Luis Enjuanes

Nidovirales: Evolving the largest RNA virus genome

Abstract This review focuses on the monophyletic group of animal RNA viruses united in the order Nidovirales. The order includes the distantly related coronaviruses, toroviruses, and roniviruses, which possess the largest known RNA genomes (from 26 to 32kb) and will therefore be called ‘large’ nidoviruses in this review. They are compared with their arterivirus cousins, which also belong to the Nidovirales despite having a much smaller genome (13–16kb). Common and unique features that have been identified for either large or all nidoviruses are outlined. These include the nidovirus genetic plan and genome diversity, the composition of the replicase machinery and virus particles, virus-specific accessory genes, the mechanisms of RNA and protein synthesis, and the origin and evolution of nidoviruses with small and large genomes. Nidoviruses employ single-stranded, polycistronic RNA genomes of positive polarity that direct the synthesis of the subunits of the replicative complex, including the RNA-dependent RNA polymerase and helicase. Replicase gene expression is under the principal control of a ribosomal frameshifting signal and a chymotrypsin-like protease, which is assisted by one or more papain-like proteases. A nested set of subgenomic RNAs is synthesized to express the 3′-proximal ORFs that encode most conserved structural proteins and, in some large nidoviruses, also diverse accessory proteins that may promote virus adaptation to specific hosts. The replicase machinery includes a set of RNA-processing enzymes some of which are unique for either all or large nidoviruses. The acquisition of these enzymes may have improved the low fidelity of RNA replication to allow genome expansion and give rise to the ancestors of small and, subsequently, large nidoviruses.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV); Announcement of the Coronavirus Study Group

During the summer of 2012, in Jeddah, Saudi Arabia, a hitherto unknown coronavirus (CoV) was isolated from the sputum of a patient with acute pneumonia and renal failure ([1][1], [2][2]). The isolate was provisionally called human coronavirus Erasmus Medical Center (EMC) ([3][3]). Shortly thereafter

A comparative sequence analysis to revise the current taxonomy of the family Coronaviridae.

Summary. The Coronaviridae family, comprising the Coronavirus and Torovirus genera, is part of the Nidovirales order that also includes two other families, Arteriviridae and Roniviridae. Based on genetic and serological relationships, groups 1, 2 and 3 were previously recognized in the Coronavirus genus. In this report we present results of comparative sequence analysis of the spike (S), envelope (E), membrane (M), and nucleoprotein (N) structural proteins, and the two most conserved replicase domains, putative RNA-dependent RNA polymerase (RdRp) and RNA helicase (HEL), aimed at a revision of the Coronaviridae taxonomy. The results of pairwise comparisons involving structural and replicase proteins of the Coronavirus genus were consistent and produced percentages of sequence identities that were distributed in discontinuous clusters. Inter-group pairwise scores formed a single cluster in the lowest percentile. No homologs of the N and E proteins have been found outside coronaviruses, and the only (very) distant homologs of S and M proteins were identified in toroviruses. Intragroup sequence conservation was higher, although for some pairs, especially those from the most diverse group 1, scores were close or even overlapped with those from the intergroup comparisons. Phylogenetic analysis of six proteins using a neighbor-joining algorithm confirmed three coronavirus groups. Comparative sequence analysis of RdRp and HEL domains were extended to include arterivirus and ronivirus homologs. The pairwise scores between sequences of the genera Coronavirus and Torovirus (22–25% and 21–25%) were found to be very close to or overlapped with the value ranges (12 to 22% and 17 to 25%) obtained for interfamily pairwise comparisons, but were much smaller than values derived from pairwise comparisons within the Coronavirus genus (63–71% and 59–67%). Phylogenetic analysis confirmed toroviruses and coronaviruses to be separated by a large distance that is comparable to those between established nidovirus families. Based on comparison of these scores with those derived from analysis of separate ranks of several multi-genera virus families, like the Picornaviridae, a revision of the Coronaviridae taxonomy is proposed. We suggest the Coronavirus and Torovirus genera to be re-defined as two subfamilies within the Coronavirdae or two families within Nidovirales, and the current three informal coronavirus groups to be converted into three genera within the Coronaviridae.

