Donald Seto

Isolation of the gene encoding the S. cerevisiae heat shock transcription factor

The yeast heat shock transcription factor gene, HSF1, has been isolated from an S. cerevisiae genomic expression library (in lambda gt11). The sequenced gene encodes an 833 amino acid protein having a mass of 93,218 daltons. Expression of specific DNA-binding activity in E. coli and of transcriptional activity in yeast confirmed the identity of the cloned gene. Southern analysis and gene-disruption experiments indicate that the heat shock transcription factor is encoded by a single-copy, essential gene. The DNA-binding domain was localized to a 118 amino acid region in the amino-terminal third of the protein. Inspection of the DNA-binding domain reveals no resemblance to any currently known secondary structural motifs implicated in DNA recognition and binding.

Evidence of Molecular Evolution Driven by Recombination Events Influencing Tropism in a Novel Human Adenovirus that Causes Epidemic Keratoconjunctivitis

In 2005, a human adenovirus strain (formerly known as HAdV-D22/H8 but renamed here HAdV-D53) was isolated from an outbreak of epidemic keratoconjunctititis (EKC), a disease that is usually caused by HAdV-D8, -D19, or -D37, not HAdV-D22. To date, a complete change of tropism compared to the prototype has never been observed, although apparent recombinant strains of other viruses from species Human adenovirus D (HAdV-D) have been described. The complete genome of HAdV-D53 was sequenced to elucidate recombination events that lead to the emergence of a viable and highly virulent virus with a modified tropism. Bioinformatic and phylogenetic analyses of this genome demonstrate that this adenovirus is a recombinant of HAdV-D8 (including the fiber gene encoding the primary cellular receptor binding site), HAdV-D22, (the ε determinant of the hexon gene), HAdV-D37 (including the penton base gene encoding the secondary cellular receptor binding site), and at least one unknown or unsequenced HAdV-D strain. Bootscanning analysis of the complete genomic sequence of this novel adenovirus, which we have re-named HAdV-D53, indicated at least five recombination events between the aforementioned adenoviruses. Intrahexon recombination sites perfectly framed the ε neutralization determinant that was almost identical to the HAdV-D22 prototype. Additional bootscan analysis of all HAdV-D hexon genes revealed recombinations in identical locations in several other adenoviruses. In addition, HAdV-D53 but not HAdV-D22 induced corneal inflammation in a mouse model. Serological analysis confirmed previous results and demonstrated that HAdV-D53 has a neutralization profile representative of the ε determinant of its hexon (HAdV-D22) and the fiber (HAdV-D8) proteins. Our recombinant hexon sequence is almost identical to the hexon sequences of the HAdV-D strain causing EKC outbreaks in Japan, suggesting that HAdV-D53 is pandemic as an emerging EKC agent. This documents the first genomic, bioinformatic, and biological descriptions of the molecular evolution events engendering an emerging pathogenic adenovirus.

Computational Analysis Identifies Human Adenovirus Type 55 as a Re-Emergent Acute Respiratory Disease Pathogen

ABSTRACT Novel human adenoviruses (HAdVs) arise from genome recombination. Analysis of HAdV type 55 from an outbreak in China shows a hexon recombination between HAdV-B11 and HAdV-B14, resulting in a genome that is 97.4% HAdV-B14. Sporadic appearances as a re-emergent pathogen and misidentification as “HAdV-B11a” are due to this partial hexon.

Computational analysis and identification of an emergent human adenovirus pathogen implicated in a respiratory fatality.

Adenoviral infections are typically acute, self-limiting, and not associated with death. However, we present the genomic and bioinformatics analysis of a novel recombinant human adenovirus (HAdV-D56) isolated in France that caused a rare neonatal fatality, and keratoconjunctivitis in three health care workers who cared for the neonate. Whole genome alignments revealed the expected diversity in the penton base, hexon, E3, and fiber coding regions, and provided evidence for extensive recombination. Bootscan analysis confirmed recombination between HAdV-D9, HAdV-D26, HAdV-D15, and HAdV-D29 in the penton base and hexon proteins, centered around hypervariable loops within the putative proteins. Protein structure analysis of the fiber coding region revealed similarity with HAdV-D8, HAdV-D9, and HAdV-D53, possibly accounting for the ocular tropism of the virus. Based on these data, this virus appears to be a new HAdV-D type (HAdV-D56), underscoring the importance of recombination events in human adenovirus evolution and the emergence of new adenovirus pathogens.

