Analysis of the Codon Usage Pattern in the Vibrio Cholerae Genome

Abstract

The codon usage in the Vibrio cholerae genome is analyzed in this paper. Although there are much more genes on the chromosome 1 than on chromosome 2, the codon usage patterns of genes on the two chromosomes are quite similar, indicating that the two chromosomes may have coexisted in the same cell for a very long history. Unlike the base frequency pattern observed in other genomes, the G+C content at the third codon position of the V. cholerae genome varies in a rather small interval. The most notable feature of codon usage of V. cholerae genome is that there is a fraction of genes show significant bias in base choice at the second codon position. The 2006 known genes can be classified into two clusters according to the base frequencies at this position. The smaller cluster contains 227 genes, most of which code for proteins involved in transport and binding functions. The encoding products of these genes have significant bias in amino acids composition as compared with other genes. The codon usage patterns for the 1836 function unknown ORFs are also analyzed, which is useful to study their functions.     

Unusual codon usage bias in low expression genes of Vibrio cholerae

Positive correlation between gene expression and synonymous codon usage bias is well documented in the literature. However, in the present study of Vibrio cholerae genome, we have identified a group of genes having unusually high codon usage bias despite being low potential expressivity. Our results suggest that codon usage in lowly expressed genes might also be selected on to preferably use non-optimal codons to maintain a low cellular concentration of the proteins that they encode. This would predict that lowly expressed genes are also biased in codon usage, but in a way that is opposite to the bias of highly expressed genes.     

Genomic and Phenotypic Diversity of Coastal Vibrio cholerae Strains Is Linked to Environmental Factors

Studies of Vibrio cholerae diversity have focused primarily on pathogenic isolates of the O1 and O139 serotypes. However, autochthonous environmental isolates of this species routinely display more extensive genetic diversity than the primarily clonal pathogenic strains. In this study, genomic and metabolic profiles of 41 non-O1/O139 environmental isolates from central California coastal waters and four clinical strains are used to characterize the core genome and metabolome of V. cholerae. Comparative genome hybridization using microarrays constructed from the fully sequenced V. cholerae O1 El Tor N16961 genome identified 2,787 core genes that approximated the projected species core genome within 1.6%. Core genes are almost universally present in strains with widely different niches, suggesting that these genes are essential for persistence in diverse aquatic environments. In contrast, the dispensable genes and phenotypic traits identified in this study should provide increased fitness for certain niche environments. Environmental parameters, measured in situ during sample collection, are correlated to the presence of specific dispensable genes and metabolic capabilities, including utilization of mannose, sialic acid, citrate, and chitosan oligosaccharides. These results identify gene content and metabolic pathways that are likely selected for in certain coastal environments and may influence V. cholerae population structure in aquatic environments.     

Codon usage and gene expression pattern of Stenotrophomonas maltophilia R551-3 for pathogenic mode of living

Stenotrophomonas maltophilia strain R551-3 is a multiple-antibiotic-resistant opportunistic human pathogen involved in nosocomial infections. It has a widely distributed GC-rich (>66%) genome. Analysis of differential expression of the genes of this genome reveals that majority of genes belonging to highly expressed category are mostly present on lagging strand without showing any strand specific codon usage bias. Relatively small number of lowly expressed genes is equally distributed on both leading and lagging strands with a difference in codon usage pattern between them. Among several multi drug resistance genes of S. maltophilia involving lowly expressed category some are predicted as horizontally transferred. It can be inferred that horizontally transferred genes may have been imported into this genome for their pathogenic mode of living. Our study may help to modify the expression level of the target genes of this human pathogen in order to control its infection.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C resonance assignments of the C-terminal domain of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> TolA protein

Vibrio cholerae is the bacterial causative agent of the human disease cholera. Non-pathogenic bacterium can be converted to pathogenic following infection by a filamentous phage, CTXΦ, that carries the cholera toxin encoding genes. A crucial step during phage infection requires a direct interaction between the CTXΦ minor coat protein (pIII^CTX) and the C-terminal domain of V. cholerae TolA protein (TolAIIIvc). In order to get a better understanding of TolA function during the infection process, we have initiated a study of the V. cholerae TolAIII domain by 2D and 3D heteronuclear NMR. With the exception of the His-tag (H123–H128), 97 % of backbone ¹H, ¹⁵N and ¹³C resonances were assigned and the side chain assignments for 92 % of the protein were obtained (BMRB deposit with accession number 25689).     

