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Category: Viruses and Viral Pathogenesis
Genetics and Reverse Genetics of Nidoviruses, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555815790/9781555814557_Chap04-1.gif /docserver/preview/fulltext/10.1128/9781555815790/9781555814557_Chap04-2.gifAbstract:
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.
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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.
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.
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.
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.
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.
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.
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.
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.
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.