Rapid generation of a mouse model for Middle East respiratory syndrome

Significance The Middle East respiratory syndrome (MERS)-coronavirus, a newly identified pathogen, causes severe pneumonia in humans, with a mortality of nearly 44%. Human-to-human spread has been demonstrated, raising the possibility that the infection could become pandemic. Mice and other small laboratory animals are not susceptible to infection. Here, we describe the development of a small-animal model for MERS, in which we use an adenovirus expressing the human host-cell receptor to sensitize mice for infection. We show that these mice are useful for determining immune responses and for evaluation of an anti-MERS vaccine and an antiviral therapy. This approach will be generally useful for the rapid (2–3 wk) development of relevant mouse and other animal models for emerging viral infections. In this era of continued emergence of zoonotic virus infections, the rapid development of rodent models represents a critical barrier to public health preparedness, including the testing of antivirus therapy and vaccines. The Middle East respiratory syndrome coronavirus (MERS-CoV) was recently identified as the causative agent of a severe pneumonia. Given the ability of coronavirus to rapidly adapt to new hosts, a major public health concern is that MERS-CoV will further adapt to replication in humans, triggering a pandemic. No small-animal model for this infection is currently available, but studies suggest that virus entry factors can confer virus susceptibility. Here, we show that mice were sensitized to MERS-CoV infection by prior transduction with adenoviral vectors expressing the human host-cell receptor dipeptidyl peptidase 4. Mice developed a pneumonia characterized by extensive inflammatory-cell infiltration with virus clearance occurring 6–8 d after infection. Clinical disease and histopathological changes were more severe in the absence of type-I IFN signaling whereas the T-cell response was required for virus clearance. Using these mice, we demonstrated the efficacy of a therapeutic intervention (poly I:C) and a potential vaccine [Venezuelan equine encephalitis replicon particles expressing MERS-CoV spike protein]. We also found little protective cross-reactivity between MERS-CoV and the severe acute respiratory syndrome-CoV. Our results demonstrate that this system will be useful for MERS-CoV studies and for the rapid development of relevant animal models for emerging respiratory viral infections.

Antigenic homology among coronaviruses related to transmissible gastroenteritis virus.

Abstract The antigenic homology of 26 coronavirus isolates, of which 22 were antigenically related to transmissible gastroenteritis virus (TGEV), was determined with 42 monoclonal antibodies. Type, group, and interspecies specific epitopes were defined. Two group specific MAbs distinguished the enteric TGEV isolates from the respiratory variants. An antigenic subsite involved in neutralization was conserved in porcine, feline, and canine coronavirus. The classification of the human coronavirus 229E in a taxonomic cluster distinct from TGEV group is suggested.

Sequence motifs involved in the regulation of discontinuous coronavirus subgenomic RNA synthesis

ABSTRACT Coronavirus transcription leads to the synthesis of a nested set of mRNAs with a leader sequence derived from the 5′ end of the genome. The mRNAs are produced by a discontinuous transcription in which the leader is linked to the mRNA coding sequences. This process is regulated by transcription-regulating sequences (TRSs) preceding each mRNA, including a highly conserved core sequence (CS) with high identity to sequences present in the virus genome and at the 3′ end of the leader (TRS-L). The role of TRSs was analyzed by reverse genetics using a full-length infectious coronavirus cDNA and site-directed mutagenesis of the CS. The canonical CS-B was nonessential for the generation of subgenomic mRNAs (sgmRNAs), but its presence led to transcription levels at least 103-fold higher than those in its absence. The data obtained are compatible with a transcription mechanism including three steps: (i) formation of 5′-3′ complexes in the genomic RNA, (ii) base-pairing scanning of the nascent negative RNA strand by the TRS-L, and (iii) template switching during synthesis of the negative strand to complete the negative sgRNA. This template switch takes place after copying the CS sequence and was predicted in silico based on high base-pairing score between the nascent negative RNA strand and the TRS-L and minimum ΔG.

The Nucleoprotein Is Required for Efficient Coronavirus Genome Replication

ABSTRACT 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.

Two Amino Acid Changes at the N-Terminus of Transmissible Gastroenteritis Coronavirus Spike Protein Result in the Loss of Enteric Tropism

