Koenraad M Van Doorslaer

Interests

Research

Papillomaviruses (PVs) are a diverse family of dsDNA viruses infecting most, if not all, amniotes. Papillomaviruses infect cutaneous or mucosal epithelia. While most infections are self-limiting, persistent infection with specific human papillomaviruses has been shown to be the causative agent for cervical cancer. All established oncogenic HPV types belong to a single viral genus (the Alphapapillomaviridae). Of note, phylogenetically, these oncogenic HPV types cluster into a so-called high-risk (HR) clade, indicating an evolutionary relationship between these viruses. Importantly, not all HPV types within this HR clade are associated with cancer.I am intrigued by the observation that only a limited subset of human papillomaviruses is oncogenic. Throughout my studies I have used a combination of biochemical assays and computational analyses to understand why evolutionarily related viruses differ in their ability to cause cancer in humans.It is improbable that the ability to cause cancer provides papillomaviruses with an evolutionary advantage. It is likely that many of the viral functions linked to oncogenesis were evolutionarily beneficial as papillomavirus adapted to novel environmental niches on the host (e.g. external genitalia vs. cervix). Papillomaviruses have evolved to usurp the cellular machinery to complete their life-cycle. The papillomaviral lifecycle perturbs the normal differentiation cycle of the infected cell, forcing cells to divide far beyond their normal lifespan. It is feasible that the continued insult provided by replicating viruses eventually results in malignant transformation of the infected cell. However, while persistent infection is key to viral oncogenesis, many long-term persisting viruses do not cause cancer. By carefully interrogating the differences between these viruses, I believe it will be possible to elucidate which viral phenotypes are associated with oncogenic progression. The pathways targeted by these viruses may represent powerful targets for therapeutic intervention.

Teaching

Virology, evolution of host-pathogen interactions

Courses

Scholarly Contributions

Books

• Van Doorslaer, K. M., & Sparza, M. (2019). Evolution of DNA viruses. Encyclopedia of Virology

Journals/Publications

• Harding, C., Larsen, B. B., Gryseels, S., Otto, H. W., Suazo, C., Kraberger, S., Upham, N. S., Worobey, M., Van Doorslaer, K., & Varsani, A. (2022). Discovery of three cycloviruses in fecal samples from silver-haired bats (Lasionycteris noctivagans) in Arizona (USA). Archives of virology, 167(12), 2771-2775.
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  Bats harbour a diverse array of viruses, some of which are zoonotic, and are one of the most speciose groups of mammals on earth. As part of an ongoing bat-associated viral diversity research project, we identified three cycloviruses (family Circoviridae) in fecal samples of silver-haired bats (Lasionycteris noctivagans) caught in Cave Creek Canyon of Arizona (USA). Two of the three identified genomes represent two new species in the genus Cyclovirus. Cycloviruses have been found in a wide range of environments and hosts; however, little is known about their biology. These new genomes of cycloviruses are the first from silver-haired bats, adding to the broader knowledge of cyclovirus diversity. With continuing studies, it is likely that additional viruses of the family Circoviridae will be identified in Arizona bat populations.
• King, K. M., Rajadhyaksha, E. V., Tobey, I. G., & Van Doorslaer, K. (2022). Synonymous nucleotide changes drive papillomavirus evolution. Tumour virus research, 14, 200248.
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  Papillomaviruses have been evolving alongside their hosts for at least 450 million years. This review will discuss some of the insights gained into the evolution of this diverse family of viruses. Papillomavirus evolution is constrained by pervasive purifying selection to maximize viral fitness. Yet these viruses need to adapt to changes in their environment, e.g., the host immune system. It has long been known that these viruses evolved a codon usage that doesn't match the infected host. Here we discuss how papillomavirus genomes evolve by acquiring synonymous changes that allow the virus to avoid detection by the host innate immune system without changing the encoded proteins and associated fitness loss. We discuss the implications of studying viral evolution, lifecycle, and cancer progression.
• King, K., Larsen, B. B., Gryseels, S., Richet, C., Kraberger, S., Jackson, R., Worobey, M., Harrison, J. S., Varsani, A., & Van Doorslaer, K. (2022). Coevolutionary Analysis Implicates Toll-Like Receptor 9 in Papillomavirus Restriction. mBio, 13(2), e0005422.
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  Upon infection, DNA viruses can be sensed by pattern recognition receptors (PRRs), leading to the activation of type I and III interferons to block infection. Therefore, viruses must inhibit these signaling pathways, avoid being detected, or both. Papillomavirus virions are trafficked from early endosomes to the Golgi apparatus and wait for the onset of mitosis to complete nuclear entry. This unique subcellular trafficking strategy avoids detection by cytoplasmic PRRs, a property that may contribute to the establishment of infection. However, as the capsid uncoats within acidic endosomal compartments, the viral DNA may be exposed to detection by Toll-like receptor 9 (TLR9). In this study, we characterized two new papillomaviruses from bats and used molecular archeology to demonstrate that their genomes altered their nucleotide compositions to avoid detection by TLR9, providing evidence that TLR9 acts as a PRR during papillomavirus infection. Furthermore, we showed that TLR9, like other components of the innate immune system, is under evolutionary selection in bats, providing the first direct evidence for coevolution between papillomaviruses and their hosts. Finally, we demonstrated that the cancer-associated human papillomaviruses show a reduction in CpG dinucleotides within a TLR9 recognition complex. Viruses must avoid detection by the innate immune system. In this study, we characterized two new papillomaviruses from bats and used molecular archeology to demonstrate that their genomes altered their nucleotide compositions to avoid detection by TLR9, providing evidence that TLR9 acts as a PRR during papillomavirus infection. Furthermore, we demonstrated that TLR9, like other components of the innate immune system, is under evolutionary selection in bats, providing the first direct evidence for coevolution between papillomaviruses and their hosts.
• Kraberger, S., Austin, C., Farkas, K., Desvignes, T., Postlethwait, J. H., Fontenele, R. S., Schmidlin, K., Bradley, R. W., Warzybok, P., Van Doorslaer, K., Davison, W., Buck, C. B., & Varsani, A. (2022). Discovery of novel fish papillomaviruses: From the Antarctic to the commercial fish market. Virology, 565, 65-72.
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  Fish papillomaviruses form a newly discovered group broadly recognized as the Secondpapillomavirinae subfamily. This study expands the documented genomes of the fish papillomaviruses from six to 16, including one from the Antarctic emerald notothen, seven from commercial market fishes, one from data mining of sea bream sequence data, and one from a western gull cloacal swab that is likely diet derived. The genomes of secondpapillomaviruses are ∼6 kilobasepairs (kb), which is substantially smaller than the ∼8 kb of terrestrial vertebrate papillomaviruses. Each genome encodes a clear homolog of the four canonical papillomavirus genes, E1, E2, L1, and L2. In addition, we identified open reading frames (ORFs) with short linear peptide motifs reminiscent of E6/E7 oncoproteins. Fish papillomaviruses are extremely diverse and phylogenetically distant from other papillomaviruses suggesting a model in which terrestrial vertebrate-infecting papillomaviruses arose after an evolutionary bottleneck event, possibly during the water-to-land transition.
• Lee, M. S., Tuohy, P. J., Kim, C. Y., Lichauco, K., Parrish, H. L., Van Doorslaer, K., & Kuhns, M. S. (2022). Enhancing and inhibitory motifs regulate CD4 activity. eLife, 11.
