-length viral cDNA into the pBeloBAC11 (13), pBAC-OC43-5'-3' was first assembled. This plasmid includes the first 720 nucleotides (nt) of the genome under the control of the CMV promoter, and the last 1,651 nt of the viral RNA, followed by a 25-bp synthetic poly(A) tail, the hepatitis delta virus ribozyme (HDV) and the bovine growth hormone termination and polyadenylation sequences (BGH) to lead to an accurate 3' end. In order to join the CMV promoter with the viral 5' end and the viral 3' end with the HDV-BGH sequences in a contiguous and very precise way, overlapping PCRs were performed.
Primers used to perform the overlapping PCRs are listed in Table 1. The vector pBAC-TGEV-5'-3', which contains the accessory sequences CMV, HDV, and BGH, was used as a template to amplify these latter sequences. After two rounds of amplification, an overlapping PCR was performed for each end of the viral genome (Fig. 1A). Both overlapping PCR amplicons were directly introduced into the pBeloBAC11 plasmid in order to create the pBAC-OC43-5'-3' (Fig. 1B). After each cloning step, the new BAC vector was sequenced to assess that no undesired mutations were introduced into the new construct.
Before performing the assembly of the full-length cDNA clone, 5' rapid amplification of cDNA ends was carried out with RNA extracted from BHK-21 cells transfected with pBAC-OC43-5'-3' in order to check that the transcription machinery was functional (data not shown). The HDV cleavage efficiency was also determined by using a reverse transcription-PCR approach and, as expected, it was estimated at approximately 60% (data not shown) (12).
The HCoV-OC43 infectious cDNA clone was assembled using a five-step cloning approach, starting with the pBAC-OC43-5'-3' as a backbone. Given the low availability of unique restriction sites in the HCoV-OC43 genome, three additional restriction sites (MluI, AsiSI, and SacII) were introduced into the sequence of the cDNA clone. This modification required the introduction of six silent point mutations (T6479C, T6482G, C12143T, T12146C, A24068G, and T24071G). These sequence changes were used as genetic markers to identify the recombinant virus (2, 19). For cDNA assembly, we took advantage of the presence of the unique SfoI restriction site in both the BAC vector and the HCoV-OC43 genome (Fig. 1B and 2A).
The genome of HCoV-OC43 was therefore amplified in five distinct overlapping regions, named A to E, which spanned the entire viral genome, except for the 5' and 3' ends (Fig. 2A). Each region was generated in such a way that it contained a given cloning site at one end and a SfoI site at the other end. Primers used for the generation of region A to E are described in Table 1. Each amplicon was first inserted into the pSMART HCKan vector in order to generate plasmids pSMART-A to -E (Fig. 2A) and positive clones with the appropriate sequence were obtained.
Viral DNA regions contained in clones pSMART-A to -E were used to fill the pBAC-OC43-5'-3' and to generate the pBAC-OC43FL (Fig. 2B). Regions A to E were respectively introduced into the BAC vector using the restriction sites they were sharing with the construct (Fig. 2A). Region E, which contained the disrupted unstable segment of ORF1a, was introduced at the last cloning step to minimize the toxicity problem usually observed with this region of the genome (6, 21). Junction sites created for each new construct were sequenced to make sure no unexpected mutations were introduced into the genome.
Recovery, replication, and neurovirulence of recombinant HCoV-OC43. One of the major advantage provided by the BAC system is that the recovery of recombinant viral particles is very efficient and simple. This was especially true for pBAC-OC43FL given that the cell line BHK-21 provided a good transfection rate and was also susceptible to infection by HCoV-OC43. For rescue of recombinant virus, 5 μg of pBAC-OC43FL were directly transfected into BHK-21 cells using Lipofectamine 2000 (Invitrogen). Virus titers obtained for the recombinant virus reached 106.5 50% tissue culture infective doses (TCID50)/ml, which are comparable to those obtained after infection with the HCoV-OC43 laboratory strain. To make sure rescued viruses were indeed recombinant particles, reverse transcription-PCR were performed and the presence of the newly introduced restriction sites, which are exclusive to the recombinant virus, was assessed (data not shown).
To test whether the recombinant virus displayed the same growth kinetics as the parental strain in HRT-18 cells, replication was assessed at different times postinfection in a multicycle growth curve. Wild-type and recombinant virus shared very similar overall patterns of replication, reaching virus titers of approximately 106.0 TCID50/ml after 4 days of infection, although the replication of the recombinant virus was slightly delayed during the first 2 days (Fig. 3A). As expected and like the wild-type HCoV-OC43 strain, recombinant virus did not form any plaques (data not shown). Taken together, these data confirm that the recombinant virus shares phenotypic properties with the wild type HCoV-OC43 strain in cell culture.
