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Biomedical subjects

Fernando Almazán

Publications and source records attributed to Fernando Almazán.

11 recordsLinked to original sources
PubMed2006

A severe acute respiratory syndrome coronavirus that lacks the E gene is attenuated in vitro and in vivo.

A deletion mutant of severe acute respiratory syndrome coronavirus (SARS-CoV) has been engineered by deleting the structural E gene in an infectious cDNA clone that was constructed as a bacterial artificial chromosome (BAC). The recombinant virus lacking the E gene (rSARS-CoV-DeltaE) was rescued in Vero E6 cells. The recovered deletion mutant grew in Vero E6, Huh-7, and CaCo-2 cells to titers 20-, 200-, and 200-fold lower than the recombinant wild-type virus, respectively, indicating that although the E protein has an effect on growth, it is not essential for virus replication. No differences in virion stability under a wide range of pH and temperature were detected between the deletion mutant and recombinant wild-type viruses. Although both viruses showed the same morphology by electron microscopy, the process of morphogenesis seemed to be less efficient with the defective virus than with the recombinant wild-type one. The rSARS-CoV-DeltaE virus replicated to titers 100- to 1,000-fold lower than the recombinant wild-type virus in the upper and lower respiratory tract of hamsters, and the lower viral load was accompanied by less inflammation in the lungs of hamsters infected with rSARS-CoV-DeltaE virus than with the recombinant wild-type virus. Therefore, the SARS-CoV that lacks the E gene is attenuated in hamsters, might be a safer research tool, and may be a good candidate for the development of a live attenuated SARS-CoV vaccine.

Animals↗
PubMed2006

Construction of a severe acute respiratory syndrome coronavirus infectious cDNA clone and a replicon to study coronavirus RNA synthesis.

The engineering of a full-length infectious cDNA clone and a functional replicon of the severe acute respiratory syndrome coronavirus (SARS-CoV) Urbani strain as bacterial artificial chromosomes (BACs) is described in this study. In this system, the viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and further amplified in the cytoplasm by the viral replicase. Both the infectious clone and the replicon were fully stable in Escherichia coli. Using the SARS-CoV replicon, we have shown that the recently described RNA-processing enzymes exoribonuclease, endoribonuclease, and 2'-O-ribose methyltransferase were essential for efficient coronavirus RNA synthesis. The SARS reverse genetic system developed as a BAC constitutes a useful tool for the study of fundamental viral processes and also for developing genetically defined vaccines.

Animals↗
PubMed2006

Recovery of a neurovirulent human coronavirus OC43 from an infectious cDNA clone.

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-OC43(FL)) 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-OC43(FL) 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.

Animals↗
PubMed2006

Biochemical aspects of coronavirus replication and virus-host interaction.

Infection by different coronaviruses (CoVs) causes alterations in the transcriptional and translational patterns, cell cycle, cytoskeleton, and apoptosis pathways of the host cells. In addition, CoV infection may cause inflammation, alter immune and stress responses, and modify the coagulation pathways. The balance between the up- and downregulated genes could explain the pathogenesis caused by these viruses. We review specific aspects of CoV-host interactions. CoV genome replication takes place in the cytoplasm in a membrane-protected microenvironment and may control the cell machinery by locating some of their proteins in the host cell nucleus. CoVs initiate translation by cap-dependent and cap-independent mechanisms. CoV transcription involves a discontinuous RNA synthesis (template switching) during the extension of a negative copy of the subgenomic mRNAs. The requirement for base-pairing during transcription has been formally demonstrated in arteriviruses and CoVs. CoV N proteins have RNA chaperone activity that may help initiate template switching. Both viral and cellular proteins are required for replication and transcription, and the role of selected proteins is addressed.

Animals↗
PubMed2005

A point mutation within the replicase gene differentially affects coronavirus genome versus minigenome replication.

During the construction of the transmissible gastroenteritis virus (TGEV) full-length cDNA clone, a point mutation at position 637 that was present in the defective minigenome DI-C was maintained as a genetic marker. Sequence analysis of the recovered viruses showed a reversion at this position to the original virus sequence. The effect of point mutations at nucleotide 637 was analyzed by reverse genetics using a TGEV full-length cDNA clone and cDNAs from TGEV-derived minigenomes. The replacement of nucleotide 637 of TGEV genome by a T, as in the DI-C sequence, or an A severely affected virus recovery from the cDNA, yielding mutant viruses with low titers and small plaques compared to those of the wild type. In contrast, T or A at position 637 was required for minigenome rescue in trans by the helper virus. No relationship between these observations and RNA secondary-structure predictions was found, indicating that mutations at nucleotide 637 most likely had an effect at the protein level. Nucleotide 637 occupies the second codon position at amino acid 108 of the pp1a polyprotein. This position is predicted to map in the N-terminal polyprotein papain-like proteinase (PLP-1) cleavage site at the p9/p87 junction. Replacement of G-637 by A, which causes a drastic amino acid change (Gly to Asp) at position 108, affected PLP-1-mediated cleavage in vitro. A correlation was found between predicted cleaving and noncleaving mutations and efficient virus rescue from cDNA and minigenome amplification, respectively.

Animals↗
PubMed2004

The nucleoprotein is required for efficient coronavirus genome replication.

The construction of a set of transmissible gastroenteritis coronavirus (TGEV)-derived replicons as bacterial artificial chromosomes is reported. These replicons were generated by sequential deletion of nonessential genes for virus replication, using a modified TGEV full-length cDNA clone containing unique restriction sites between each pair of consecutive genes. Efficient activity of TGEV replicons was associated with the presence of the nucleoprotein provided either in cis or in trans. TGEV replicons were functional in several cell lines, including the human cell line 293T, in which no or very low cytopathic effect was observed, and expressed high amounts of heterologous protein.

Base Sequence↗
PubMed2003

Transmissible gastroenteritis coronavirus gene 7 is not essential but influences in vivo virus replication and virulence.

Transmissible gastroenteritis coronavirus (TGEV) contains eight overlapping genes that are expressed from a 3'-coterminal nested set of leader-containing mRNAs. To facilitate the genetic manipulation of the viral genome, genes were separated by duplication of transcription regulating sequences (TRSs) and introduction of unique restriction endonuclease sites at the 5' end of each gene using an infectious cDNA clone. The recombinant TGEV (rTGEV) replicated in cell culture with similar efficiency to the wild-type virus and stably maintained the modifications introduced into the genome. In contrast, the rTGEV replication level in the lungs and gut of infected piglets and virulence were significantly reduced. rTGEV in which gene 7 expression was abrogated (rTGEV-delta7) were recovered from cDNA constructs, indicating that TGEV gene 7 was a nonessential gene for virus replication. Interestingly, in vivo infections with rTGEV-delta7 showed an additional reduction in virus replication in the lung and gut, and in virulence, indicating that TGEV gene 7 influences virus pathogenesis.

Animals↗
PubMed2002

Stabilization of a full-length infectious cDNA clone of transmissible gastroenteritis coronavirus by insertion of an intron.

The stable propagation of a full-length transmissible gastroenteritis coronavirus (TGEV) cDNA in Escherichia coli cells as a bacterial artificial chromosome has been considerably improved by the insertion of an intron to disrupt a toxic region identified in the viral genome. The viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and the intron was efficiently removed during translocation of this RNA to the cytoplasm. The insertion in two different positions allowed stable plasmid amplification for at least 200 generations. Infectious TGEV was efficiently recovered from cells transfected with the modified cDNAs.

Animals↗