ICTV Virus Taxonomy Profile: Herpesviridae 2021.

Members of the family Herpesviridae have enveloped, spherical virions with characteristic complex structures consisting of symmetrical and non-symmetrical components. The linear, double-stranded DNA genomes of 125-241 kbp contain 70-170 genes, of which 43 have been inherited from an ancestral herpesvirus. In general, herpesviruses have coevolved with and are highly adapted to their hosts, which comprise many mammalian, avian and reptilian species. Following primary infection, they are able to establish lifelong latent infection, during which there is limited viral gene expression. Severe disease is usually observed only in the foetus, the very young, the immunocompromised or following infection of an alternative host. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Herpesviridae, which is available at ictv.global/report/herpesviridae.

Mitochondrial DNA Profiling Reveals Two Lineages of Sun Bears in East and West Malaysia.

Sun bear populations are fragmented and at risk from habitat loss and exploitation for body parts. These threats are made worse by significant gaps in knowledge of sun bear population genetic diversity, population connectivity, and taxonomically significant management units. Using a complete sun bear mitochondrial genome, we developed a set of mitochondrial markers to assess haplotype variation and the evolutionary history of sun bears from Peninsular (West) Malaysia and Sabah (East Malaysia). Genetic samples from 28 sun bears from Peninsular Malaysia, 36 from Sabah, and 18 from Thailand were amplified with primers targeting a 1800 bp region of the mitochondrial genome including the complete mitochondrial control region and adjacent genes. Sequences were analyzed using phylogenetic methods. We identified 51 mitochondrial haplotypes among 82 sun bears. Phylogenetic and network analyses provided strong support for a deep split between Malaysian sun bears and sun bears in East Thailand and Yunnan province in China. The Malaysian lineage was further subdivided into two clades: Peninsular Malaysian and Malaysian Borneo (Sabah). Sun bears from Thailand occurred in both Sabah and Peninsular Malaysian clades. Our study supports recent findings that sun bears from Sundaland form a distinct clade from those in China and Indochina with Thailand possessing lineages from the three clades. Importantly, we demonstrate a more recent and clear genetic delineation between sun bears from the Malay Peninsula and Sabah indicating historical barriers to gene flow within the Sundaic region.

Towards the knowledge-based design of universal influenza epitope ensemble vaccines.

MOTIVATION: Influenza A viral heterogeneity remains a significant threat due to unpredictable antigenic drift in seasonal influenza and antigenic shifts caused by the emergence of novel subtypes. Annual review of multivalent influenza vaccines targets strains of influenza A and B likely to be predominant in future influenza seasons. This does not induce broad, cross protective immunity against emergent subtypes. Better strategies are needed to prevent future pandemics. Cross-protection can be achieved by activating CD8+ and CD4+ T cells against highly conserved regions of the influenza genome. We combine available experimental data with informatics-based immunological predictions to help design vaccines potentially able to induce cross-protective T-cells against multiple influenza subtypes. RESULTS: To exemplify our approach we designed two epitope ensemble vaccines comprising highly conserved and experimentally verified immunogenic influenza A epitopes as putative non-seasonal influenza vaccines; one specifically targets the US population and the other is a universal vaccine. The USA-specific vaccine comprised 6 CD8+ T cell epitopes (GILGFVFTL, FMYSDFHFI, GMDPRMCSL, SVKEKDMTK, FYIQMCTEL, DTVNRTHQY) and 3 CD4+ epitopes (KGILGFVFTLTVPSE, EYIMKGVYINTALLN, ILGFVFTLTVPSERG). The universal vaccine comprised 8 CD8+ epitopes: (FMYSDFHFI, GILGFVFTL, ILRGSVAHK, FYIQMCTEL, ILKGKFQTA, YYLEKANKI, VSDGGPNLY, YSHGTGTGY) and the same 3 CD4+ epitopes. Our USA-specific vaccine has a population protection coverage (portion of the population potentially responsive to one or more component epitopes of the vaccine, PPC) of over 96 and 95% coverage of observed influenza subtypes. The universal vaccine has a PPC value of over 97 and 88% coverage of observed subtypes. AVAILABILITY AND IMPLEMENTATION: http://imed.med.ucm.es/Tools/episopt.html CONTACT: d.r.flower@aston.ac.uk.