Abstract To study the molecular basis of TGEV tropism, a collection of recombinants between the PUR46-MAD strain of transmissible gastroenteritis coronavirus (TGEV) infecting the enteric and respiratory tracts and the PTV strain, which only infects the respiratory tract, was generated. The recombinant isolation frequency was about 10−9recombinants per nucleotide and was 3.7-fold higher at the 5′-end of the S gene than in other areas of the genome. Thirty recombinants were plaque purified and characterized phenotypically and genetically. All recombinant viruses had a single crossover and had inherited the 5′- and 3′-halves of their genome from the enteric and respiratory parents, respectively. Recombinant viruses were classified into three groups, named 1 to 3, according to the location of the crossover. Group 1 recombinants had the crossover in the S gene, while in Groups 2 and 3 the crossovers were located in ORF1b and ORF1a, respectively. The tropism of the recombinants was studied. Recombinants of Group 1 had enteric and respiratory tropism, while Group 2 recombinants infected the respiratory, but not the enteric, tract. Viruses of both groups differed by two nucleotide changes at positions 214 and 655. Both changes may be in principle responsible for the loss of enteric tropism but only the change in nucleotide 655 was specifically found in the respiratory isolates and most likely this single nucleotide change, which leads to a substitution in amino acid 219 of the S protein, was responsible for the loss of enteric tropism in the closely related PUR-46 isolates. The available data indicate that in order to infect enteric tract cells with TGEV, two different domains of the S protein, mapping between amino acids 522 and 744 and around amino acid 219, respectively, are involved. The first domain binds to porcine aminopeptidase N, the cellular receptor for TGEV. In the other domain maps a second factor of undefined nature but which may be the binding site for a coreceptor essential for the enteric tropism of TGEV.

Genetic evolution and tropism of transmissible gastroenteritis coronaviruses

Abstract Transmissible gastroenteritis virus (TGEV) is an enteropathogenic coronavirus isolated for the first time in 1946. Nonenteropathogenic porcine respiratory coronaviruses (PRCVs) have been derived from TGEV. The genetic relationship among six European PRCVs and five coronaviruses of the TGEV antigenic cluster has been determined based on their RNA sequences. The S protein of six PRCVs have an identical deletion of 224 amino acids starting at position 21. The deleted area includes the antigenic sites C and B of TGEV S glycoprotein. Interestingly, two viruses (NEB72 and TOY56) with respiratory tropism have S proteins with a size similar to the enteric viruses. NEB72 and TOY56 viruses have in the S protein 2 and 15 specific amino acid differences with the enteric viruses. Four of the residues changed (aa 219 of NEB72 isolate and as 92, 94, and 218 of TOY56) are located within the deletion present in the PRCVs and may be involved in the receptor binding site (RBS) conferring enteric tropism to TGEVs. A second RBS used by the virus to infect ST cells might be located in a conserved area between sites A and D of the S glycoprotein, since monoclonal antibodies specific for these sites inhibit the binding of the virus to ST cells. An evolutionary tree relating 13 enteric and respiratory isolates has been proposed. According to this tree, a main virus lineage evolved from a recent progenitor virus which was circulating around 1941. From this, secondary lineages originated PUR46, NEB72, TOY56, MIL65, BR170, and the PRCVs, in this order. Least squares estimation of the origin of TGEV-related coronaviruses showed a significant constancy in the fixation of mutations with time, that is, the existence of a well-defined molecular clock. A mutation fixation rate of 7 ± 2 × 10−4 nucleotide substitutions per site and per year was calculated for TGEV-related viruses. This rate falls in the range reported for other RNA viruses. Point mutations and probably recombination events have occurred during TGEV evolution.

Generation of a Replication-Competent, Propagation-Deficient Virus Vector Based on the Transmissible Gastroenteritis Coronavirus Genome

ABSTRACT Replication-competent propagation-deficient virus vectors based on the transmissible gastroenteritis coronavirus (TGEV) genome that are deficient in the essential E gene have been developed by complementation within E+ packaging cell lines. Cell lines expressing the TGEV E protein were established using the noncytopathic Sindbis virus replicon pSINrep21. In addition, cell lines stably expressing the E gene under the CMV promoter have been developed. The Sindbis replicon vector and the ectopic TGEV E protein did not interfere with the rescue of infectious TGEV from full-length cDNA. Recombinant TGEV deficient in the nonessential 3a and 3b genes and the essential E gene (rTGEV-Δ3abΔE) was successfully rescued in these cell lines. rTGEV-Δ3abΔE reached high titers (107 PFU/ml) in baby hamster kidney cells expressing porcine aminopeptidase N (BHK-pAPN), the cellular receptor for TGEV, using Sindbis replicon and reached titers up to 5 × 105 PFU/ml in cells stably expressing E protein under the control of the CMV promoter. The virus titers were proportional to the E protein expression level. The rTGEV-Δ3abΔE virions produced in the packaging cell line showed the same morphology and stability under different pHs and temperatures as virus derived from the full-length rTGEV genome, although a delay in virus assembly was observed by electron microscopy and virus titration in the complementation system in relation to the wild-type virus. These viruses were stably grown for >10 passages in the E+ packaging cell lines. The availability of packaging cell lines will significantly facilitate the production of safe TGEV-derived vectors for vaccination and possibly gene therapy.