Vibrio vulnificus Phage PV94 Is Closely Related to Temperate Phages of V. cholerae and Other Vibrio Species

Background

Vibrio vulnificus is an important pathogen which can cause serious infections in humans. Yet, there is limited knowledge on its virulence factors and the question whether temperate phages might be involved in pathogenicity, as is the case with V. cholerae. Thus far, only two phages (SSP002 and VvAW1) infecting V. vulnificus have been genetically characterized. These phages were isolated from the environment and are not related to Vibrio cholerae phages. The lack of information on temperate V. vulnificus phages prompted us to isolate those phages from lysogenic strains and to compare them with phages of other Vibrio species.

Results

In this study the temperate phage PV94 was isolated from a V. vulnificus biotype 1 strain by mitomycin C induction. PV94 is a myovirus whose genome is a linear double-stranded DNA of 33,828 bp with 5′-protruding ends. Sequence analysis of PV94 revealed a modular organization of the genome. The left half of the genome comprising the immunity region and genes for the integrase, terminase and replication proteins shows similarites to V. cholerae kappa phages whereas the right half containing genes for structural proteins is closely related to a prophage residing in V. furnissii NCTC 11218.

Conclusion

We present the first genomic sequence of a temperate phage isolated from a human V. vulnificus isolate. The sequence analysis of the PV94 genome demonstrates the wide distribution of closely related prophages in various Vibrio species. Moreover, the mosaicism of the PV94 genome indicates a high degree of horizontal genetic exchange within the genus Vibrio, by which V. vulnificus might acquire virulence-associated genes from other species.     

Non-O1/Non-O139 Vibrio cholerae Carrying Multiple Virulence Factors and V. cholerae O1 in the Chesapeake Bay,Maryland

Non-O1/non-O139 Vibrio cholerae inhabits estuarine and coastal waters globally, but its clinical significance has not been sufficiently investigated, despite the fact that it has been associated with septicemia and gastroenteritis. The emergence of virulent non-O1/non-O139 V. cholerae is consistent with the recognition of new pathogenic variants worldwide. Oyster, sediment, and water samples were collected during a vibrio surveillance program carried out from 2009 to 2012 in the Chesapeake Bay, Maryland. V. cholerae O1 was detected by a direct fluorescent-antibody (DFA) assay but was not successfully cultured, whereas 395 isolates of non-O1/non-O139 V. cholerae were confirmed by multiplex PCR and serology. Only a few of the non-O1/non-O139 V. cholerae isolates were resistant to ampicillin and/or penicillin. Most of the isolates were sensitive to all antibiotics tested, and 77 to 90% carried the El Tor variant hemolysin gene hlyA_ET, the actin cross-linking repeats in toxin gene rtxA, the hemagglutinin protease gene hap, and the type 6 secretion system. About 19 to 21% of the isolates carried the neuraminidase-encoding gene nanH and/or the heat-stable toxin (NAG-ST), and only 5% contained a type 3 secretion system. None of the non-O1/non-O139 V. cholerae isolates contained Vibrio pathogenicity island-associated genes. However, ctxA, ace, or zot was present in nine isolates. Fifty-five different genotypes showed up to 12 virulence factors, independent of the source of isolation, and represent the first report of both antibiotic susceptibility and virulence associated with non-O1/non-O139 V. cholerae from the Chesapeake Bay. Since these results confirm the presence of potentially pathogenic non-O1/non-O139 V. cholerae, monitoring for total V. cholerae, regardless of serotype, should be done within the context of public health.     