Sequence-independent characterization of viruses based on the pattern of viral small RNAs produced by the host.

Virus surveillance in vector insects is potentially of great benefit to public health. Large-scale sequencing of small and long RNAs has previously been used to detect viruses, but without any formal comparison of different strategies. Furthermore, the identification of viral sequences largely depends on similarity searches against reference databases. Here, we developed a sequence-independent strategy based on virus-derived small RNAs produced by the host response, such as the RNA interference pathway. In insects, we compared sequences of small and long RNAs, demonstrating that viral sequences are enriched in the small RNA fraction. We also noted that the small RNA size profile is a unique signature for each virus and can be used to identify novel viral sequences without known relatives in reference databases. Using this strategy, we characterized six novel viruses in the viromes of laboratory fruit flies and wild populations of two insect vectors: mosquitoes and sandflies. We also show that the small RNA profile could be used to infer viral tropism for ovaries among other aspects of virus biology. Additionally, our results suggest that virus detection utilizing small RNAs can also be applied to vertebrates, although not as efficiently as to plants and insects.

Zika virus: a previously slow pandemic spreads rapidly through the Americas.

Zika virus (family Flaviviridae) is an emerging arbovirus. Spread by Aedes mosquitoes, it was first discovered in Uganda in 1947, and later in humans elsewhere in sub-Saharan Africa, arriving in south-east Asia at latest by the mid-twentieth century. In the twenty-first century, it spread across the Pacific islands reaching South America around 2014. Since then it has spread rapidly northwards reaching Mexico in November 2015. Its clinical profile is that of a dengue-like febrile illness, but associations with Guillain-Barré syndrome and microcephaly have appeared recently. The final geographical range and ultimate clinical impact of Zika virus are still a matter for speculation.

The serology of Ebolavirus - a wider geographical range, a wider genus of viruses or a wider range of virulence?

Viruses of the genus Ebolavirus are the causative agents of Ebola virus disease (EVD), of which there have been only 25 recorded outbreaks since the discovery of Zaire and Sudan ebolaviruses in the late 1970s. Until the west African outbreak commencing in late 2013, EVD was confined to an area of central Africa stretching from the coast of Gabon through the Congo river basin and eastward to the Great Lakes. Nevertheless, population serological studies since 1976, most of which were carried out in the first two decades after that date, have suggested a wider distribution and more frequent occurrence across tropical Africa. We review this body of work, discussing the various methods employed over the years and the degree to which they can currently be regarded as reliable. We conclude that there is adequate evidence for a wider geographical range of exposure to Ebolavirus or related filoviruses and discuss three possibilities that could account for this: (a) EVD outbreaks have been misidentified as other diseases in the past; (b) unidentified, and clinically milder, species of the genus Ebolavirus circulate over a wider range than the most pathogenic species; and (c) EVD may be subclinical with a frequency high enough that smaller outbreaks may be unidentified. We conclude that the second option is the most likely and therefore predict the future discovery of other, less virulent, members of the genus Ebolavirus.

The Genome of a Tortoise Herpesvirus (Testudinid Herpesvirus 3) Has a Novel Structure and Contains a Large Region That Is Not Required for Replication In Vitro or Virulence In Vivo.

