The genetic content of wild-type human cytomegalovirus was investigated by sequencing the 235 645 bp genome of a low passage strain (Merlin). Substantial regions of the genome (genes RL1-UL11, UL105-UL112 and UL120-UL150) were also sequenced in several other strains, including two that had not been passaged in cell culture. Comparative analyses, which employed the published genome sequence of a high passage strain (AD169), indicated that Merlin accurately reflects the wild-type complement of 165 genes, containing no obvious mutations other than a single nucleotide substitution that truncates gene UL128. A sizeable subset of genes exhibits unusually high variation between strains, and comprises many, but not all, of those that encode proteins known or predicted to be secreted or membrane-associated. In contrast to unpassaged strains, all of the passaged strains analysed have visibly disabling mutations in one or both of two groups of genes that may influence cell tropism. One comprises UL128, UL130 and UL131A, which putatively encode secreted proteins, and the other contains RL5A, RL13 and UL9, which are members of the RL11 glycoprotein gene family. The case in support of a lack of protein-coding potential in the region between UL105 and UL111A was also strengthened.

Human cytomegalovirus (HCMV) in clinical material cannot replicate efficiently in vitro until it has adapted by mutation. Consequently, wild-type HCMV differ fundamentally from the passaged strains used for research. To generate a genetically intact source of HCMV, we cloned strain Merlin into a self-excising BAC. The Merlin BAC clone had mutations in the RL13 gene and UL128 locus that were acquired during limited replication in vitro prior to cloning. The complete wild-type HCMV gene complement was reconstructed by reference to the original clinical sample. Characterization of viruses generated from repaired BACs revealed that RL13 efficiently repressed HCMV replication in multiple cell types; moreover, RL13 mutants rapidly and reproducibly emerged in transfectants. Virus also acquired mutations in genes UL128, UL130, or UL131A, which inhibited virus growth specifically in fibroblast cells in wild-type form. We further report that RL13 encodes a highly glycosylated virion envelope protein and thus has the potential to modulate tropism. To overcome rapid emergence of mutations in genetically intact HCMV, we developed a system in which RL13 and UL131A were conditionally repressed during virus propagation. This technological advance now permits studies to be undertaken with a clonal, characterized HCMV strain containing the complete wild-type gene complement and promises to enhance the clinical relevance of fundamental research on HCMV.

Ostreid herpesvirus 1 (OsHV-1) is the only member of the Herpesviridae that has an invertebrate host and is associated with sporadic mortality in the Pacific oyster (Crassostrea gigas) and other bivalve species. Cryo-electron microscopy of purified capsids revealed the distinctive T=16 icosahedral structure characteristic of herpesviruses, although the preparations examined lacked pentons. The gross genome organization of OsHV-1 was similar to that of certain mammalian herpesviruses (including herpes simplex virus and human cytomegalovirus), consisting of two invertible unique regions (U L , 167?8 kbp; U S , 3?4 kbp) each flanked by inverted repeats (TR L /IR L , 7?6 kbp; TR S /IR S , 9?8 kbp), with an additional unique sequence (X, 1?5 kbp) between IR L and IR S . Of the 124 unique genes predicted from the 207 439 bp genome sequence, 38 were members of 12 families of related genes and encoded products related to helicases, inhibitors of apoptosis, deoxyuridine triphosphatase and RING-finger proteins, in addition to membrane-associated proteins. Eight genes in three of the families appeared to be fragmented. Other genes that did not belong to the families were predicted to encode DNA polymerase, the two subunits of ribonucleotide reductase, a helicase, a primase, the ATPase subunit of terminase, a RecB-like protein, additional RING-like proteins, an ion channel and several other membrane-associated proteins. Sequence comparisons showed that OsHV-1 is at best tenuously related to the two classes of vertebrate herpesviruses (those associated with mammals, birds and reptiles, and those associated with bony fish and amphibians). OsHV-1 thus represents a third major class of the herpesviruses. INTRODUCTIONViruses are assigned to the family Herpesviridae on the basis of morphological criteria and have been identified in a wide range of vertebrates and one invertebrate, the Pacific oyster, Crassostrea gigas (Minson et al., 2000). The vertebrate herpesviruses fall into two major phylogenetic groups. Those in the first group have mammalian or avian hosts and are classified into three subfamilies (Alphaherpesvirinae, Betaherpesvirinae and Gammaherpesvirinae) that share extensive genetic relationships . Reptilian herpesviruses probably also belong among the Alphaherpesvirinae (Nigro et al., 2004;Quackenbush et al., 1998;Une et al., 2000;Yu et al., 2001). Viruses in the second group infect amphibians or bony fish and again are interrelated (Bernard & Mercier, 1993;Davison, 1998; Davison et al., 1999). Genetic evidence for a common evolutionary origin for the two groups is tenuous, however, since not a single herpesvirus-specific gene is detectably conserved in both. The only completely sequenced lower vertebrate herpesvirus, channel catfish virus (CCV), does share a few genes with the higher vertebrate group, but these have counterparts in other organisms (Davison, 1992). The conserved gene that comes closest to being herpesvirus specific encodes the putative ATPase subunit of the terminase, an enzyme complex involved in p...

