The Genome of Salmonid Herpesvirus 1

Salmonid herpesvirus 1 (SalHV-1) is a pathogen of the rainbow trout (Oncorhynchus mykiss). Restriction endonuclease mapping, cosmid cloning, DNA hybridization, and targeted DNA sequencing experiments showed that the genome is 174.4 kbp in size, consisting of a long unique region (U_L; 133.4 kbp) linked to a short unique region (U_S; 25.6 kbp) which is flanked by an inverted repeat (R_S; 7.7 kbp). U_S is present in virion DNA in either orientation, but U_L is present in a single orientation. This structure is characteristic of the Varicellovirus genus of the subfamily Alphaherpesvirinae but has evidently evolved independently, since an analysis of randomly sampled DNA sequence data showed that SalHV-1 shares at least 18 genes with channel catfish virus (CCV), a fish herpesvirus whose complete sequence is known and which is unrelated to mammalian herpesviruses. The use of oligonucleotide probes demonstrated that in comparison with CCV, the conserved SalHV-1 genes are located in U_L in at least five rearranged blocks. Large-scale gene rearrangements of this type are also characteristic of the three mammalian herpesvirus subfamilies. The junction between two SalHV-1 gene blocks was confirmed by sequencing a 4,245-bp region which contains the dUTPase gene, part of a putative spliced DNA polymerase gene, and one other complete gene. The implications of these findings in herpesvirus taxonomy are discussed.Herpesviruses are a large group of complex, double-stranded DNA viruses which infect vertebrates from teleost (bony) fish to humans. They exhibit narrow host specificites, most infecting only a single species in nature, and are thus considered likely to have evolved with their hosts. Comparisons of primary amino acid sequences predicted from complete genome sequences have shown that mammalian herpesviruses are genetically very divergent but nonetheless share a set of about 40 homologous genes, thus providing compelling evidence that these viruses evolved from a single ancestral herpesvirus (reviewed in reference 7). Moreover, genetic comparisons support the division of the family into three subfamilies, Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae, as proposed previously from biological criteria (15). The order of genes is largely conserved within each subfamily, whereas members of different subfamilies are more distantly related and exhibit several large-scale genomic rearrangements (4, 9). Viral phylogenies derived from rigorous sequence comparisons generally fit well with host phylogenies deduced from the fossil record, thus supporting the view that mammalian herpesviruses have cospeciated with their hosts, and this has allowed a time frame to be assigned (13, 14). Moreover, limited sequence data also indicate that avian herpesviruses fit readily into the subfamily Alphaherpesvirinae.Nearly all research on herpesviruses has involved mammalian (and, to a lesser extent, avian) herpesviruses, and little is known about the many herpesviruses which infect cold-blooded vertebrates. The most extensively studied member of the latter group, channel catfish virus (CCV; ictalurid herpesvirus 1), was initially classified as a herpesvirus on the basis of its virion morphology and as a member of the Alphaherpesvirinae on the basis of its biological properties (15). Analysis of the complete genome sequence (6) indicated, however, that CCV has no specific relationship with mammalian herpesviruses at the level of primary amino acid sequence, in that no counterpart of a protein which is encoded only by mammalian herpesviruses, such as a structural protein, was detected in CCV. Thus CCV cannot be accommodated by the current taxonomy. The virus does encode several enzymes which are also specified by mammalian herpesviruses, such as DNA polymerase, dUTPase, and thymidine kinase. The genes encoding these proteins, however, are ubiquitous and could quite possibly have been acquired independently by the mammalian and fish herpesvirus lineages. Moreover, the CCV enzymes are no more closely related to their counterparts in other herpesviruses than to those in other organisms.These findings may be interpreted in two ways. First, CCV and mammalian herpesviruses arose independently and have convergently acquired similar virion morphologies. Second, they evolved from an ancestral herpesvirus but have diverged so extensively over the 400 million years since their hosts separated that little sequence evidence remains. Several lines of evidence support the latter view, but it is fair to say that the case is not yet overwhelming. The best genetic indication for divergence rests in a single highly conserved protein which is encoded by two exons in the mammalian herpesviruses and three in CCV (open reading frames [ORFs] 62, 69, and 71). This protein apparently has a distant relative in bacteriophage T4 which functions as a subunit of the terminase involved in DNA packaging, but the fact that no cellular counterpart has yet been discovered highlights it as the best candidate for a gene which may have been inherited from a common ancestor rather than acquired via independent capture events. Moreover, despite the lack of conservation of the amino acid sequences of structural proteins, structural and functional congruences have been detected. Thus, the detailed three-dimensional structure of the CCV capsid is strikingly similar to that of herpes simplex virus type 1 (3). Also, local sequence features of the putative scaffold protein involved in CCV capsid formation suggest that it may be autoproteolytically processed via a pathway that is otherwise found only in mammalian herpesviruses (8).Evidence for a herpesvirus lineage that lies outside the current taxonomic scheme has prompted investigations of its extent. Comparisons of CCV with salmonid herpesviruses appear useful in this respect, since the fossil record indicates that the three main subgroups of euteleosts (salmoniforms, neoteleosts, and ostariophysans, the latter including catfish) diverged around 130 million years ago (1). Salmonid fish are host to several herpesviruses, the principal of which are salmonid herpesviruses 1 and 2 (SalHV-1 and SalHV-2) (reviewed in reference 19). SalHV-1 was isolated on several occasions from a rainbow trout (Oncorhynchus mykiss) hatchery in the state of Washington in association with excessive mortality in young fish (20). The virus causes disease when injected into young rainbow trout maintained at 6 to 9°C but not in other salmonid species. SalHV-2 was isolated from Oncorhynchus masou, a landlocked Japanese form of Pacific salmon (11). It is serologically distinct from and has a wider host range than SalHV-1, causing virulent disease in the young of several Oncorhynchus species, including the rainbow trout. It also exhibits a higher temperature optimum for growth in cell culture than SalHV-1.Partial sequence data for two genes have previously indicated that SalHV-2 is related to CCV (2). In this report, I describe the genome structure and gene arrangement of SalHV-1 and show that this virus is evolutionarily related to SalHV-2 and CCV. The data indicate that the processes which have resulted in the generation of certain genome structures and large-scale gene rearrangements during mammalian herpesvirus evolution have parallels in fish herpesvirus evolution. They also imply that fish herpesviruses occupy a distinct evolutionary space of an size equivalent to that occupied by mammalian herpesviruses and urge an accommodation in the herpesvirus taxonomy.     