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  CD4 T cells use T cell receptor (TCR)-CD3 complexes, and CD4, to respond to peptide antigens within MHCII molecules (pMHCII). We report here that, through ~435 million years of evolution in jawed vertebrates, purifying selection has shaped motifs in the extracellular, transmembrane, and intracellular domains of eutherian CD4 that enhance pMHCII responses, and covary with residues in an intracellular motif that inhibits responses. Importantly, while CD4 interactions with the Src kinase, Lck, are viewed as key to pMHCII responses, our data indicate that CD4-Lck interactions derive their importance from the counterbalancing activity of the inhibitory motif, as well as motifs that direct CD4-Lck pairs to specific membrane compartments. These results have implications for the evolution and function of complex transmembrane receptors and for biomimetic engineering.
• Paietta, E. N., Kraberger, S., Custer, J. M., Vargas, K. L., Van Doorslaer, K., Yoder, A. D., & Varsani, A. (2022). Identification of diverse papillomaviruses in captive black-and-white ruffed lemurs (Varecia variegata). Archives of virology, 168(1), 13.
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  Papillomaviruses (PVs) are host-species-specific and tissue-specific viruses that infect a diverse array of vertebrate hosts, including humans and non-human primates, with varying pathogenic outcomes. Although primate PVs have been studied extensively, no complete genome sequences of PVs from lemurs have been determined to date. Saliva samples from three critically endangered, captive black-and-white ruffed lemurs (Varecia variegata variegata) at the Duke Lemur Center (USA) were analyzed, using high-throughput sequencing, for the presence of oral papillomaviruses. We identified three PVs from two individuals, one of which had a coinfection with two different PVs. Two of the three PVs share 99.6% nucleotide sequence identity, and we have named these isolates "Varecia variegata papillomavirus 1" (VavPV1). The third PV shares ~63% nucleotide sequence identity with VavPV1, and thus, we have named it "Varecia variegata papillomavirus 2" (VavPV2). Based on their E1 + E2 + L1 protein sequence phylogeny, the VavPVs form a distinct clade. This clade likely represents a novel genus, with VavPV1 and VavPV2 belonging to two distinct species. Our findings represent the first complete genome sequences of PVs found in lemuriform primates, with their presence suggesting the potential existence of diverse PVs across the over 100 species of lemurs.
• Van Doorslaer, K. (2022). Revisiting Papillomavirus Taxonomy: A Proposal for Updating the Current Classification in Line with Evolutionary Evidence. Viruses, 14(10).
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  Papillomaviruses infect a wide array of animal hosts and are responsible for roughly 5% of all human cancers. Comparative genomics between different virus types belonging to specific taxonomic groupings (e.g., species, and genera) has the potential to illuminate physiological differences between viruses with different biological outcomes. Likewise, extrapolation of features between related viruses can be very powerful but requires a solid foundation supporting the evolutionary relationships between viruses. The current papillomavirus classification system is based on pairwise sequence identity. However, with the advent of metagenomics as facilitated by high-throughput sequencing and molecular tools of enriching circular DNA molecules using rolling circle amplification, there has been a dramatic increase in the described diversity of this viral family. Not surprisingly, this resulted in a dramatic increase in absolute number of viral types (i.e., sequences sharing
• Walker, P. J., Siddell, S. G., Lefkowitz, E. J., Mushegian, A. R., Adriaenssens, E. M., Alfenas-Zerbini, P., Dempsey, D. M., Dutilh, B. E., García, M. L., Curtis Hendrickson, R., Junglen, S., Krupovic, M., Kuhn, J. H., Lambert, A. J., Łobocka, M., Oksanen, H. M., Orton, R. J., Robertson, D. L., Rubino, L., , Sabanadzovic, S., et al. (2022). Recent changes to virus taxonomy ratified by the International Committee on Taxonomy of Viruses (2022). Archives of virology, 167(11), 2429-2440.
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  This article reports the changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses (ICTV) in March 2022. The entire ICTV was invited to vote on 174 taxonomic proposals approved by the ICTV Executive Committee at its annual meeting in July 2021. All proposals were ratified by an absolute majority of the ICTV members. Of note, the Study Groups have started to implement the new rule for uniform virus species naming that became effective in 2021 and mandates the binomial 'Genus_name species_epithet' format with or without Latinization. As a result of this ratification, the names of 6,481 virus species (more than 60 percent of all species names currently recognized by ICTV) now follow this format.
• Zerbini, F. M., Siddell, S. G., Mushegian, A. R., Walker, P. J., Lefkowitz, E. J., Adriaenssens, E. M., Alfenas-Zerbini, P., Dutilh, B. E., García, M. L., Junglen, S., Krupovic, M., Kuhn, J. H., Lambert, A. J., Łobocka, M., Oksanen, H. M., Robertson, D. L., Rubino, L., Sabanadzovic, S., Simmonds, P., , Suzuki, N., et al. (2022). Differentiating between viruses and virus species by writing their names correctly. Archives of virology.
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  Following the results of the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021, a standard two-part "binomial nomenclature" is now the norm for naming virus species. Adoption of the new nomenclature is still in its infancy; thus, it is timely to reiterate the distinction between "virus" and "virus species" and to provide guidelines for naming and writing them correctly. Following the results of the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021, a standard two-part "binomial nomenclature" is now the norm for naming virus species. Adoption of the new nomenclature is still in its infancy; thus, it is timely to reiterate the distinction between "virus" and "virus species" and to provide guidelines for naming and writing them correctly.Following the results of the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021, a standard two-part "binomial nomenclature" is now the norm for naming virus species. Adoption of the new nomenclature is still in its infancy; thus, it is timely to reiterate the distinction between "virus" and "virus species" and to provide guidelines for naming and writing them correctly.Following the results of the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021, a standard two-part "binomial nomenclature" is now the norm for naming virus species. Adoption of the new nomenclature is still in its infancy; thus, it is timely to reiterate the distinction between "virus" and "virus species" and to provide guidelines for naming and writing them correctly.Following the results of the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021, a standard two-part "binomial nomenclature" is now the norm for naming virus species. Adoption of the new nomenclature is still in its infancy; thus, it is timely to reiterate the distinction between "virus" and "virus species" and to provide guidelines for naming and writing them correctly.Following the results of the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021, a standard two-part "binomial nomenclature" is now the norm for naming virus species. Adoption of the new nomenclature is still in its infancy; thus, it is timely to reiterate the distinction between "virus" and "virus species" and to provide guidelines for naming and writing them correctly.Following the results of the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021, a standard two-part "binomial nomenclature" is now the norm for naming virus species. Adoption of the new nomenclature is still in its infancy; thus, it is timely to reiterate the distinction between "virus" and "virus species" and to provide guidelines for naming and writing them correctly.Following the results of the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021, a standard two-part "binomial nomenclature" is now the norm for naming virus species. Adoption of the new nomenclature is still in its infancy; thus, it is timely to reiterate the distinction between "virus" and "virus species" and to provide guidelines for naming and writing them correctly.Following the results of the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021, a standard two-part "binomial nomenclature" is now the norm for naming virus species. Adoption of the new nomenclature is still in its infancy; thus, it is timely to reiterate the distinction between "virus" and "virus species" and to provide guidelines for naming and writing them correctly.Following the results of the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021, a standard two-part "binomial nomenclature" is now the norm for naming virus species. Adoption of the new nomenclature is still in its infancy; thus, it is timely to reiterate the distinction between "virus" and "virus species" and to provide guidelines for naming and writing them correctly.Following the results of the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021, a standard two-part "binomial nomenclature" is now the norm for naming virus species. Adoption of the new nomenclature is still in its infancy; thus, it is timely to reiterate the distinction between "virus" and "virus species" and to provide guidelines for naming and writing them correctly.Following the results of the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021, a standard two-part "binomial nomenclature" is now the norm for naming virus species. Adoption