To determine whether the HCoV-OC43 strain, as well as the recombinant virus, displayed the same phenotype in vivo, their neurovirulence was assessed in mice. C57BL/6 and BALB/c mice were infected intracerebrally with either virus (8). In C57BL/6 mice, a survival rate of 30% was achieved for both viruses, whereas 70 and 80% of BALB/c mice survived the infection with the recombinant virus and the parent strain, respectively (Fig. 3B). Both recombinant and wild-type virus displayed very similar levels of virulence following injection into the brain. Furthermore, both viruses showed very similar patterns of replication in mouse brain (data not shown).
Overall, the virus recovered from the infectious cDNA clone exhibited the same genotypic and phenotypic properties as the HCoV-OC43 from ATCC, both in vitro and in vivo.
The cDNA infectious clones represent very interesting tools for the study of potential vaccine candidates, like genetically attenuated viruses, but also allow multiple possibilities for the characterization of any viral functions and mechanisms by reverse genetics. The pBAC-OC43FL will be invaluable in the characterization of the adaptation of the HCoV-OC43 virus in a neural environment and will provide an opportunity to better understand the biology of the HCoV-OC43, as well as other related coronaviruses, such as SARS-CoV.
ACKNOWLEDGMENTS
We are grateful to Francine Lambert for technical assistance with cell and virus culture. We thank R. C. Levesque (Universite Laval, Quebec, Canada) for providing vector pBeloBAC11 and Luc Gagnon (Ecopia Biosciences, Inc., Montreal, Quebec, Canada) for his valuable advice regarding cloning techniques in the BAC vector.
This work was supported by grant MT-9203 from Canadian Institutes for Health Research (Institute of Infection and Immunity) to P.J.T., who is the holder of a Tier-1 Canada Research Chair, and by a grant from the Department of Education and Science of Spain (BIO2004-00636) to L.E. J.R.S.-J. acknowledges a studentship from the Fonds Quebecois de Recherche sur la Nature et les Technologies.
Supplemental material for this article may be found at http://jvi.asm.org/.
REFERENCES
Almazán, F., C. Galán, and L. Enjuanes. 2004. The nucleoprotein is required for efficient coronavirus genome replication. J. Virol. 78:12683-12688.
Almazán, F., J. M. González, Z. Penzes, A. Izeta, E. Calvo, J. Plana-Durán, and L. Enjuanes. 2000. Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proc. Natl. Acad. Sci. USA 97:5516-5521.
Arbour, N., R. Day, J. Newcombe, and P. J. Talbot. 2000. Neuroinvasion by human respiratory coronaviruses. J. Virol. 74:8913-8921.
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.
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.
Gonzalez, J. M., Z. Penzes, F. Almazán, 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.
Gorbalenya, A. E., E. J. Snijder, and W. J. Spaan. 2004. Severe acute respiratory syndrome coronavirus phylogeny: toward consensus. J. Virol. 78:7863-7866.
Jacomy, H., and P. J. Talbot. 2003. Vacuolating encephalitis in mice infected by human coronavirus OC43. Virology 315:20-33.
Masters, P. S. 1999. Reverse genetics of the largest RNA viruses. Adv. Virus Res. 53:245-264.
Myint, S. H. 1994. Human coronaviruses: a brief review. Rev. Med. Virol. 4:35-46.
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.
Sharmeen, L., M. Y. P. Kuo, G. Dinter-Gottlieb, and J. Taylor. 1988. Antigenomic RNA of human hepatitis delta virus can undergo self-cleavage. J. Virol. 62:2674-2679.
Shizuya, H., B. Birren, U.-J. Kim, V. Mancino, T. Slepak, Y. Tachiiri, and M. Simon. 1992. Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc. Natl. Acad. Sci. USA 89:8794-8797.
Snijder, E. J., P. J. Bredenbeek, J. C. Dobbe, V. Thiel, J. Ziebuhr, L. L. Poon, Y. Guan, M. Rozanov, W. J. M. Spaan, and A. E. Gorbalenya. 2003. Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J. Mol. Biol. 331:991-1004.
Sola, I., S. Alonso, S. Zúiga, M. Balasch, J. Plana-Durán, and L. Enjuanes. 2003. Engineering the transmissible gastroenteritis virus genome as an expression vector inducing lactogenic immunity. J. Virol. 77:4357-4369.
Sola, I., J. L. Moreno, S. Zúiga, S. Alonso, and L. Enjuanes. 2005. Role of nucleotides immediately flanking the transcription-regulating sequence core in coronavirus subgenomic mRNA synthesis. J. Virol. 79:2506-2516.
St-Jean, J. R., H. Jacomy, M. Desforges, A. Vabret, F. Freymuth, and P. J. Talbot. 2004. Human respiratory coronavirus OC43: genetic stability and neuroinvasion. J. Virol. 78:8824-8834.
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.
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.
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.
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.
Zúiga, S., I. Sola, S. Alonso, and L. Enjuanes. 2004. Sequence motifs involved in the regulation of discontinuous coronavirus subgenomic RNA synthesis. J. Virol. 78:980-994.(Julien R. St-Jean, Marc D)