UNLABELLED: Testudinid herpesvirus 3 (TeHV-3) is the causative agent of a lethal disease affecting several tortoise species. The threat that this virus poses to endangered animals is focusing efforts on characterizing its properties, in order to enable the development of prophylactic methods. We have sequenced the genomes of the two most studied TeHV-3 strains (1976 and 4295). TeHV-3 strain 1976 has a novel genome structure and is most closely related to a turtle herpesvirus, thus supporting its classification into genus Scutavirus, subfamily Alphaherpesvirinae, family Herpesviridae. The sequence of strain 1976 also revealed viral counterparts of cellular interleukin-10 and semaphorin, which have not been described previously in members of subfamily Alphaherpesvirinae. TeHV-3 strain 4295 is a mixture of three forms (m1, m2, and M), in which, in comparison to strain 1976, the genomes exhibit large, partially overlapping deletions of 12.5 to 22.4 kb. Viral subclones representing these forms were isolated by limiting dilution assays, and each replicated in cell culture comparably to strain 1976. With the goal of testing the potential of the three forms as attenuated vaccine candidates, strain 4295 was inoculated intranasally into Hermann's tortoises (Testudo hermanni). All inoculated subjects died, and PCR analyses demonstrated the ability of the m2 and M forms to spread and invade the brain. In contrast, the m1 form was detected in none of the organs tested, suggesting its potential as the basis of an attenuated vaccine candidate. Our findings represent a major step toward characterizing TeHV-3 and developing prophylactic methods against it. IMPORTANCE: Testudinid herpesvirus 3 (TeHV-3) causes a lethal disease in tortoises, several species of which are endangered. We have characterized the viral genome and used this information to take steps toward developing an attenuated vaccine. We have sequenced the genomes of two strains (1976 and 4295), compared their growth in vitro, and investigated the pathogenesis of strain 4295, which consists of three deletion mutants. The major findings are that (i) TeHV-3 has a novel genome structure, (ii) its closest relative is a turtle herpesvirus, (iii) it contains interleukin-10 and semaphorin genes (the first time these have been reported in an alphaherpesvirus), (iv) a sizeable region of the genome is not required for viral replication in vitro or virulence in vivo, and (v) one of the components of strain 4295, which has a deletion of 22.4 kb, exhibits properties indicating that it may serve as the starting point for an attenuated vaccine.

Non-random escape pathways from a broadly neutralizing human monoclonal antibody map to a highly conserved region on the hepatitis C virus E2 glycoprotein encompassing amino acids 412-423.

A challenge for hepatitis C virus (HCV) vaccine development is to define epitopes that are able to elicit protective antibodies against this highly diverse virus. The E2 glycoprotein region located at residues 412-423 is conserved and antibodies to 412-423 have broadly neutralizing activities. However, an adaptive mutation, N417S, is associated with a glycan shift in a variant that cannot be neutralized by a murine but by human monoclonal antibodies (HMAbs) against 412-423. To determine whether HCV escapes from these antibodies, we analyzed variants that emerged when cell culture infectious HCV virions (HCVcc) were passaged under increasing concentrations of a specific HMAb, HC33.1. Multiple nonrandom escape pathways were identified. Two pathways occurred in the context of an N-glycan shift mutation at N417T. At low antibody concentrations, substitutions of two residues outside of the epitope, N434D and K610R, led to variants having improved in vitro viral fitness and reduced sensitivity to HC33.1 binding and neutralization. At moderate concentrations, a S419N mutation occurred within 412-423 in escape variants that have greatly reduced sensitivity to HC33.1 but compromised viral fitness. Importantly, the variants generated from these pathways differed in their stability. N434D and K610R-associated variants were stable and became dominant as the virions were passaged. The S419N mutation reverted back to N419S when immune pressure was reduced by removing HC33.1. At high antibody concentrations, a mutation at L413I was observed in variants that were resistant to HC33.1 neutralization. Collectively, the combination of multiple escape pathways enabled the virus to persist under a wide range of antibody concentrations. Moreover, these findings pose a different challenge to vaccine development beyond the identification of highly conserved epitopes. It will be necessary for a vaccine to induce high potency antibodies that prevent the formation of escape variants, which can co-exist with lower potency or levels of neutralizing activities.

Rosen's (M,R) system as an X-machine.

Robert Rosen's (M,R) system is an abstract biological network architecture that is allegedly both irreducible to sub-models of its component states and non-computable on a Turing machine. (M,R) stands as an obstacle to both reductionist and mechanistic presentations of systems biology, principally due to its self-referential structure. If (M,R) has the properties claimed for it, computational systems biology will not be possible, or at best will be a science of approximate simulations rather than accurate models. Several attempts have been made, at both empirical and theoretical levels, to disprove this assertion by instantiating (M,R) in software architectures. So far, these efforts have been inconclusive. In this paper, we attempt to demonstrate why - by showing how both finite state machine and stream X-machine formal architectures fail to capture the self-referential requirements of (M,R). We then show that a solution may be found in communicating X-machines, which remove self-reference using parallel computation, and then synthesise such machine architectures with object-orientation to create a formal basis for future software instantiations of (M,R) systems.