of the new nomenclature is still in its infancy; thus, it is timely to reiterate the distinction between "virus" and "virus species" and to provide guidelines for naming and writing them correctly.Following the results of the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021, a standard two-part "binomial nomenclature" is now the norm for naming virus species. Adoption of the new nomenclature is still in its infancy; thus, it is timely to reiterate the distinction between "virus" and "virus species" and to provide guidelines for naming and writing them correctly.Following the results of the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021, a standard two-part "binomial nomenclature" is now the norm for naming virus species. Adoption of the new nomenclature is still in its infancy; thus, it is timely to reiterate the distinction between "virus" and "virus species" and to provide guidelines for naming and writing them correctly.Following the results of the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021, a standard two-part "binomial nomenclature" is now the norm for naming virus species. Adoption of the new nomenclature is still in its infancy; thus, it is timely to reiterate the distinction between "virus" and "virus species" and to provide guidelines for naming and writing them correctly.Following the results of the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021, a standard two-part "binomial nomenclature" is now the norm for naming virus species. Adoption of the new nomenclature is still in its infancy; thus, it is timely to reiterate the distinction between "virus" and "virus species" and to provide guidelines for naming and writing them correctly.Following the results of the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021, a standard two-part "binomial nomenclature" is now the norm for naming virus species. Adoption of the new nomenclature is still in its infancy; thus, it is timely to reiterate the distinction between "virus" and "virus species" and to provide guidelines for naming and writing them correctly.Following the results of the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021, a standard two-part "binomial nomenclature" is now the norm for naming virus species. Adoption of the new nomenclature is still in its infancy; thus, it is timely to reiterate the distinction between "virus" and "virus species" and to provide guidelines for naming and writing them correctly.Following the results of the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021, a standard two-part "binomial nomenclature" is now the norm for naming virus species. Adoption of the new nomenclature is still in its infancy; thus, it is timely to reiterate the distinction between "virus" and "virus species" and to provide guidelines for naming and writing them correctly.Following the results of the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021, a standard two-part "binomial nomenclature" is now the norm for naming virus species. Adoption of the new nomenclature is still in its infancy; thus, it is timely to reiterate the distinction between "virus" and "virus species" and to provide guidelines for naming and writing them correctly.Following the results of the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021, a standard two-part "binomial nomenclature" is now the norm for naming virus species. Adoption of the new nomenclature is still in its infancy; thus, it is timely to reiterate the distinction between "virus" and "virus species" and to provide guidelines for naming and writing them correctly.
• Coursey, T. L., Van Doorslaer, K., & McBride, A. A. (2021). Regulation of Human Papillomavirus 18 Genome Replication, Establishment, and Persistence by Sequences in the Viral Upstream Regulatory Region. Journal of virology, 95(19), e0068621.
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  During persistent human papillomavirus infection, the viral genome replicates as an extrachromosomal plasmid that is efficiently partitioned to daughter cells during cell division. We have previously shown that an element which overlaps the human papillomavirus 18 (HPV18) transcriptional enhancer promotes stable DNA replication of replicons containing the viral replication origin. Here, we perform comprehensive analyses to elucidate the function of this maintenance element. We conclude that no unique element or binding site in this region is absolutely required for persistent replication and partitioning and instead propose that the overall chromatin architecture of this region is important to promote efficient use of the replication origin. These results have important implications for the genome partitioning mechanism of papillomaviruses. Persistent infection with oncogenic human papillomaviruses (HPVs) is responsible for ∼5% of human cancers. The viral DNA replicates as an extrachromosomal plasmid and is partitioned to daughter cells in dividing keratinocytes. Using a complementation assay that allows us to separate viral transcription and replication, we provide insight into viral sequences that are required for long-term replication and persistence in keratinocytes. Understanding how viral genomes replicate persistently for such long periods of time will guide the development of antiviral therapies.
• Fontenele, R. S., Salywon, A. M., Majure, L. C., Cobb, I. N., Bhaskara, A., Avalos-Calleros, J. A., Argüello-Astorga, G. R., Schmidlin, K., Khalifeh, A., Smith, K., Schreck, J., Lund, M. C., Köhler, M., Wojciechowski, M. F., Hodgson, W. C., Puente-Martinez, R., Van Doorslaer, K., Kumari, S., Oyeniran, K. A., , Vernière, C., et al. (2021). New World Cactaceae Plants Harbor Diverse Geminiviruses. Viruses, 13(4).
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  The family Cactaceae comprises a diverse group of typically succulent plants that are native to the American continent but have been introduced to nearly all other continents, predominantly for ornamental purposes. Despite their economic, cultural, and ecological importance, very little research has been conducted on the viral community that infects them. We previously identified a highly divergent geminivirus that is the first known to infect cacti. Recent research efforts in non-cultivated and asymptomatic plants have shown that the diversity of this viral family has been under-sampled. As a consequence, little is known about the effects and interactions of geminiviruses in many plants, such as cacti. With the objective to expand knowledge on the diversity of geminiviruses infecting cacti, we used previously acquired high-throughput sequencing results to search for viral sequences using BLASTx against a viral RefSeq protein database. We identified two additional sequences with similarity to geminiviruses, for which we designed abutting primers and recovered full-length genomes. From 42 cacti and five scale insects, we derived 42 complete genome sequences of a novel geminivirus species that we have tentatively named Opuntia virus 2 (OpV2) and 32 genomes of an Opuntia-infecting becurtovirus (which is a new strain of the spinach curly top Arizona virus species). Interspecies recombination analysis of the OpV2 group revealed several recombinant regions, in some cases spanning half of the genome. Phylogenetic analysis demonstrated that OpV2 is a novel geminivirus more closely related to viruses of the genus , which was further supported by the detection of three recombination events between curtoviruses and OpV2. Both OpV2 and Opuntia becurtoviruses were identified in mixed infections, which also included the previously characterized Opuntia virus 1. Viral quantification of the co-infected cactus plants compared with single infections did not show any clear trend in viral dynamics that might be associated with the mixed infections. Using experimental -mediated inoculations, we found that the initial accumulation of OpV2 is facilitated by co-infection with OpV1. This study shows that the diversity of geminiviruses that infect cacti is under-sampled and that cacti harbor diverse geminiviruses. The detection of the Opuntia becurtoviruses suggests spill-over events between viruses of cultivated species and native vegetation. The threat this poses to cacti needs to be further investigated.
• Kraberger, S., Serieys, L. E., Richet, C., Fountain-Jones, N. M., Baele, G., Bishop, J. M., Nehring, M., Ivan, J. S., Newkirk, E. S., Squires, J. R., Lund, M. C., Riley, S. P., Wilmers, C. C., van Helden, P. D., Van Doorslaer, K., Culver, M., VandeWoude, S., Martin, D. P., & Varsani, A. (2021). Complex evolutionary history of felid anelloviruses. Virology, 562, 176-189.
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  Anellovirus infections are highly prevalent in mammals, however, prior to this study only a handful of anellovirus genomes had been identified in members of the Felidae family. Here we characterise anelloviruses in pumas (Puma concolor), bobcats (Lynx rufus), Canada lynx (Lynx canadensis), caracals (Caracal caracal) and domestic cats (Felis catus). The complete anellovirus genomes (n = 220) recovered from 149 individuals were diverse. ORF1 protein sequence similarity network analysis coupled with phylogenetic analysis, revealed two distinct clusters that are populated by felid-derived anellovirus sequences, a pattern mirroring that observed for the porcine anelloviruses. Of the two-felid dominant anellovirus groups, one includes sequences from bobcats, pumas, domestic cats and an ocelot, and the other includes sequences from caracals, Canada lynx, domestic cats and pumas. Coinfections of diverse anelloviruses appear to be common among the felids. Evidence of recombination, both within and between felid-specific anellovirus groups, supports a long coevolution history between host and virus.