Ribotyping and TCP gene cluster analysis of environmental and clinical Vibrio cholerae strains isolated in Iran

The aim of this study was to investigate the presence of TCP gene clusters among clinical and environmental Vibrio cholerae isolates and to explore the genetic relatedness of isolates using ribotyping technique. A total of 50 V. cholerae strains (30 clinical and 20 environmental) were included in this study. Three clinical isolates were negative for TCP cluster genes while the cluster was absent in all of the environmental strains. Ribotyping of rRNA genes with BglI produced 18 different ribotype patterns, three of which belonged to clinical O1 serotype isolates. The remaining 15 ribotypes belonged to clinical non-O1, non-O139 serogroups (two patterns) and environmental non-O1, non-O139 serogroups (13 patterns). Clinical V. cholerae O1 strains from 2004 through 2006 and several environmental non-O1, non-O139 V. cholerae strains from 2006 showed 67.3 % similarity and fell within one single gene cluster. Ribotyping analysis made it possible to further comprehend the close originality of clinical isolates as very little changes have been occurred within rRNA genes of different genotypes of V. cholerae strains through years. In conclusion, ribotyping analysis of environmental V. cholerae isolates showed a substantial genomic diversity supporting the fact that genetic changes within bacterial genome occurs during years in the environment, while only little changes may arise within the genome of clinical isolates.     

Detecting anomalous gene clusters and pathogenicity islands in diverse bacterial genomes.

A gene in a genome is defined as putative alien (pA) if its codon usage difference from the average gene exceeds a high threshold and codon usage differences from ribosomal protein genes, chaperone genes and protein-synthesis-processing factors are also high. pA gene clusters in bacterial genomes are relevant for detecting genomic islands (GIs), including pathogenicity islands (PAIs). Four other analyses appropriate to this task are G+C genome variation (the standard method); genomic signature divergences (dinucleotide bias); extremes of codon bias; and anomalies of amino acid usage. For example, the cagA domain of Helicobacter pylori is highly deviant in its genome signature and codon bias from the rest of the genome. Using these methods we can detect two potential PAIs in the Neisseria meningitidis genome, which contain hemagglutinin and/or hemolysin-related genes. Additionally, G+C variation and genome signature differences of the Mycobacterium tuberculosis genome indicate two pA gene clusters.     

Comparative evolutionary genomics of <Emphasis Type="Italic">Corynebacterium</Emphasis> with special reference to codon and amino acid usage diversities

The present study has been aimed to the comparative analysis of high GC composition containing Corynebacterium genomes and their evolutionary study by exploring codon and amino acid usage patterns. Phylogenetic study by MLSA approach, indel analysis and BLAST matrix differentiated Corynebacterium species in pathogenic and non-pathogenic clusters. Correspondence analysis on synonymous codon usage reveals that, gene length, optimal codon frequencies and tRNA abundance affect the gene expression of Corynebacterium. Most of the optimal codons as well as translationally optimal codons are C ending i.e. RNY (R-purine, N-any nucleotide base, and Y-pyrimidine) and reveal translational selection pressure on codon bias of Corynebacterium. Amino acid usage is affected by hydrophobicity, aromaticity, protein energy cost, etc. Highly expressed genes followed the cost minimization hypothesis and are less diverged at their synonymous positions of codons. Functional analysis of core genes shows significant difference in pathogenic and non-pathogenic Corynebacterium. The study reveals close relationship between non-pathogenic and opportunistic pathogenic Corynebaterium as well as between molecular evolution and survival niches of the organism.     

Isolation and Characterization of the New Mosaic Filamentous Phage VFJ Φ of Vibrio cholerae

Filamentous phages have distinguished roles in conferring many pathogenicity and survival related features to Gram-negative bacteria including the medically important Vibrio cholerae, which carries factors such as cholera toxin on phages. A novel filamentous phage, designated VFJΦ, was isolated in this study from an ampicillin and kanamycin-resistant O139 serogroup V. cholerae strain ICDC-4470. The genome of VFJΦ is 8555 nucleotides long, including 12 predicted open reading frames (ORFs), which are organized in a modular structure. VFJΦ was found to be a mosaic of two groups of V. cholerae phages. A large part of the genome is highly similar to that of the fs2 phage, and the remaining 700 bp is homologous to VEJ and VCYΦ. This 700 bp region gave VFJΦ several characteristics that are not found in fs2 and other filamentous phages. In its native host ICDC-4470 and newly-infected strain N16961, VFJΦ was found to exist as a plasmid but did not integrate into the host chromosome. It showed a relatively wide host range but did not infect the classical biotype O1 V. cholerae strains. After infection, the host strains exhibited obvious inhibition of both growth and flagellum formation and had acquired a low level of ampicillin resistance and a high level of kanamycin resistance. The antibiotic resistances were not directly conferred to the hosts by phage-encoded genes and were not related to penicillinase. The discovery of VFJΦ updates our understanding of filamentous phages as well as the evolution and classification of V. cholerae filamentous phage, and the study provides new information on the interaction between phages and their host bacteria.     