An N-terminal extension to the hepatitis B virus core protein forms a poorly ordered trimeric spike in assembled virus-like particles.

Virus-like particles composed of the core antigen of hepatitis B virus (HBcAg) have been shown to be an effective platform for the display of foreign epitopes in vaccine development. Heterologous sequences have been successfully inserted at both amino and carboxy termini as well as internally at the major immunodominant epitope. We used cryogenic electron microscopy (CryoEM) and three-dimensional image reconstruction to investigate the structure of VLPs assembled from an N-terminal extended HBcAg that contained a polyhistidine tag. The insert was seen to form a trimeric spike on the capsid surface that was poorly resolved, most likely owing to it being flexible. We hypothesise that the capacity of N-terminal inserts to form trimers may have application in the development of multivalent vaccines to trimeric antigens. Our analysis also highlights the value of tools for local resolution assessment in studies of partially disordered macromolecular assemblies by cryoEM.

Stability of the parainfluenza virus 5 genome revealed by deep sequencing of strains isolated from different hosts and following passage in cell culture.

UNLABELLED: The strain diversity of a rubulavirus, parainfluenza virus 5 (PIV5), was investigated by comparing 11 newly determined and 6 previously published genome sequences. These sequences represent 15 PIV5 strains, of which 6 were isolated from humans, 1 was from monkeys, 2 were from pigs, and 6 were from dogs. Strain diversity is remarkably low, regardless of host, year of isolation, or geographical origin; a total of 7.8% of nucleotides are variable, and the average pairwise difference between strains is 2.1%. Variation is distributed unevenly across the PIV5 genome, but no convincing evidence of selection for antibody-mediated evasion in hemagglutinin-neuraminidase was found. The finding that some canine and porcine, but not primate, strains are mutated in the SH gene, and do not produce SH, raised the possibility that dogs (or pigs) may not be the natural host of PIV5. The genetic stability of PIV5 was also demonstrated during serial passage of one strain (W3) in Vero cells at a high multiplicity of infection, under conditions of competition with large proportions of defective interfering genomes. A similar observation was made for a strain W3 mutant (PIV5VΔC) lacking V gene function, in which the dominant changes were related to pseudoreversion in this gene. The mutations detected in PIV5VΔC during pseudoreversion, and also those characterizing the SH gene in canine and porcine strains, predominantly involved U-to-C transitions. This suggests an important role for biased hypermutation via an adenosine deaminase, RNA-specific (ADAR)-like activity. IMPORTANCE: Here we report the sequence variation of 16 different isolates of parainfluenza virus 5 (PIV5) that were isolated from a number of species, including humans, monkeys, dogs, and pigs, over 4 decades. Surprisingly, strain diversity was remarkably low, regardless of host, year of isolation, or geographical origin. Variation was distributed unevenly across the PIV5 genome, but no convincing evidence of immune or host selection was found. This overall genome stability of PIV5 was also observed when the virus was grown in the laboratory, and the genome stayed remarkably constant even during the selection of virus mutants. Some of the canine isolates had lost their ability to encode one of the viral proteins, termed SH, suggesting that although PIV5 commonly infects dogs, dogs may not be the natural host for PIV5.

Evolution and diversity in human herpes simplex virus genomes.

Herpes simplex virus 1 (HSV-1) causes a chronic, lifelong infection in >60% of adults. Multiple recent vaccine trials have failed, with viral diversity likely contributing to these failures. To understand HSV-1 diversity better, we comprehensively compared 20 newly sequenced viral genomes from China, Japan, Kenya, and South Korea with six previously sequenced genomes from the United States, Europe, and Japan. In this diverse collection of passaged strains, we found that one-fifth of the newly sequenced members share a gene deletion and one-third exhibit homopolymeric frameshift mutations (HFMs). Individual strains exhibit genotypic and potential phenotypic variation via HFMs, deletions, short sequence repeats, and single-nucleotide polymorphisms, although the protein sequence identity between strains exceeds 90% on average. In the first genome-scale analysis of positive selection in HSV-1, we found signs of selection in specific proteins and residues, including the fusion protein glycoprotein H. We also confirmed previous results suggesting that recombination has occurred with high frequency throughout the HSV-1 genome. Despite this, the HSV-1 strains analyzed clustered by geographic origin during whole-genome distance analysis. These data shed light on likely routes of HSV-1 adaptation to changing environments and will aid in the selection of vaccine antigens that are invariant worldwide.