• Nguyen, J. T., Allen, C. T., Dodge, J. T., Van Doorslaer, K., McBride, A. A., Pavletic, S. Z., & Mays, J. W. (2021). HPV32-related Heck's disease in a chronic graft-versus-host disease patient with long-term successful KTP laser treatment: A rare case report. Clinical case reports, 9(5), e04253.
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  We recently identified and treated a rare case of oral focal epithelial hyperplasia (FEH) in an adult patient with chronic graft-vs-host disease. This is the first report linking KTP laser therapy to successful long-term treatment HPV32 FEH.
• Schmidlin, K., Kraberger, S., Cook, C., DeNardo, D. F., Fontenele, R. S., Van Doorslaer, K., Martin, D. P., Buck, C. B., & Varsani, A. (2021). A novel lineage of polyomaviruses identified in bark scorpions. Virology, 563, 58-63.
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  Polyomaviruses are non-enveloped viruses with circular double-stranded DNA genomes (~4-7 kb). Initially identified in mammals, polyomaviruses have now been identified in birds and a few fish species. Although fragmentary polyomavirus-like sequences have been detected as apparent 'hitchhikers' in shotgun genomics datasets of various arthropods, the possible diversity of these viruses in invertebrates remains unclear. Scorpions are predatory arachnids that are among the oldest terrestrial animals. Using high-throughput sequencing and traditional molecular techniques we determine the genome sequences of eight novel polyomaviruses in scorpions (Centruroides sculpturatus) from the greater Phoenix area, Arizona, USA. Analysis of Centruroides transcriptomic datasets elucidated the splicing of the viral late gene array, which is more complex than that of vertebrate polyomaviruses. Phylogenetic analysis provides further evidence of co-divergence of polyomaviruses with their hosts, suggesting that at least one ancestral species of polyomaviruses was circulating amongst the primitive common ancestors of arthropods and chordates.
• Walker, P. J., Siddell, S. G., Lefkowitz, E. J., Mushegian, A. R., Adriaenssens, E. M., Alfenas-Zerbini, P., Davison, A. J., Dempsey, D. M., Dutilh, B. E., García, M. L., Harrach, B., Harrison, R. L., Hendrickson, R. C., Junglen, S., Knowles, N. J., Krupovic, M., Kuhn, J. H., Lambert, A. J., Łobocka, M., , Nibert, M. L., et al. (2021). Changes to virus taxonomy and to the International Code of Virus Classification and Nomenclature ratified by the International Committee on Taxonomy of Viruses (2021). Archives of virology, 166(9), 2633-2648.
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  This article reports the changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses (ICTV) in March 2021. The entire ICTV was invited to vote on 290 taxonomic proposals approved by the ICTV Executive Committee at its meeting in October 2020, as well as on the proposed revision of the International Code of Virus Classification and Nomenclature (ICVCN). All proposals and the revision were ratified by an absolute majority of the ICTV members. Of note, ICTV mandated a uniform rule for virus species naming, which will follow the binomial 'genus-species' format with or without Latinized species epithets. The Study Groups are requested to convert all previously established species names to the new format. ICTV has also abolished the notion of a type species, i.e., a species chosen to serve as a name-bearing type of a virus genus. The remit of ICTV has been clarified through an official definition of 'virus' and several other types of mobile genetic elements. The ICVCN and ICTV Statutes have been amended to reflect these changes.
• Fontenele, R. S., Salywon, A. M., Majure, L. C., Cobb, I. N., Bhaskara, A., Avalos-Calleros, J. A., Argüello-Astorga, G. R., Schmidlin, K., Khalifeh, A., Smith, K., Schreck, J., Lund, M. C., Köhler, M., Wojciechowski, M. F., Hodgson, W. C., Puente-Martinez, R., Van Doorslaer, K., Kumari, S., Vernière, C., , Filloux, D., et al. (2020). A Novel Divergent Geminivirus Identified in Asymptomatic New World Cactaceae Plants. Viruses, 12(4).
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  Cactaceae comprise a diverse and iconic group of flowering plants which are almost exclusively indigenous to the New World. The wide variety of growth forms found amongst the cacti have led to the trafficking of many species throughout the world as ornamentals. Despite the evolution and physiological properties of these plants having been extensively studied, little research has focused on cactus-associated viral communities. While only single-stranded RNA viruses had ever been reported in cacti, here we report the discovery of cactus-infecting single-stranded DNA viruses. These viruses all apparently belong to a single divergent species of the family and have been tentatively named Opuntia virus 1 (OpV1). A total of 79 apparently complete OpV1 genomes were recovered from 31 different cactus plants (belonging to 20 different cactus species from both the Cactoideae and Opuntioideae clades) and from nine cactus-feeding cochineal insects ( sp.) sampled in the USA and Mexico. These 79 OpV1 genomes all share > 78.4% nucleotide identity with one another and < 64.9% identity with previously characterized geminiviruses. Collectively, the OpV1 genomes display evidence of frequent recombination, with some genomes displaying up to five recombinant regions. In one case, recombinant regions span ~40% of the genome. We demonstrate that an infectious clone of an OpV1 genome can replicate in and In addition to expanding the inventory of viruses that are known to infect cacti, the OpV1 group is so distantly related to other known geminiviruses that it likely represents a new geminivirus genus. It remains to be determined whether, like its cactus hosts, its geographical distribution spans the globe.
• Jackson, R., Maarsingh, J. D., Herbst-Kralovetz, M. M., & Van Doorslaer, K. (2020). 3D Oral and Cervical Tissue Models for Studying Papillomavirus Host-Pathogen Interactions. Current protocols in microbiology, 59(1), e129.
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  Human papillomavirus (HPV) infection occurs in differentiating epithelial tissues. Cancers caused by high-risk types (e.g., HPV16 and HPV18) typically occur at oropharyngeal and anogenital anatomical sites. The HPV life cycle is differentiation-dependent, requiring tissue culture methodology that is able to recapitulate the three-dimensional (3D) stratified epithelium. Here we report two distinct and complementary methods for growing differentiating epithelial tissues that mimic many critical morphological and biochemical aspects of in vivo tissue. The first approach involves growing primary human epithelial cells on top of a dermal equivalent consisting of collagen fibers and living fibroblast cells. When these cells are grown at the liquid-air interface, differentiation occurs and allows for epithelial stratification. The second approach uses a rotating wall vessel bioreactor. The low-fluid-shear microgravity environment inside the bioreactor allows the cells to use collagen-coated microbeads as a growth scaffold and self-assemble into 3D cellular aggregates. These approaches are applied to epithelial cells derived from HPV-positive and HPV-negative oral and cervical tissues. The second part of the article introduces potential downstream applications for these 3D tissue models. We describe methods that will allow readers to start successfully culturing 3D tissues from oral and cervical cells. These tissues have been used for microscopic visualization, scanning electron microscopy, and large omics-based studies to gain insights into epithelial biology, the HPV life cycle, and host-pathogen interactions. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Establishing human primary cell-derived 3D organotypic raft cultures Support Protocol 1: Isolation of epithelial cells from patient-derived tissues Support Protocol 2: Growth and maintenance of primary human epithelial cells in monolayer culture Support Protocol 3: PCR-based HPV screening of primary cell cultures Basic Protocol 2: Establishing human 3D cervical tissues using the rotating wall vessel bioreactor Support Protocol 4: Growth and maintenance of human A2EN cells in monolayer culture Support Protocol 5: Preparation of the slow-turning lateral vessel bioreactor Support Protocol 6: Preparation of Cytodex-3 microcarrier beads Basic Protocol 3: Histological assessment of 3D organotypic raft tissues Basic Protocol 4: Spatial analysis of protein expression in 3D organotypic raft cultures Basic Protocol 5: Immunofluorescence imaging of RWV-derived 3D tissues Basic Protocol 6: Ultrastructural visualization and imaging of RWV-derived 3D tissues Basic Protocol 7: Characterization of gene expression by RT-qPCR.