Impact of translational selection on codon usage bias in the archaeon Methanococcus maripaludis

Patterns of codon usage have been extensively studied among Bacteria and Eukaryotes, but there has been little investigation of species from the third domain of life, the Archaea. Here, we examine the nature of codon usage bias in a methanogenic archaeon, Methanococcus maripaludis. Genome-wide patterns of codon usage are dominated by a strong A + T bias, presumably largely reflecting mutation patterns. Nevertheless, there is variation among genes in the use of a subset of putatively translationally optimal codons, which is strongly correlated with gene expression level. In comparison with Bacteria such as Escherichia coli, the strength of selected codon usage bias in highly expressed genes in M. maripaludis seems surprisingly high given its moderate growth rate. However, the pattern of selected codon usage differs between M. maripaludis and E. coli: in the archaeon, strongly selected codon usage bias is largely restricted to twofold degenerate amino acids (AAs). Weaker bias among the codons for fourfold degenerate AAs is consistent with the small number of tRNA genes in the M. maripaludis genome.     

Role of the Vibrio cholerae Matrix Protein Bap1 in Cross-Resistance to Antimicrobial Peptides

Outer membrane vesicles (OMVs) that are released from Gram-negative pathogenic bacteria can serve as vehicles for the translocation of effectors involved in infectious processes. In this study we have investigated the role of OMVs of the Vibrio cholerae O1 El Tor A1552 strain in resistance to antimicrobial peptides (AMPs). To assess this potential role, we grew V. cholerae with sub-lethal concentrations of Polymyxin B (PmB) or the AMP LL-37 and analyzed the OMVs produced and their effects on AMP resistance. Our results show that growing V. cholerae in the presence of AMPs modifies the protein content of the OMVs. In the presence of PmB, bacteria release OMVs that are larger in size and contain a biofilm-associated extracellular matrix protein (Bap1). We demonstrated that Bap1 binds to the OmpT porin on the OMVs through the LDV domain of OmpT. In addition, OMVs from cultures incubated in presence of PmB also provide better protection for V. cholerae against LL-37 compared to OMVs from V. cholerae cultures grown without AMPs or in presence of LL-37. Using a bap1 mutant we showed that cross-resistance between PmB and LL-37 involved the Bap1 protein, whereby Bap1 on OMVs traps LL-37 with no subsequent degradation of the AMP.     

Genome-wide analysis of codon usage bias patterns in an enterotoxigenic <Emphasis Type="Italic">Escherichia coli</Emphasis> F18 strain

Enterogenic Escherichia coli (ETEC) F18 strains are the main pathogenic bacteria causing severe diarrhea in humans and domestic animals. However, the information about synonymous codon usage pattern of ETEC F18 genome remains unclear. We conducted a genome-wide analysis of synonymous codon usage patterns in the ETEC F18 strain SRA: SAMN02471895. After filtering of the complete genome sequence, 4327 coding sequences were analyzed using multivariate statistical methods to calculate synonymous codon usage patterns and to evaluate the influence of various factors in shaping the codon usage. The mean GC content was 51.38%, with a slight preference for G/C-ending codons. Twenty-two codons were determined as ‘‘optimal codons”. ENC plots showed some of the genes were on or close to the expected curve, while only points with low-ENC values were below the curve. PR2 analysis showed that GC and AT were not used proportionally, suggesting major roles for mutational pressure and natural selection in shaping usage. Neutrality plots showed a significant correlation between GC12 and GC3, suggesting that mutational pressure is responsible for nucleotide composition in shaping the strength of codon usage. Translational selection was the main factor shaping the codon usage pattern of ETEC F18 genome, while other factors such as protein length, GRAVY and ARO values also influenced codon usage to some extent. We analyzed the codon usage pattern systematically and identified the factors shaping codon usage bias in the ETEC F18 genome. Such information further elucidates the mechanisms of synonymous codon usage bias and provides the basis of molecular genetic engineering and evolutionary studies.     