Viral genotype correlates with distinct liver gene transcription signatures in chronic hepatitis C virus infection.

BACKGROUND: Chronic hepatitis C virus (HCV) infection of the liver with either genotype 1 or genotype 3 gives rise to distinct pathologies, and the two viral genotypes respond differently to antiviral therapy. METHODS: To understand these clinical differences, we compared gene transcription profiles in liver biopsies from patients infected with either gt1 or gt3, and uninfected controls. RESULTS: Gt1-infected biopsies displayed elevated levels of transcripts regulated by type I and type III interferons (IFN), including genes that predict response to IFN-α therapy. In contrast, genes controlled by IFN-γ were induced in gt3-infected biopsies. Moreover, IFN-γ levels were higher in gt3-infected biopsies. Analysis of hepatocyte-derived cell lines confirmed that the genes upregulated in gt3 infection were preferentially induced by IFN-γ. The transcriptional profile of gt3 infection was unaffected by IFNL4 polymorphisms, providing a rationale for the reduced predictive power of IFNL genotyping in gt3-infected patients. CONCLUSIONS: The interactions between HCV genotypes 1 and 3 and hepatocytes are distinct. These unique interactions provide avenues to explore the biological mechanisms that drive viral genotype-specific differences in disease progression and treatment response. A greater understanding of the distinct host-pathogen interactions of the different HCV genotypes is required to facilitate optimal management of HCV infection.

The 2014 Ebola virus disease outbreak in West Africa.

On 23 March 2014, the World Health Organization issued its first communiqué on a new outbreak of Ebola virus disease (EVD), which began in December 2013 in Guinée Forestière (Forested Guinea), the eastern sector of the Republic of Guinea. Located on the Atlantic coast of West Africa, Guinea is the first country in this geographical region in which an outbreak of EVD has occurred, leaving aside the single case reported in Ivory Coast in 1994. Cases have now also been confirmed across Guinea as well as in the neighbouring Republic of Liberia. The appearance of cases in the Guinean capital, Conakry, and the transit of another case through the Liberian capital, Monrovia, presents the first large urban setting for EVD transmission. By 20 April 2014, 242 suspected cases had resulted in a total of 147 deaths in Guinea and Liberia. The causative agent has now been identified as an outlier strain of Zaire Ebola virus. The full geographical extent and degree of severity of the outbreak, its zoonotic origins and its possible spread to other continents are sure to be subjects of intensive discussion over the next months.

IL-10 encoded by viruses: a remarkable example of independent acquisition of a cellular gene by viruses and its subsequent evolution in the viral genome.

Many viruses have evolved strategies to deregulate the host immune system. These strategies include mechanisms to subvert or recruit the host cytokine network. IL-10 is a pleiotropic cytokine that has both immunostimulatory and immunosuppressive properties. However, its key features relate mainly to its capacity to exert potent immunosuppressive effects. Several viruses have been shown to upregulate the expression of cellular IL-10 (cIL-10) with, in some cases, enhancement of infection by suppression of immune functions. Other viruses encode functional orthologues of cIL-10, called viral IL-10s (vIL-10s). The present review is devoted to these virokines. To date, vIL-10 orthologues have been reported for 12 members of the family Herpesviridae, two members of the family Alloherpesviridae and seven members of the family Poxviridae. Study of vIL-10s demonstrated several interesting aspects on the origin and the evolution of these viral genes, e.g. the existence of multiple (potentially up to nine) independent gene acquisition events at different times during evolution, viral gene acquisition resulting from recombination with cellular genomic DNA or cDNA derived from cellular mRNA and the evolution of cellular sequence in the viral genome to restrict the biological activities of the viral orthologues to those beneficial for the virus life cycle. Here, various aspects of the vIL-10s described to date are reviewed, including their genetic organization, protein structure, origin, evolution, biological properties and potential in applied research.

Comparative genomics of carp herpesviruses.