• King, K., & Van Doorslaer, K. M. (2018). Building (Viral) phylogenetic trees using a maximum likelihood approach. Current Protocols in Microbiology.
• O'Brien, H., Alvin, J. W., Menghani, S. V., Sanchez-Rosario, Y., Van Doorslaer, K., & Johnson, M. D. (2020). Rules of Expansion: an Updated Consensus Operator Site for the CopR-CopY Family of Bacterial Copper Exporter System Repressors. mSphere, 5(3).
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  Copper is broadly toxic to bacteria. As such, bacteria have evolved specialized copper export systems ( operons) often consisting of a DNA-binding/copper-responsive regulator (which can be a repressor or activator), a copper chaperone, and a copper exporter. For those bacteria using DNA-binding copper repressors, few studies have examined the regulation of this operon regarding the operator DNA sequence needed for repressor binding. In (the pneumococcus), CopY is the copper repressor for the operon. Previously, homologs of pneumococcal CopY have been characterized to bind a 10-base consensus sequence T/GACANNTGTA known as the box. Using this motif, we sought to determine whether genes outside the operon are also regulated by the CopY repressor, which was previously shown in We found that CopY did not bind to operators upstream of these candidate genes During this process, we found that the box sequence is necessary but not sufficient for CopY binding. Here, we propose an updated operator sequence for the operon to be ATTGACAAATGTAGAT binding CopY with a dissociation constant ( ) of ∼28 nM. We demonstrate strong cross-species interaction between some CopY proteins and CopY operators, suggesting strong evolutionary conservation. Taken together with our binding studies and bioinformatics data, we propose the consensus operator RNYKACANNYGTMRNY for the bacterial CopR-CopY copper repressor homologs. Many Gram-positive bacteria respond to copper stress by upregulating a copper export system controlled by a copper-sensitive repressor, CopR-CopY. The previous operator sequence for this family of proteins had been identified as TACANNTGTA. Here, using several recombinant proteins and mutations in various DNA fragments, we define those 10 bases as necessary but not sufficient for binding and in doing so, refine the operon operator to the 16-base sequence RNYKACANNTGTMRNY. Due to the sheer number of repressors that have been said to bind to the original 10 bases, including many antibiotic resistance repressors such as BlaI and MecI, we feel that this study highlights the need to reexamine many of these sites of the past and use added stringency for verifying operators in the future.
• Pace, J., Youens-Clark, K., Freeman, C., Hurwitz, B., & Van Doorslaer, K. (2020). PuMA: A papillomavirus genome annotation tool. Virus evolution, 6(2), veaa068.
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  High-throughput sequencing technologies provide unprecedented power to identify novel viruses from a wide variety of (environmental) samples. The field of 'viral metagenomics' has dramatically expanded our understanding of viral diversity. Viral metagenomic approaches imply that many novel viruses will not be described by researchers who are experts on (the genomic organization of) that virus family. We have developed the papillomavirus annotation tool (PuMA) to provide researchers with a convenient and reproducible method to annotate and report novel papillomaviruses. PuMA currently correctly annotates 99% of the papillomavirus genes when benchmarked against the 655 reference genomes in the papillomavirus episteme. Compared to another viral annotation pipeline, PuMA annotates more viral features while being more accurate. To demonstrate its general applicability, we also developed a preliminary version of PuMA that can annotate polyomaviruses. PuMA is available on GitHub (https://github.com/KVD-lab/puma) and through the iMicrobe online environment (https://www.imicrobe.us/#/apps/puma).
• Payne, N., Kraberger, S., Fontenele, R. S., Schmidlin, K., Bergeman, M. H., Cassaigne, I., Culver, M., Varsani, A., & Van Doorslaer, K. (2020). Novel Circoviruses Detected in Feces of Sonoran Felids. Viruses, 12(9).
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  Sonoran felids are threatened by drought and habitat fragmentation. Vector range expansion and anthropogenic factors such as habitat encroachment and climate change are altering viral evolutionary dynamics and exposure. However, little is known about the diversity of viruses present in these populations. Small felid populations with lower genetic diversity are likely to be most threatened with extinction by emerging diseases, as with other selective pressures, due to having less adaptive potential. We used a metagenomic approach to identify novel circoviruses, which may have a negative impact on the population viability, from confirmed bobcat () and puma () scats collected in Sonora, Mexico. Given some circoviruses are known to cause disease in their hosts, such as porcine and avian circoviruses, we took a non-invasive approach using scat to identify circoviruses in free-roaming bobcats and puma. Three circovirus genomes were determined, and, based on the current species demarcation, they represent two novel species. Phylogenetic analyses reveal that one circovirus species is more closely related to rodent associated circoviruses and the other to bat associated circoviruses, sharing highest genome-wide pairwise identity of approximately 70% and 63%, respectively. At this time, it is unknown whether these scat-derived circoviruses infect felids, their prey, or another organism that might have had contact with the scat in the environment. Further studies should be conducted to elucidate the host of these viruses and assess health impacts in felids.
• Uhlorn, B. L., Jackson, R., Li, S., Bratton, S. M., Van Doorslaer, K., & Campos, S. K. (2020). Vesicular trafficking permits evasion of cGAS/STING surveillance during initial human papillomavirus infection. PLoS pathogens, 16(11), e1009028.
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  Oncogenic human papillomaviruses (HPVs) replicate in differentiating epithelium, causing 5% of cancers worldwide. Like most other DNA viruses, HPV infection initiates after trafficking viral genome (vDNA) to host cell nuclei. Cells possess innate surveillance pathways to detect microbial components or physiological stresses often associated with microbial infections. One of these pathways, cGAS/STING, induces IRF3-dependent antiviral interferon (IFN) responses upon detection of cytosolic DNA. Virion-associated vDNA can activate cGAS/STING during initial viral entry and uncoating/trafficking, and thus cGAS/STING is an obstacle to many DNA viruses. HPV has a unique vesicular trafficking pathway compared to many other DNA viruses. As the capsid uncoats within acidic endosomal compartments, minor capsid protein L2 protrudes across vesicular membranes to facilitate transport of vDNA to the Golgi. L2/vDNA resides within the Golgi lumen until G2/M, whereupon vesicular L2/vDNA traffics along spindle microtubules, tethering to chromosomes to access daughter cell nuclei. L2/vDNA-containing vesicles likely remain intact until G1, following nuclear envelope reformation. We hypothesize that this unique vesicular trafficking protects HPV from cGAS/STING surveillance. Here, we investigate cGAS/STING responses to HPV infection. DNA transfection resulted in acute cGAS/STING activation and downstream IFN responses. In contrast, HPV infection elicited minimal cGAS/STING and IFN responses. To determine the role of vesicular trafficking in cGAS/STING evasion, we forced premature viral penetration of vesicular membranes with membrane-perturbing cationic lipids. Such treatment renders a non-infectious trafficking-defective mutant HPV infectious, yet susceptible to cGAS/STING detection. Overall, HPV evades cGAS/STING by its unique subcellular trafficking, a property that may contribute to establishment of infection.
• Kim, W. J., Mai, A., Weyand, N. J., Rendón, M. A., Van Doorslaer, K., & So, M. (2019). Neisseria gonorrhoeae evades autophagic killing by downregulating CD46-cyt1 and remodeling lysosomes. PLoS pathogens, 15(2), e1007495.