Genotypes Associated with Virulence in Environmental Isolates of Vibrio cholerae

Vibrio cholerae is an autochthonous inhabitant of riverine and estuarine environments and also is a facultative pathogen for humans. Genotyping can be useful in assessing the risk of contracting cholera, intestinal, or extraintestinal infections via drinking water and/or seafood. In this study, environmental isolates of V. cholerae were examined for the presence of ctxA, hlyA, ompU, stn/sto, tcpA, tcpI, toxR, and zot genes, using multiplex PCR. Based on tcpA and hlyA gene comparisons, the strains could be grouped into Classical and El Tor biotypes. The toxR, hlyA, and ompU genes were present in 100, 98.6, and 87.0% of the V. cholerae isolates, respectively. The CTX genetic element and toxin-coregulated pilus El Tor (tcpA ET) gene were present in all toxigenic V. cholerae O1 and V. cholerae O139 strains examined in this study. Three of four nontoxigenic V. cholerae O1 strains contained tcpA ET. Interestingly, among the isolates of V. cholerae non-O1/non-O139, two had tcpA Classical, nine contained tcpA El Tor, three showed homology with both biotype genes, and four carried the ctxA gene. The stn/sto genes were present in 28.2% of the non-O1/non-O139 strains, in 10.5% of the toxigenic V. cholerae O1, and in 14.3% of the O139 serogroups. Except for stn/sto genes, all of the other genes studied occurred with high frequency in toxigenic V. cholerae O1 and O139 strains. Based on results of this study, surveillance of non-O1/non-O139 V. cholerae in the aquatic environment, combined with genotype monitoring using ctxA, stn/sto, and tcpA ET genes, could be valuable in human health risk assessment.     

Molecular Characterization of Vibrio cholerae O1 Reveals Continuous Evolution of Its New Variants in India

Vibrio cholerae, the causing agent of cholera is still a major health challenge in most of the developing countries. In this study, V. cholerae strains collected from different cholera outbreaks in India over a period of past 7 years were found to have various toxigenic, pathogenic and regulatory genes viz. ctxAB, zot, tcp, hlyA, ace, ompU, ompW, rfbO1, toxT and toxR. The biotype specific genes rstR and rtxC revealed the El Tor biotype in majority of the isolates. However, variants among the isolates were found having genotype of both the biotypes. Sequencing of ctxB gene revealed the presence of altered ctxB of classical biotype with additional variations in isolates of 2007. Mismatch amplification mutation assay PCR also confirmed the isolates belonging to classical biotype. Antibiogram of the isolates revealed resistance for nalidixic acid, co-trimoxazole, streptomycin, and polymyxin B and susceptibility for tetracycline among most of the isolates from India. However, V. cholerae isolates from a recent outbreak in Eastern India were resistant to tetracycline. The study corroborated the continuous emergence and wide-spread of multidrug resistant El Tor variant strains in the Indian subcontinent.     

Crystal Structures of a CTXφ pIII Domain Unbound and in Complex with a Vibrio cholerae TolA Domain Reveal Novel Interaction Interfaces

Vibrio cholerae colonize the small intestine where they secrete cholera toxin, an ADP-ribosylating enzyme that is responsible for the voluminous diarrhea characteristic of cholera disease. The genes encoding cholera toxin are located on the genome of the filamentous bacteriophage, CTXφ, that integrates as a prophage into the V. cholerae chromosome. CTXφ infection of V. cholerae requires the toxin-coregulated pilus and the periplasmic protein TolA. This infection process parallels that of Escherichia coli infection by the Ff family of filamentous coliphage. Here we demonstrate a direct interaction between the N-terminal domain of the CTXφ minor coat protein pIII (pIII-N1) and the C-terminal domain of TolA (TolA-C) and present x-ray crystal structures of pIII-N1 alone and in complex with TolA-C. The structures of CTXφ pIII-N1 and V. cholerae TolA-C are similar to coliphage pIII-N1 and E. coli TolA-C, respectively, yet these proteins bind via a distinct interface that in E. coli TolA corresponds to a colicin binding site. Our data suggest that the TolA binding site on pIII-N1 of CTXφ is accessible in the native pIII protein. This contrasts with the Ff family phage, where the TolA binding site on pIII is blocked and requires a pilus-induced unfolding event to become exposed. We propose that CTXφ pIII accesses the periplasmic TolA through retraction of toxin-coregulated pilus, which brings the phage through the outer membrane pilus secretin channel. These data help to explain the process by which CTXφ converts a harmless marine microbe into a deadly human pathogen.     