Examining the Role of Transmission of Chelonid Alphaherpesvirus 5

Marine turtle fibropapillomatosis (FP) is a devastating neoplastic disease characterized by single or multiple cutaneous and visceral fibrovascular tumors. Chelonid alphaherpesvirus 5 (ChHV5) has been identified as the most likely etiologic agent. From 2010 to 2013, the presence of ChHV5 DNA was determined in apparently normal skin, tumors and swab samples (ocular, nasal and cloacal) collected from 114 olive ridley (Lepidochelys olivacea) and 101 green (Chelonia mydas) turtles, with and without FP tumors, on the Pacific coasts of Costa Rica and Nicaragua. For nesting olive ridley turtles from Costa Rica without FP, 13.5% were found to be positive for ChHV5 DNA in at least one sample, while in Nicaragua, all olive ridley turtles had FP tumors, and 77.5% tested positive for ChHV5 DNA. For green turtles without FP, 19.8% were found to be positive for ChHV5 DNA in at least one of the samples. In turtles without FP tumors, ChHV5 DNA was detected more readily in skin biopsies than swabs. Juvenile green turtles caught at the foraging site had a higher prevalence of ChHV5 DNA than adults. The presence of ChHV5 DNA in swabs suggests a possible route of viral transmission through viral secretion and excretion via corporal fluids.     

Complete Genome Sequence of Equine Herpesvirus Type 9

Equine herpesvirus type 9 (EHV-9), which we isolated from a case of epizootic encephalitis in a herd of Thomson''s gazelles (Gazella thomsoni) in 1993, has been known to cause fatal encephalitis in Thomson''s gazelle, giraffe, and polar bear in natural infections. Our previous report indicated that EHV-9 was similar to the equine pathogen equine herpesvirus type 1 (EHV-1), which mainly causes abortion, respiratory infection, and equine herpesvirus myeloencephalopathy. We determined the genome sequence of EHV-9. The genome has a length of 148,371 bp and all 80 of the open reading frames (ORFs) found in the genome of EHV-1. The nucleotide sequences of the ORFs in EHV-9 were 86 to 95% identical to those in EHV-1. The whole genome sequence should help to reveal the neuropathogenicity of EHV-9.     