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  The Gram-negative human pathogen N. gonorrhoeae (Ngo) quickly attaches to epithelial cells, and large numbers of the bacteria remain on the cell surface for prolonged periods. Ngo invades cells but few viable intracellular bacteria are recovered until later stages of infection, leading to the assumption that Ngo is a weak invader. On the cell surface, Ngo quickly recruits CD46-cyt1 to the epithelial cell cortex directly beneath the bacteria and causes its cleavage by metalloproteinases and Presenilin/γSecretease; how these interactions affect the Ngo lifecycle is unknown. Here, we show Ngo induces an autophagic response in the epithelial cell through CD46-cyt1/GOPC, and this response kills early invaders. Throughout infection, the pathogen slowly downregulates CD46-cyt1 and remodeling of lysosomes, another key autophagy component, and these activities ultimately promote intracellular survival. We present a model on the dynamics of Ngo infection and describe how this dual interference with the autophagic pathway allows late invaders to survive within the cell.
• O\textquoterightBrien, H., Alvin, J. W., Menghani, S. V., Van, D. K., & Johnson, M. (2019). Characterization of consensus operator site for Streptococcus pneumoniae copper repressor, CopY. bioRxiv.
• Pace, J., Youens-Clark, K. .., Freeman, C., Hurwitz, B., & Van Doorslaer, K. (2019). PuMA: a papillomavirus genome annotation tool. bioRxiv.
• Van Arsdale, A., Patterson, N. E., Maggi, E. C., Agoni, L., Van Doorslaer, K., Harmon, B., Nevadunsky, N., Kuo, D. Y., Einstein, M. H., Lenz, J., & Montagna, C. (2020). Insertional oncogenesis by HPV70 revealed by multiple genomic analyses in a clinically HPV-negative cervical cancer. Genes, chromosomes & cancer, 59(2), 84-95.
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  Cervical carcinogenesis, the second leading cause of cancer death in women worldwide, is caused by multiple types of human papillomaviruses (HPVs). To investigate a possible role for HPV in a cervical carcinoma that was HPV-negative by PCR testing, we performed HPV DNA hybridization capture plus massively parallel sequencing. This detected a subgenomic, URR-E6-E7-E1 segment of HPV70 DNA, a type not generally associated with cervical cancer, inserted in an intron of the B-cell lymphoma/leukemia 11B (BCL11B) gene in the human genome. Long range DNA sequencing confirmed the virus and flanking BCL11B DNA structures including both insertion junctions. Global transcriptomic analysis detected multiple, alternatively spliced, HPV70-BCL11B, fusion transcripts with fused open reading frames. The insertion and fusion transcripts were present in an intraepithelial precursor phase of tumorigenesis. These results suggest oncogenicity of HPV70, identify novel BCL11B variants with potential oncogenic implications, and underscore the advantages of thorough genomic analyses to elucidate insights into HPV-associated tumorigenesis.
• Van Doorslaer, K., & Dillner, J. (2019). The Launch of an International Animal Papillomavirus Reference Center. Viruses, 11(1).
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  The is a family of DNA viruses [...].
• King, K. M., & Van Doorslaer, K. (2018). Building (Viral) Phylogenetic Trees Using a Maximum Likelihood Approach. Current protocols in microbiology, 51(1), e63.
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  Phylogenetic analyses allow for inferring a hypothesis about the evolutionary history of a set of homologous molecular sequences. This hypothesis can be used as the basis for further molecular and computational studies. In this unit, we offer one specific method to construct a Maximum Likelihood phylogenetic tree. We outline how to identify homologous sequences and construct a multiple sequence alignment. Following alignment, sequences are screened for potentially confounding factors such as recombination and genetic saturation. Finally, a Maximum Likelihood phylogenetic tree can be constructed implementing a rigorously tested model of evolution. The workflow outlined in this unit provides sufficient background for inferring a robust phylogenetic tree starting from a particular gene of interest. © 2018 by John Wiley & Sons, Inc.
• Pastrana, D. V., Peretti, A., Welch, N. L., Borgogna, C., Olivero, C., Badolato, R., Notarangelo, L. D., Gariglio, M., FitzGerald, P. C., McIntosh, C. E., Reeves, J., Starrett, G. J., Bliskovsky, V., Velez, D., Brownell, I., Yarchoan, R., Wyvill, K. M., Uldrick, T. S., Maldarelli, F., , Lisco, A., et al. (2018). Metagenomic Discovery of 83 New Human Papillomavirus Types in Patients with Immunodeficiency. mSphere, 3(6).
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  Several immunodeficiencies are associated with high susceptibility to persistent and progressive human papillomavirus (HPV) infection leading to a wide range of cutaneous and mucosal lesions. However, the HPV types most commonly associated with such clinical manifestations in these patients have not been systematically defined. Here, we used virion enrichment, rolling circle amplification, and deep sequencing to identify circular DNA viruses present in skin swabs and/or wart biopsy samples from 48 patients with rare genetic immunodeficiencies, including patients with warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome, or epidermodysplasia verruciformis (EV). Their profiles were compared with the profiles of swabs from 14 healthy adults and warts from 6 immunologically normal children. Individual patients were typically infected with multiple HPV types; up to 26 different types were isolated from a single patient (multiple anatomical sites, one time point). Among these, we identified the complete genomes of 83 previously unknown HPV types and 35 incomplete genomes representing possible additional new types. HPV types in the genus were common in WHIM patients, whereas EV patients mainly shed HPVs from the genus Preliminary evidence based on three WHIM patients treated with plerixafor, a leukocyte mobilizing agent, suggest that longer-term therapy may correlate with decreased HPV diversity and increased predominance of HPV types associated with childhood skin warts. Although some members of the viral family cause benign skin warts (papillomas), many human papillomavirus (HPV) infections are not associated with visible symptoms. For example, most healthy adults chronically shed () virions from apparently healthy skin surfaces. To further explore the diversity of papillomaviruses, we performed viromic surveys on immunodeficient individuals suffering from florid skin warts. Our results nearly double the number of known HPV types and suggest that WHIM syndrome patients are uniquely susceptible to HPV-associated skin warts. Preliminary results suggest that treatment with the drug plerixafor may promote resolution of the unusual HPV skin warts observed in WHIM patients.
• Smeele, Z. E., Burns, J. M., Van Doorslaer, K. M., Fontenele, R. S., Waits, K., Stainton, D., Shero, M. R., Beltran, R. S., Kirkham, A. L., Berngartt, R., Kraberger, S., & Varsani, A. (2018). Diverse papillomaviruses identified in Weddell seals. Journal of General Virology.
• Van Doorslaer, K., Chen, Z., Bernard, H. U., Chan, P. K., DeSalle, R., Dillner, J., Forslund, O., Haga, T., McBride, A. A., Villa, L. L., Burk, R. D., & Ictv Report Consortium, . (2018). ICTV Virus Taxonomy Profile: Papillomaviridae. The Journal of general virology, 99(8), 989-990.
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  The Papillomaviridae is a family of small, non-enveloped viruses with double-stranded DNA genomes of 5 748 to 8 607 bp. Their classification is based on pairwise nucleotide sequence identity across the L1 open reading frame. Members of the Papillomaviridae primarily infect mucosal and keratinised epithelia, and have been isolated from fish, reptiles, birds and mammals. Despite a long co-evolutionary history with their hosts, some papillomaviruses are pathogens of their natural host species. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Papillomaviridae, which is available at http://www.ictv.global/report/papillomaviridae.
• Van Doorslaer, K., Kraberger, S., Austin, C., Farkas, K., Bergeman, M., Paunil, E., Davison, W., & Varsani, A. (2018). Fish polyomaviruses belong to two distinct evolutionary lineages. The Journal of general virology, 99(4), 567-573.