BackgroundThe near exclusive use of praziquantel (PZQ) for treatment of human schistosomiasis has raised concerns about the possible emergence of drug-resistant schistosomes.Methodology/Principal FindingsWe measured susceptibility to PZQ of isolates of Schistosoma mansoni obtained from patients from Kisumu, Kenya continuously exposed to infection as a consequence of their occupations as car washers or sand harvesters. We used a) an in vitro assay with miracidia, b) an in vivo assay targeting adult worms in mice and c) an in vitro assay targeting adult schistosomes perfused from mice. In the miracidia assay, in which miracidia from human patients were exposed to PZQ in vitro, reduced susceptibility was associated with previous treatment of the patient with PZQ. One isolate (“KCW”) that was less susceptible to PZQ and had been derived from a patient who had never fully cured despite multiple treatments was studied further. In an in vivo assay of adult worms, the KCW isolate was significantly less susceptible to PZQ than two other isolates from natural infections in Kenya and two lab-reared strains of S. mansoni. The in vitro adult assay, based on measuring length changes of adults following exposure to and recovery from PZQ, confirmed that the KCW isolate was less susceptible to PZQ than the other isolates tested. A sub-isolate of KCW maintained separately and tested after three years was susceptible to PZQ, indicative that the trait of reduced sensitivity could be lost if selection was not maintained.Conclusions/SignificanceIsolates of S. mansoni from some patients in Kisumu have lower susceptibility to PZQ, including one from a patient who was never fully cured after repeated rounds of treatment administered over several years. As use of PZQ continues, continued selection for worms with diminished susceptibility is possible, and the probability of emergence of resistance will increase as large reservoirs of untreated worms diminish. The potential for rapid emergence of resistance should be an important consideration of treatment programs.