Clustering of Genes Coding for DNA Binding Proteins in a Regionof Atypical Evolution of the Human Genome

Comparison of the human and mouse genomes has revealed that significant variations in evolutionary rates exist among genomic regions and that a large part of this variation is interchromosomal. We confirm in this work, using a large collection of introns, that human chromosome 19 is the one that shows the highest divergence with respect to mouse. To search for other differences among chromosomes, we examine the distribution of gene functions in human and mouse chromosomes using the Gene Ontology definitions. We found by correspondence analysis that among the strongest clusterings of gene functions in human chromosomes is a group of genes coding for DNA binding proteins in chromosome 19. Interestingly, chromosome 19 also has a very high GC content, a feature that has been proposed to promote an opening of the chromatin, thereby facilitating binding of proteins to the DNA helix. In the mouse genome, however, a similar aggregation of genes coding for DNA binding proteins and high GC content cannot be found. This suggests that the distribution of genes coding for DNA binding proteins and the variations of the chromatin accessibility to these proteins are different in the human and mouse genomes. It is likely that the overall high synonymous and intron rates in chromosome 19 are a by-product of the high GC content of this chromosome.Department of Physiology and Molecular Biodiversity, Institut de Biologia Molecular de Barcelona, CSIC, Jordi Girona 18, 08034 Barcelona, Spain     

Genome Sequence Analysis Indicates that the Model Eukaryote Nematostella vectensis Harbors Bacterial Consorts

Analysis of the genome sequence of the starlet sea anemone, Nematostella vectensis, reveals many genes whose products are phylogenetically closer to proteins encoded by bacteria or bacteriophages than to any metazoan homologs. One explanation for such sequence affinities could be that these genes have been horizontally transferred from bacteria to the Nematostella lineage. We show, however, that bacterium-like and phage-like genes sequenced by the N. vectensis genome project tend to cluster on separate scaffolds, which typically do not include eukaryotic genes and differ from the latter in their GC contents. Moreover, most of the bacterium-like genes in N. vectensis either lack introns or the introns annotated in such genes are false predictions that, when translated, often restore the missing portions of their predicted protein products. In a freshwater cnidarian, Hydra, for which a proteobacterial endosymbiont is known, these gene features have been used to delineate the DNA of that endosymbiont sampled by the genome sequencing project. We predict that a large fraction of bacterium-like genes identified in the N. vectensis genome similarly are drawn from the contemporary bacterial consorts of the starlet sea anemone. These uncharacterized bacteria associated with N. vectensis are a proteobacterium and a representative of the phylum Bacteroidetes, each represented in the database by an apparently random sample of informational and operational genes. A substantial portion of a putative bacteriophage genome was also detected, which would be especially unlikely to have been transferred to a eukaryote.     

Fibropapillomatosis and the Chelonid Alphaherpesvirus 5 in Green Turtles from West Africa

EcoHealth - Fibropapillomatosis (FP) is a tumorigenic panzootic disease of sea turtles, most common in green turtles (Chelonia mydas). FP is linked to the chelonid alphaherpesvirus 5 (ChAHV5) and...     

水稻基因组中的tRNA和rRNA基因

在最近完成测序的水稻籼稻和粳稻两个亚种基因组中,各找到564和519个较为可靠的tRNA基因,进一步证实了于2002年发表的基于基因组序列草图的分析结果。修正的摆动假设,即至少需要46种tRNA基因才能译出61种可能的反密码子,在这两个亚种中均准确成立。在这46种tRNA中,有些在籼稻和粳稻中的序列均全同。有18种水稻tRNA与拟南芥中的相应序列全同。在籼稻基因组序列中还发现了384个5S rRNA基因,一批17S和5．8S rRNA基因以及一个25S rRNA基因。这些rRNA基因的不完备是由于它们通常以串接阵列形式存在于异染色质区域,而后者在全基因组霰弹法测序中不易完整测出。在tRNA和rRNA基因序列之间发现了多处互补片段,这将有助于研究它们的进化和相互作用。     

Systematic Excision of Vector Sequences from the BAC-Cloned Herpesvirus Genome during Virus Reconstitution

Recently the mouse cytomegalovirus (MCMV) genome was cloned as an infectious bacterial artificial chromosome (BAC) (M. Messerle, I. Crnkovic, W. Hammerschmidt, H. Ziegler, and U. H. Koszinowski, Proc. Natl. Acad. Sci. USA 94:14759-14763, 1997). The virus obtained from this construct is attenuated in vivo due to deletion of viral sequences and insertion of the BAC vector. We reconstituted the full-length MCMV genome and flanked the BAC vector with identical viral sequences. This new construct represents a versatile basis for construction of MCMV mutants since virus generated from the construct loses the bacterial sequences and acquires wild-type properties.     