Molecular evolution of human adenoviruses

The recent emergence of highly virulent human adenoviruses (HAdVs) with new tissue tropisms underscores the need to determine their ontogeny. Here we report complete high quality genome sequences and analyses for all the previously unsequenced HAdV serotypes (n = 20) within HAdV species D. Analysis of nucleotide sequence variability for these in conjunction with another 40 HAdV prototypes, comprising all seven HAdV species, confirmed the uniquely hypervariable regions within species. The mutation rate among HAdV-Ds was low when compared to other HAdV species. Homologous recombination was identified in at least two of five examined hypervariable regions for every virus, suggesting the evolution of HAdV-Ds has been highly dependent on homologous recombination. Patterns of alternating GC and AT rich motifs correlated well with hypervariable region recombination sites across the HAdV-D genomes, suggesting foci of DNA instability lead to formulaic patterns of homologous recombination and confer agility to adenovirus evolution.

Using the whole genome sequence to characterize and name human adenoviruses.

We propose that human adenoviruses (HAdVs) be identified, characterized, and typed on the basis of complete genome sequence analyses rather than serological approaches. This idea has recently percolated through the community of adenovirologists. As a result, an open-floor discussion took place at the Ninth International Adenovirus Meeting (Dobogokő, Hungary, April 2009) on the need for a paradigm shift in recognizing and naming HAdVs in the future. An ad hoc committee then met to formulate the principles of the new approach. Recommendations 1 through 4 originated during the open-floor discussion and were discussed by the committee; recommendations 5 and 6 were developed by the authors to avoid conflicting claims and to deal with recombinants. These were reaffirmed at an NIH-sponsored and user-driven workshop of the Human Adenovirus Working Group, which met at NCBI (Bethesda, MD) on February 3, 2011. “Type” will succeed “serotype,” reflecting the prevalence of genome sequence data usage; type is already in usage per International Committee on Taxonomy of Viruses (ICTV) definitions. Previously named HAdVs will transition to the new format; e.g., “serotype HAdV-C1” will become “type HAdV-C1,” where the letter “C” indicates the adenovirus species (currently species A through G). Each adenovirus type will have a unique, consecutively assigned number; i.e., there will not be an HAdV-D54 and an HAdV-C54. Acceptance of a new type will require analysis of the complete genome sequence, including phylogenomics. Serum neutralization will continue to be used as an additional criterion, per the ICTV definition. This should be by actual serum neutralization and hemagglutination inhibition, rather than by imputed derivation by limited DNA sequencing of the epitopes. Naming priority will follow the order in which genome sequence data are released in the public sequence databases. Adherence to the Bermuda principles, whereby sequences are released as soon as possible, will be encouraged (see http://www.ornl.gov/sci/techresources/Human_Genome/publicat/hgn/v7n6/19intern.shtml). Recombination is an accepted feature of HAdV evolution and will be accommodated. Recombinants will be classified as novel types if there are sufficient genomic, biological, or pathogenic differences from related types. Until a new recombinant genome is peer-reviewed and published, a provisional standardized name that reflects critical serological markers and similarities to established types in species, penton base, and serology-based motifs in hexon and fiber, plus the year and place of isolation, will be used for the recombinant. Thus, the provisional name for HAdV-D53 is HAdV-D/Hannover/[unique identifier]/2005/P37/H22/F8. “P” refers to the penton base, “H” to the hexon loop 1 region, and “F” to the fiber knob; numbers refer to the established type with the highest level of nucleotide identity in the respective regions. The unique identifier is a lab designation or strain name, e.g., 2005-IAI-1 for HAdV-D53 isolate 1 and 2005-IAI-2 for HAdV-D53 isolate 2 should both genomes be deposited in GenBank. Discoverers of “candidate” novel HAdV types should submit a FASTA file with the genome nucleotide sequence and the “evidence” to vog.hin.mln.ibcn@semoneg with the subject line “Human Adenovirus Working Group.” This file will be forwarded with submitter information removed to the NIH Human Adenovirus Working Group for a rapid, preliminary, confidential data review to coordinate the assignment of type number. This is not meant as a peer review but will prevent multiple genomes being assigned the same type number. Detailed information, including contact information for the working group members and current recommendations on typing parameters, may be found at http://hadvwg.gmu.edu. HAdV typing criteria are being refined, and input from the community is appreciated. Revisions to the criteria used in establishing HAdV types must avoid compromising the stability and utility of the current system while accommodating changes in technology that reflect the nature of the data available. The effective replacement of serology by DNA sequencing and bioinformatics has recently been observed with the characterization of HAdV-G52, HAdV-D53, HAdV-D54, HAdV-B55, and HAdV-D56. These advances set the scene for the recognition of new HAdV types in the future.