In silico prediction of drug targets in <Emphasis Type="Italic">Vibrio cholerae</Emphasis>

Identification of potential drug targets is the first step in the process of modern drug discovery, subjected to their validation and drug development. Whole genome sequences of a number of organisms allow prediction of potential drug targets using sequence comparison approaches. Here, we present a subtractive approach exploiting the knowledge of global gene expression along with sequence comparisons to predict the potential drug targets more efficiently. Based on the knowledge of 155 known virulence and their coexpressed genes mined from microarray database in the public domain, 357 coexpressed probable virulence genes for Vibrio cholerae were predicted. Based on screening of Database of Essential Genes using blastn, a total of 102 genes out of these 357 were enlisted as vitally essential genes, and hence good putative drug targets. As the effective drug target is a protein which is only present in the pathogen, similarity search of these 102 essential genes against human genome sequence led to subtraction of 66 genes, thus leaving behind a subset of 36 genes whose products have been called as potential drug targets. The gene ontology analysis using Blast2GO of these 36 genes revealed their roles in important metabolic pathways of V. cholerae or on the surface of the pathogen. Thus, we propose that the products of these genes be evaluated as target sites of drugs against V. cholerae in future investigations.     

The Effect of Multiple Evolutionary Selections on Synonymous Codon Usage of Genes in the Mycoplasma bovis Genome

Mycoplasma bovis is a major pathogen causing arthritis, respiratory disease and mastitis in cattle. A better understanding of its genetic features and evolution might represent evidences of surviving host environments. In this study, multiple factors influencing synonymous codon usage patterns in M. bovis (three strains’ genomes) were analyzed. The overall nucleotide content of genes in the M. bovis genome is AT-rich. Although the G and C contents at the third codon position of genes in the leading strand differ from those in the lagging strand (p<0.05), the 59 synonymous codon usage patterns of genes in the leading strand are highly similar to those in the lagging strand. The over-represented codons and the under-represented codons were identified. A comparison of the synonymous codon usage pattern of M. bovis and cattle (susceptible host) indicated the independent formation of synonymous codon usage of M. bovis. Principal component analysis revealed that (i) strand-specific mutational bias fails to affect the synonymous codon usage pattern in the leading and lagging strands, (ii) mutation pressure from nucleotide content plays a role in shaping the overall codon usage, and (iii) the major trend of synonymous codon usage has a significant correlation with the gene expression level that is estimated by the codon adaptation index. The plot of the effective number of codons against the G+C content at the third codon position also reveals that mutation pressure undoubtedly contributes to the synonymous codon usage pattern of M. bovis. Additionally, the formation of the overall codon usage is determined by certain evolutionary selections for gene function classification (30S protein, 50S protein, transposase, membrane protein, and lipoprotein) and translation elongation region of genes in M. bovis. The information could be helpful in further investigations of evolutionary mechanisms of the Mycoplasma family and heterologous expression of its functionally important proteins.     

Viral Proteins Originated De Novo by Overprinting Can Be Identified by Codon Usage: Application to the “Gene Nursery” of Deltaretroviruses

A well-known mechanism through which new protein-coding genes originate is by modification of pre-existing genes, e.g. by duplication or horizontal transfer. In contrast, many viruses generate protein-coding genes de novo, via the overprinting of a new reading frame onto an existing (“ancestral”) frame. This mechanism is thought to play an important role in viral pathogenicity, but has been poorly explored, perhaps because identifying the de novo frames is very challenging. Therefore, a new approach to detect them was needed. We assembled a reference set of overlapping genes for which we could reliably determine the ancestral frames, and found that their codon usage was significantly closer to that of the rest of the viral genome than the codon usage of de novo frames. Based on this observation, we designed a method that allowed the identification of de novo frames based on their codon usage with a very good specificity, but intermediate sensitivity. Using our method, we predicted that the Rex gene of deltaretroviruses has originated de novo by overprinting the Tax gene. Intriguingly, several genes in the same genomic region have also originated de novo and encode proteins that regulate the functions of Tax. Such “gene nurseries” may be common in viral genomes. Finally, our results confirm that the genomic GC content is not the only determinant of codon usage in viruses and suggest that a constraint linked to translation must influence codon usage.