Connexin Genes in the Mouse and Human Genome

Gap junctions serve for direct intercellular communication by docking of two hemichannels in adjacent cells thereby forming conduits between the cytoplasmic compartments of adjacent cells. Connexin genes code for subunit proteins of gap junction channels and are members of large gene families in mammals. So far, 17 connexin (Cx) genes have been described and characterized in the murine genome. For most of them, orthologues in the human genome have been found (see White and Paul 1999; Manthey et al. 1999; Teubner et al. 2001; Söhl et al. 2001). We have recently performed searches for connexin genes in murine and human gene libraries available at EMBL/Heidelberg, NCBI and the Celera company that have increased the number of identified connexins to 19 in mouse and 20 in humans. For one mouse connexin gene and two human connexin genes we did not find orthologues in the other genome. Here we present a short overview on distinct connexin genes which we found in the mouse and human genome and which may include all members of this gene family, if no further connexin gene will be discovered in the remaining non-sequenced parts (about 1-5%) of the genomes.     

Inferring Bacterial Genome Flux While Considering Truncated Genes

Bacterial gene content variation during the course of evolution has been widely acknowledged and its pattern has been actively modeled in recent years. Gene truncation or gene pseudogenization also plays an important role in shaping bacterial genome content. Truncated genes could also arise from small-scale lateral gene transfer events. Unfortunately, the information of truncated genes has not been considered in any existing mathematical models on gene content variation. In this study, we developed a model to incorporate truncated genes. Maximum-likelihood estimates (MLEs) of the new model reveal fast rates of gene insertions/deletions on recent branches, suggesting a fast turnover of many recently transferred genes. The estimates also suggest that many truncated genes are in the process of being eliminated from the genome. Furthermore, we demonstrate that the ignorance of truncated genes in the estimation does not lead to a systematic bias but rather has a more complicated effect. Analysis using the new model not only provides more accurate estimates on gene gains/losses (or insertions/deletions), but also reduces any concern of a systematic bias from applying simplified models to bacterial genome evolution. Although not a primary purpose, the model incorporating truncated genes could be potentially used for phylogeny reconstruction using gene family content.GENE content variation as a key feature of bacterial genome evolution has been well recognized (Garcia-Vallvé et al. 2000; Ochman and Jones 2000; Snel et al. 2002; Welch et al. 2002; Kunin and Ouzounis 2003; Fraser-Liggett 2005; Tettelin et al. 2005) and gained increasing attention in recent years. Various methods have been employed to study the variation of gene content in the form of gene insertions/deletions (or gene gains/losses); there are studies of population dynamics (Nielsen and Townsend 2004), birth-and-death evolutionary models (Berg and Kurland 2002; Novozhilov et al. 2005), phylogeny-dependent studies including parsimony methods (Mirkin et al. 2003; Daubin et al. 2003a,b; Hao and Golding 2004), and maximum-likelihood methods (Hao and Golding 2006, 2008b; Cohen et al. 2008; Cohen and Pupko 2010; Spencer and Sangaralingam 2009). The pattern of gene presence/absence also contains phylogenetic signals (Fitz-Gibbon and House 1999; Snel et al. 1999; Tekaia et al. 1999) and has been used for phylogenetic reconstruction (Dutilh et al. 2004; Gu and Zhang 2004; Huson and Steel 2004; Zhang and Gu 2004; Spencer et al. 2007a,b). All these studies make use of the binary information of gene presence or absence and neglect the existence of gene segments or truncated genes.Bacterial genomes are known to harbor pseudogenes. An intracellular species Mycobacterium leprae is an extreme case for both the proportion and the number of pseudogenes: estimated as 40% of the 3.2-Mb genome and 1116 genes (Cole et al. 2001). In free-living bacteria, pseudogenes can make up to 8% of the annotated genes in the genome (Lerat and Ochman 2004). Many pseudogenes result from the degradation of native functional genes (Cole et al. 2001; Mira et al. 2001). Pseudogenes could also result from the degradation of transferred genes and might even be acquired directly via lateral gene transfer. For instance, in plant mitochondrial genomes, which have an α-proteobacterial ancestry, most, if not all, of the laterally transferred genes are pseudogenes (Richardson and Palmer 2007). Furthermore, evidence has been documented that gene transfer could take place at the subgenic level in a wide range of organisms, e.g., among bacteria (Miller et al. 2005; Choi and Kim 2007; Chan et al. 2009), between ancient duplicates in archaea (Archibald and Roger 2002), between different organelles (Hao and Palmer 2009; Hao 2010), and between eukaryotes (Keeling and Palmer 2001). A large fraction of pseudogenes have been shown to arise from failed lateral transfer events (Liu et al. 2004) and most of them are transient in bacterial genomes (Lerat and Ochman 2005). Zhaxybayeva et al. (2007) reported that genomes with truncated homologs might erroneously lead to false inferences of “gene gain” rather than multiple instances of “gene loss.” This raises the question of how a false diagnosis of gene absence affects the estimation of insertion/deletion rates. Recently, we showed that the effect of a false diagnosis of gene absence on estimation of insertion/deletion rates is not systematic, but rather more complicated (Hao and Golding 2008a). To further address the problem, a study incorporating the information of truncated genes is highly desirable. This will not only yield more accurate estimates of the rates of gene insertions/deletions, but also provide a quantitative view of the effect of truncated genes on rate estimation, which has been understudied in bacterial genome evolution.In this study, we developed a model that considers the information of truncated genes and makes use of a parameter-rich time-reversible rate matrix. Rate variation among genes is allowed in the model by incorporating a discrete Γ-distribution. We also allow rates to vary on different parts of the phylogeny (external branches vs. internal branches). Consistent with previous studies, the rates of gene insertions/deletions are comparable to or larger than the rates of nucleotide substitution and the rates of gene insertions/deletions are further inflated in closely related groups and on external branches, suggesting high rates of gene turnover of recently transferred genes. The results from the new model also suggest that many recently truncated genes are in the process of being rapidly deleted from the genome. Some other interesting estimates in the model are also presented and discussed. One implication of the study, though not primary, is that the state of truncated genes could serve as an additional phylogenetic character for phylogenetic reconstruction using gene family content.     