Deep sequencing was used to bring high resolution to the human cytomegalovirus (HCMV) transcriptome at the stage when infectious virion production is under way, and major findings were confirmed by extensive experimentation using conventional techniques. The majority (65.1%) of polyadenylated viral RNA transcription is committed to producing four noncoding transcripts (RNA2.7, RNA1.2, RNA4.9, and RNA5.0) that do not substantially overlap designated protein-coding regions. Additional noncoding RNAs that are transcribed antisense to protein-coding regions map throughout the genome and account for 8.7% of transcription from these regions. RNA splicing is more common than recognized previously, which was evidenced by the identification of 229 potential donor and 132 acceptor sites, and it affects 58 proteincoding genes. The great majority (94) of 96 splice junctions most abundantly represented in the deep-sequencing data was confirmed by RT-PCR or RACE or supported by involvement in alternative splicing. Alternative splicing is frequent and particularly evident in four genes (RL8A, UL74A, UL124, and UL150A) that are transcribed by splicing from any one of many upstream exons. The analysis also resulted in the annotation of four previously unrecognized protein-coding regions (RL8A, RL9A, UL150A, and US33A), and expression of the UL150A protein was shown in the context of HCMV infection. The overall conclusion, that HCMV transcription is complex and multifaceted, has implications for the potential sophistication of virus functionality during infection. The study also illustrates the key contribution that deep sequencing can make to the genomics of nuclear DNA viruses.T he genetic repertoire of human cytomegalovirus (HCMV; species Human herpesvirus 5) is incompletely understood. Most bioinformatic investigations have focused on identifying open reading frames (ORFs) that are conserved in other organisms or that exhibit pattern-based similarities (e.g., in nucleotide or codon bias) to recognized protein-coding regions (CRs) (1). Our current map of the wild-type HCMV genome, based on strain Merlin, contains 166 protein-coding genes (2-5). It is entirely possible that additional, small protein-coding genes will be found. Candidates involve ORFs that overlap recognized CRs and for which there is some evidence for expression (6), ORFs highlighted in pattern-based bioinformatic (7) or proteomic analyses (8), and ORFs whose expression is presently unsuspected.Recognition of many protein-coding genes has been supplemented by information on protein expression and function. However, HCMV also specifies polyadenylated (polyA) transcripts that, because they lack sizeable, conserved ORFs, appear unlikely to function via translation. One class consists of noncoding, nonoverlapping transcripts (NNTs) that do not substantially overlap the designated CRs of other genes. These include an abundant 2.7-kb RNA (β2.7 or RNA2.7) (9), a 1.1-kb spliced RNA and associated 5-kb stable intron (RNA5.0) (10, 11), and a 1.2-kb RNA (RNA1.2) (12). RNA...

Mutations that occurred during adaptation of human cytomegalovirus to cell culture were monitored by isolating four strains from clinical samples, passaging them in various cell types and sequencing ten complete virus genomes from the final passages. Mutational dynamics were assessed by targeted sequencing of intermediate passages and the original clinical samples. Gene RL13 and the UL128 locus (UL128L, consisting of genes UL128, UL130 and UL131A) mutated in all strains. Mutations in RL13 occurred in fibroblast, epithelial and endothelial cells, whereas those in UL128L were limited to fibroblasts and detected later than those in RL13. In addition, a region containing genes UL145, UL144, UL142, UL141 and UL140 mutated in three strains. All strains exhibited numerous mutations in other regions of the genome, with a preponderance in parts of the inverted repeats. An investigation was carried out on the kinetic growth yields of viruses derived from selected passages that were predominantly non-mutated in RL13 and UL128L (RL13+UL128L+), or that were largely mutated in RL13 (RL13−UL128L+) or both RL13 and UL128L (RL13−UL128L−). RL13−UL128L− viruses produced greater yields of infectious progeny than RL13−UL128L+ viruses, and RL13−UL128L+ viruses produced greater yields than RL13+UL128L+ viruses. These results suggest strongly that RL13 and UL128L exert at least partially independent suppressive effects on growth in fibroblasts. As all isolates proved genetically unstable in all cell types tested, caution is advised in choosing and monitoring strains for experimental studies of vulnerable functions, particularly those involved in cell tropism, immune evasion or growth temperance.

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During gradient purification of herpes simplex virus type 1 (HSV-1) two bands of particles were observed: a sharp lower band and a more diffuse upper band. The lower band contained almost exclusively HSV-1 virions (H particles) whereas the upper band consisted of membrane-enclosed particles (L particles). These L particles resembled the virions in appearance, but lacked the viral nucleocapsid and were not infectious. Many polypeptides of the viral envelope and the tegument were common to both types of particles. The H particles had polypeptide profiles typical of HSV virions. The L particles contained at least three phosphoproteins (175K, 92K and 55K) and a further two phosphorylated polypeptides not normally observed in virion profiles which comigrated with the 134K and 60K glycoproteins. This clearly indicates that the novel L particles were not merely virions which had formed without the inclusion of a nucleocapsid or virions which had subsequently lost their nucleocapsid during preparative handling. Thus these novel L particles are genuine products of the infectious processes occurring when HSV-1 replicates.