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  The Polyomaviridae is a diverse family of circular double-stranded DNA viruses. Polyomaviruses have been isolated from a wide array of animal hosts. An understanding of the evolutionary and ecological dynamics of these viruses is essential to understanding the pathogenicity of polyomaviruses. Using a high throughput sequencing approach, we identified a novel polyomavirus in an emerald notothen (Trematomus bernacchii) sampled in the Ross sea (Antarctica), expanding the known number of fish-associated polyomaviruses. Our analysis suggests that polyomaviruses belong to three main evolutionary clades; the first clade is made up of all recognized terrestrial polyomaviruses. The fish-associated polyomaviruses are not monophyletic, and belong to two divergent evolutionary lineages. The fish viruses provide evidence that the evolution of the key viral large T protein involves gain and loss of distinct domains.
• Van Doorslaer, K., Chen, D., Chapman, S., Khan, J., & McBride, A. A. (2017). Persistence of an Oncogenic Papillomavirus Genome Requires cis Elements from the Viral Transcriptional Enhancer. mBio, 8(6).
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  Human papillomavirus (HPV) genomes are replicated and maintained as extrachromosomal plasmids during persistent infection. The viral E2 proteins are thought to promote stable maintenance replication by tethering the viral DNA to host chromatin. However, this has been very difficult to prove genetically, as the E2 protein is involved in transcriptional regulation and initiation of replication, as well as its assumed role in genome maintenance. This makes mutational analysis of viral trans factors and cis elements in the background of the viral genome problematic and difficult to interpret. To circumvent this problem, we have developed a complementation assay in which the complete wild-type HPV18 genome is transfected into primary human keratinocytes along with subgenomic or mutated replicons that contain the minimal replication origin. The wild-type genome provides the E1 and E2 proteins in trans, allowing us to determine additional cis elements that are required for long-term replication and partitioning of the replicon. We found that, in addition to the core replication origin (and the three E2 binding sites located therein), additional sequences from the transcriptional enhancer portion of the URR (upstream regulatory region) are required in cis for long-term genome replication.IMPORTANCE Human papillomaviruses infect cutaneous and mucosal epithelial cells of the host, and this results in very-long-lived, persistent infection. The viral genomes are small, circular, double-stranded DNA molecules that replicate extrachromosomally in concert with cellular DNA. This replication strategy requires that the virus has a robust mechanism to partition and retain the viral genomes in dividing cells. This has been difficult to study, because viral transcription, replication, and partitioning are regulated by the same viral proteins and involve overlapping elements in the viral genome. We developed a complementation assay that allows us to separate these functions and define the elements required for long-term replication and stable maintenance replication of the HPV genome. This has important implications, as disruption of viral maintenance replication can eliminate viral genomes from infected cells, thus curing persistent HPV infection.
• Van Doorslaer, K., Li, Z., Xirasagar, S., Maes, P., Kaminsky, D., Liou, D., Sun, Q., Kaur, R., Huyen, Y., & McBride, A. A. (2017). The Papillomavirus Episteme: a major update to the papillomavirus sequence database. Nucleic acids research, 45(D1), D499-D506.
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  The Papillomavirus Episteme (PaVE) is a database of curated papillomavirus genomic sequences, accompanied by web-based sequence analysis tools. This update describes the addition of major new features. The papillomavirus genomes within PaVE have been further annotated, and now includes the major spliced mRNA transcripts. Viral genes and transcripts can be visualized on both linear and circular genome browsers. Evolutionary relationships among PaVE reference protein sequences can be analysed using multiple sequence alignments and phylogenetic trees. To assist in viral discovery, PaVE offers a typing tool; a simplified algorithm to determine whether a newly sequenced virus is novel. PaVE also now contains an image library containing gross clinical and histopathological images of papillomavirus infected lesions. Database URL: https://pave.niaid.nih.gov/.
• Van Doorslaer, K., Ruoppolo, V., Schmidt, A., Lescroël, A., Jongsomjit, D., Elrod, M., Kraberger, S., Stainton, D., Dugger, K. M., Ballard, G., Ainley, D. G., & Varsani, A. (2017). Unique genome organization of non-mammalian papillomaviruses provides insights into the evolution of viral early proteins. Virus evolution, 3(2), vex027.
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  The family Papillomaviridae contains more than 320 papillomavirus types, with most having been identified as infecting skin and mucosal epithelium in mammalian hosts. To date, only nine non-mammalian papillomaviruses have been described from birds (n = 5), a fish (n = 1), a snake (n = 1), and turtles (n = 2). The identification of papillomaviruses in sauropsids and a sparid fish suggests that early ancestors of papillomaviruses were already infecting the earliest Euteleostomi. The Euteleostomi clade includes more than 90 per cent of the living vertebrate species, and progeny virus could have been passed on to all members of this clade, inhabiting virtually every habitat on the planet. As part of this study, we isolated a novel papillomavirus from a 16-year-old female Adélie penguin (Pygoscelis adeliae) from Cape Crozier, Ross Island (Antarctica). The new papillomavirus shares ∼64 per cent genome-wide identity to a previously described Adélie penguin papillomavirus. Phylogenetic analyses show that the non-mammalian viruses (expect the python, Morelia spilota, associated papillomavirus) cluster near the base of the papillomavirus evolutionary tree. A papillomavirus isolated from an avian host (Northern fulmar; Fulmarus glacialis), like the two turtle papillomaviruses, lacks a putative E9 protein that is found in all other avian papillomaviruses. Furthermore, the Northern fulmar papillomavirus has an E7 more similar to the mammalian viruses than the other avian papillomaviruses. Typical E6 proteins of mammalian papillomaviruses have two Zinc finger motifs, whereas the sauropsid papillomaviruses only have one such motif. Furthermore, this motif is absent in the fish papillomavirus. Thus, it is highly likely that the most recent common ancestor of the mammalian and sauropsid papillomaviruses had a single motif E6. It appears that a motif duplication resulted in mammalian papillomaviruses having a double Zinc finger motif in E6. We estimated the divergence time between Northern fulmar-associated papillomavirus and the other Sauropsid papillomaviruses be to around 250 million years ago, during the Paleozoic-Mesozoic transition and our analysis dates the root of the papillomavirus tree between 400 and 600 million years ago. Our analysis shows evidence for niche adaptation and that these non-mammalian viruses have highly divergent E6 and E7 proteins, providing insights into the evolution of the early viral (onco-)proteins.
• Warren, C. J., Westrich, J. A., Doorslaer, K. V., & Pyeon, D. (2017). Roles of APOBEC3A and APOBEC3B in Human Papillomavirus Infection and Disease Progression. Viruses, 9(8).
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  The apolipoprotein B messenger RNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family of cytidine deaminases plays an important role in the innate immune response to viral infections by editing viral genomes. However, the cytidine deaminase activity of APOBEC3 enzymes also induces somatic mutations in host genomes, which may drive cancer progression. Recent studies of human papillomavirus (HPV) infection and disease outcome highlight this duality. HPV infection is potently inhibited by one family member, APOBEC3A. Expression of APOBEC3A and APOBEC3B is highly elevated by the HPV oncoproteins E6 and E7 during persistent virus infection and disease progression. Furthermore, there is a high prevalence of APOBEC3A and APOBEC3B mutation signatures in HPV-associated cancers. These findings suggest that induction of an APOBEC3-mediated antiviral response during HPV infection may inadvertently contribute to cancer mutagenesis and virus evolution. Here, we discuss current understanding of APOBEC3A and APOBEC3B biology in HPV restriction, evolution, and associated cancer mutagenesis.