Detection of Prokaryotic Genes in the Amphimedon queenslandica Genome

Horizontal gene transfer (HGT) is common between prokaryotes and phagotrophic eukaryotes. In metazoans, the scale and significance of HGT remains largely unexplored but is usually linked to a close association with parasites and endosymbionts. Marine sponges (Porifera), which host many microorganisms in their tissues and lack an isolated germ line, are potential carriers of genes transferred from prokaryotes. In this study, we identified a number of potential horizontally transferred genes within the genome of the sponge, Amphimedon queenslandica. We further identified homologs of some of these genes in other sponges. The transferred genes, most of which possess catalytic activity for carbohydrate or protein metabolism, have assimilated host genome characteristics and are actively expressed. The diversity of functions contributed by the horizontally transferred genes is likely an important factor in the adaptation and evolution of A. queenslandica. These findings highlight the potential importance of HGT on the success of sponges in diverse ecological niches.     

Silage Collected from Dairy Farms Harbors an Abundance of Listeriaphages with Considerable Host Range and Genome Size Diversity

Since the food-borne pathogen Listeria monocytogenes is common in dairy farm environments, it is likely that phages infecting this bacterium (“listeriaphages”) are abundant on dairy farms. To better understand the ecology and diversity of listeriaphages on dairy farms and to develop a diverse phage collection for further studies, silage samples collected on two dairy farms were screened for L. monocytogenes and listeriaphages. While only 4.5% of silage samples tested positive for L. monocytogenes, 47.8% of samples were positive for listeriaphages, containing up to >1.5 × 10⁴ PFU/g. Host range characterization of the 114 phage isolates obtained, with a reference set of 13 L. monocytogenes strains representing the nine major serotypes and four lineages, revealed considerable host range diversity; phage isolates were classified into nine lysis groups. While one serotype 3c strain was not lysed by any phage isolates, serotype 4 strains were highly susceptible to phages and were lysed by 63.2 to 88.6% of phages tested. Overall, 12.3% of phage isolates showed a narrow host range (lysing 1 to 5 strains), while 28.9% of phages represented broad host range (lysing ≥11 strains). Genome sizes of the phage isolates were estimated to range from approximately 26 to 140 kb. The extensive host range and genomic diversity of phages observed here suggest an important role of phages in the ecology of L. monocytogenes on dairy farms. In addition, the phage collection developed here has the potential to facilitate further development of phage-based biocontrol strategies (e.g., in silage) and other phage-based tools.