Three alloherpesviruses are known to cause disease in cyprinid fish: cyprinid herpesviruses 1 and 3 (CyHV1 and CyHV3) in common carp and koi and cyprinid herpesvirus 2 (CyHV2) in goldfish. We have determined the genome sequences of CyHV1 and CyHV2 and compared them with the published CyHV3 sequence. The CyHV1 and CyHV2 genomes are 291,144 and 290,304 bp, respectively, in size, and thus the CyHV3 genome, at 295,146 bp, remains the largest recorded among the herpesviruses. Each of the three genomes consists of a unique region flanked at each terminus by a sizeable direct repeat. The CyHV1, CyHV2, and CyHV3 genomes are predicted to contain 137, 150, and 155 unique, functional protein-coding genes, respectively, of which six, four, and eight, respectively, are duplicated in the terminal repeat. The three viruses share 120 orthologous genes in a largely colinear arrangement, of which up to 55 are also conserved in the other member of the genus Cyprinivirus, anguillid herpesvirus 1. Twelve genes are conserved convincingly in all sequenced alloherpesviruses, and two others are conserved marginally. The reference CyHV3 strain has been reported to contain five fragmented genes that are presumably nonfunctional. The CyHV2 strain has two fragmented genes, and the CyHV1 strain has none. CyHV1, CyHV2, and CyHV3 have five, six, and five families of paralogous genes, respectively. One family unique to CyHV1 is related to cellular JUNB, which encodes a transcription factor involved in oncogenesis. To our knowledge, this is the first time that JUNB-related sequences have been reported in a herpesvirus.

The family Parvoviridae.

A set of proposals to rationalize and extend the taxonomy of the family Parvoviridae is currently under review by the International Committee on Taxonomy of Viruses (ICTV). Viruses in this family infect a wide range of hosts, as reflected by the longstanding division into two subfamilies: the Parvovirinae, which contains viruses that infect vertebrate hosts, and the Densovirinae, encompassing viruses that infect arthropod hosts. Using a modified definition for classification into the family that no longer demands isolation as long as the biological context is strong, but does require a near-complete DNA sequence, 134 new viruses and virus variants were identified. The proposals introduce new species and genera into both subfamilies, resolve one misclassified species, and improve taxonomic clarity by employing a series of systematic changes. These include identifying a precise level of sequence similarity required for viruses to belong to the same genus and decreasing the level of sequence similarity required for viruses to belong to the same species. These steps will facilitate recognition of the major phylogenetic branches within genera and eliminate the confusion caused by the near-identity of species and viruses. Changes to taxon nomenclature will establish numbered, non-Latinized binomial names for species, indicating genus affiliation and host range rather than recapitulating virus names. Also, affixes will be included in the names of genera to clarify subfamily affiliation and reduce the ambiguity that results from the vernacular use of "parvovirus" and "densovirus" to denote multiple taxon levels.

Modelling the structure of full-length Epstein-Barr virus nuclear antigen 1.

Epstein-Barr virus is a clinically important human virus associated with several cancers and is the etiologic agent of infectious mononucleosis. The viral nuclear antigen-1 (EBNA1) is central to the replication and propagation of the viral genome and likely contributes to tumourigenesis. We have compared EBNA1 homologues from other primate lymphocryptoviruses and found that the central glycine/alanine repeat (GAr) domain as well as predicted cellular protein (USP7 and CK2) binding sites are present in homologues in the Old World primates, but not the marmoset, suggesting that these motifs may have co-evolved. Using the resolved structure of the C-terminal one-third of EBNA1 (homodimerization and DNA binding domain), we have gone on to develop monomeric and dimeric models in silico of the full-length protein. The C-terminal domain is predicted to be structurally highly similar between homologues, indicating conserved function. Zinc could be stably incorporated into the model, bonding with two N-terminal cysteines predicted to facilitate multimerisation. The GAr contains secondary structural elements in the models, while the protein binding regions are unstructured, irrespective of the prediction approach used and sequence origin. These intrinsically disordered regions may facilitate the diversity observed in partner interactions. We hypothesize that the structured GAr could mask the disordered regions, thereby protecting the protein from default degradation. In the dimer conformation, the C-terminal tails of each monomer wrap around a proline-rich protruding loop of the partner monomer, providing dimer stability, a feature which could be exploited in therapeutic design.

High-resolution human cytomegalovirus transcriptome.

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 protein-coding 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.