Computational Analysis of Two Species C Human Adenoviruses Provides Evidence of a Novel Virus

ABSTRACT Human adenovirus C (HAdV-C) species are a common cause of respiratory infections and can occasionally produce severe clinical manifestations. A deeper understanding of the variation and evolution in species HAdV-C is especially important since these viruses, including HAdV-C6, are used as gene delivery vectors for human gene therapy and in other biotechnological applications. Here, the full-genome analysis of the prototype HAdV-C6 and a recently identified virus provisionally termed HAdV-C57 are reported. Although the genomes of all species HAdV-C members are very similar to each other, the E3 region, hexon and fiber (ten proteins total) present a wide range of identity values at the amino acid level. Studies of these viruses in comparison to the other three HAdV-C prototypes (1, 2, and 5) comprise a comprehensive analysis of the diversity and conservation within HAdV-C species. HAdV-C6 contains a recombination event within the constant region of the hexon gene. HAdV-C57 is a recombinant virus with a fiber gene nearly identical to HAdV-C6 and a unique hexon distinguished by its loop 2 motif.

Molecular evolution of human species D adenoviruses

Adenoviruses are medium-sized double stranded DNA viruses that infect vertebrates. Human adenoviruses cause an array of diseases. Currently there are 56 human adenovirus types recognized and characterized within seven species (A-G). Of those types, a majority belongs to species D. In this review, the genomic conservation and diversity are examined among human adenoviruses within species D, particularly in contrast to other human adenovirus species. Specifically, homologous recombination is presented as a driving force for the molecular evolution of human adenoviruses and the emergence of new adenovirus pathogens.

Computational analysis of human adenovirus type 22 provides evidence for recombination among species D human adenoviruses in the penton base gene.

ABSTRACT Recombination in human adenoviruses (HAdV) may confer virulence upon an otherwise nonvirulent strain. The genome sequence of species D HAdV type 22 (HAdV-D22) revealed evidence for recombination with HAdV-D19 and HAdV-D37 within the capsid penton base gene. Bootscan analysis demonstrated that recombination sites within the penton base gene frame the coding sequences for the two external hypervariable loops in the protein. A similar pattern of recombination was evident within other HAdV-D types but not other HAdV species. Further study of recombination among HAdVs is needed to better predict possible recombination events among wild-type viruses and adenoviral gene therapy vectors.

Genetic Analysis of a Novel Human Adenovirus With a Serologically Unique Hexon and a Recombinant Fiber Gene

In February of 1996 a human adenovirus (formerly known as Ad-Cor-96-487) was isolated from the stool of an AIDS patient who presented with severe chronic diarrhea. To characterize this apparently novel pathogen of potential public health significance, the complete genome of this adenovirus was sequenced to elucidate its origin. Bioinformatic and phylogenetic analyses of this genome demonstrate that this virus, heretofore referred to as HAdV-D58, contains a novel hexon gene as well as a recombinant fiber gene. In addition, serological analysis demonstrated that HAdV-D58 has a different neutralization profile than all previously characterized HAdVs. Bootscan analysis of the HAdV-D58 fiber gene strongly suggests one recombination event.