• Buck, C. B., Van Doorslaer, K., Peretti, A., Geoghegan, E. M., Tisza, M. J., An, P., Katz, J. P., Pipas, J. M., McBride, A. A., Camus, A. C., McDermott, A. J., Dill, J. A., Delwart, E., Ng, T. F., Farkas, K., Austin, C., Kraberger, S., Davison, W., Pastrana, D. V., & Varsani, A. (2016). The Ancient Evolutionary History of Polyomaviruses. PLoS pathogens, 12(4), e1005574.
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  Polyomaviruses are a family of DNA tumor viruses that are known to infect mammals and birds. To investigate the deeper evolutionary history of the family, we used a combination of viral metagenomics, bioinformatics, and structural modeling approaches to identify and characterize polyomavirus sequences associated with fish and arthropods. Analyses drawing upon the divergent new sequences indicate that polyomaviruses have been gradually co-evolving with their animal hosts for at least half a billion years. Phylogenetic analyses of individual polyomavirus genes suggest that some modern polyomavirus species arose after ancient recombination events involving distantly related polyomavirus lineages. The improved evolutionary model provides a useful platform for developing a more accurate taxonomic classification system for the viral family Polyomaviridae.
• Harrison, J. S., Jacobs, T. M., Houlihan, K., Van Doorslaer, K., & Kuhlman, B. (2016). UbSRD: The Ubiquitin Structural Relational Database. Journal of molecular biology, 428(4), 679-687.
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  The structurally defined ubiquitin-like homology fold (UBL) can engage in several unique protein-protein interactions and many of these complexes have been characterized with high-resolution techniques. Using Rosetta's structural classification tools, we have created the Ubiquitin Structural Relational Database (UbSRD), an SQL database of features for all 509 UBL-containing structures in the PDB, allowing users to browse these structures by protein-protein interaction and providing a platform for quantitative analysis of structural features. We used UbSRD to define the recognition features of ubiquitin (UBQ) and SUMO observed in the PDB and the orientation of the UBQ tail while interacting with certain types of proteins. While some of the interaction surfaces on UBQ and SUMO overlap, each molecule has distinct features that aid in molecular discrimination. Additionally, we find that the UBQ tail is malleable and can adopt a variety of conformations upon binding. UbSRD is accessible as an online resource at rosettadesign.med.unc.edu/ubsrd.
• Van Doorslaer, K. M., Porter, S., McKinney, C., Stepp, W. H., & McBride, A. A. (2016). Novel recombinant papillomavirus genomes expressing selectable genes.. Scientific Reports, 6.
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  Papillomaviruses infect and replicate in keratinocytes, but viral proteins are initially expressed at low levels and there is no effective and quantitative method to determine the efficiency of infection on a cell-to-cell basis. Here we describe human papillomavirus (HPV) genomes that express marker proteins (antibiotic resistance genes and Green Fluorescent Protein), and can be used to elucidate early stages in HPV infection of primary keratinocytes. To generate these recombinant genomes, the late region of the oncogenic HPV18 genome was replaced by CpG free marker genes. Insertion of these exogenous genes did not affect early replication, and had only minimal effects on early viral transcription. When introduced into primary keratinocytes, the recombinant marker genomes gave rise to drug-resistant keratinocyte colonies and cell lines, which maintained the extrachromosomal recombinant genome long-term. Furthermore, the HPV18 "marker" genomes could be packaged into viral particles (quasivirions) and used to infect primary human keratinocytes in culture. This resulted in the outgrowth of drug-resistant keratinocyte colonies containing replicating HPV18 genomes. In summary, we describe HPV18 marker genomes that can be used to quantitatively investigate many aspects of the viral life cycle.
• Van Doorslaer, K., & McBride, A. A. (2016). Molecular archeological evidence in support of the repeated loss of a papillomavirus gene. Scientific Reports, 6.
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  It is becoming clear that, in addition to gene gain, the loss of genes may be an important evolutionary mechanism for many organisms. However, gene loss is often associated with an increased mutation rate, thus quickly erasing evidence from the genome. The analysis of evolutionarily related sequences can provide empirical evidence for gene loss events. This paper analyzes the sequences of over 300 genetically distinct papillomaviruses and provides evidence for a role of gene loss during the evolution of certain papillomavirus genomes. Phylogenetic analysis suggests that the viral E6 gene was lost at least twice. Despite belonging to distant papillomaviral genera, these viruses lacking a canonical E6 protein may potentially encode a highly hydrophobic protein from an overlapping open reading frame, which we designate E10. Evolutionary pressure working on this alternative frame, may explain why, despite having lost the E6 open reading frame between 20 and 60 million years ago, evidence of an E6-like protein is conserved.
• Van Doorslaer, K., & McBride, A. A. (2016). Molecular archeological evidence in support of the repeated loss of a papillomavirus gene. Scientific reports, 6, 33028.
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  It is becoming clear that, in addition to gene gain, the loss of genes may be an important evolutionary mechanism for many organisms. However, gene loss is often associated with an increased mutation rate, thus quickly erasing evidence from the genome. The analysis of evolutionarily related sequences can provide empirical evidence for gene loss events. This paper analyzes the sequences of over 300 genetically distinct papillomaviruses and provides evidence for a role of gene loss during the evolution of certain papillomavirus genomes. Phylogenetic analysis suggests that the viral E6 gene was lost at least twice. Despite belonging to distant papillomaviral genera, these viruses lacking a canonical E6 protein may potentially encode a highly hydrophobic protein from an overlapping open reading frame, which we designate E10. Evolutionary pressure working on this alternative frame, may explain why, despite having lost the E6 open reading frame between 20 and 60 million years ago, evidence of an E6-like protein is conserved.
• Van Doorslaer, K., Chen, Z., & McBride, A. A. (2016). Detection and Genotyping of Human Papillomaviruses from Archival Formalin-Fixed Tissue Samples. Current Protocols in Microbiology, 14B--9.
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  Pathology departments routinely process and store formalin-fixed, paraffin-embedded (FFPE) tissue samples for clinical diagnosis. These collections often contain decades' worth of samples and represent a treasure trove of specimens that can be analyzed for retrospective epidemiological studies, diagnostics, and pathogen discovery. Accurate amplification and sequencing of DNA from these samples is critical for the usability of these FFPE samples. Here we present a collection of protocols that describe extraction of DNA from FFPE tissues, PCR amplification of human papillomavirus DNA, and subsequent genotyping of the infecting viru
• Van Doorslaer, K., Chen, Z., & McBride, A. A. (2016). Detection and Genotyping of Human Papillomaviruses from Archival Formalin-Fixed Tissue Samples. Current protocols in microbiology, 43, 14B.9.1-14B.9.20.
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  Pathology departments routinely process and store formalin-fixed, paraffin-embedded (FFPE) tissue samples for clinical diagnosis. These collections often contain decades' worth of samples and represent a treasure trove of specimens that can be analyzed for retrospective epidemiological studies, diagnostics, and pathogen discovery. Accurate amplification and sequencing of DNA from these samples is critical for the usability of these FFPE samples. Here we present a collection of protocols that describe extraction of DNA from FFPE tissues, PCR amplification of human papillomavirus DNA, and subsequent genotyping of the infecting virus. © 2016 by John Wiley & Sons, Inc.
• Van Doorslaer, K., Li, Z., Xirasagar, S., Maes, P., Kaminsky, D., Liou, D., Sun, Q., Kaur, R., Huyen, Y., & McBride, A. A. (2016). The Papillomavirus Episteme: a major update to the papillomavirus sequence database. Nucleic Acids Research, gkw879.
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  The Papillomavirus Episteme (PaVE) is a database of curated papillomavirus genomic sequences, accompanied by web-based sequence analysis tools. This update describes the addition of major new features. The papillomavirus genomes within PaVE have been further annotated, and now includes the major spliced mRNA transcripts. Viral genes and transcripts can be visualized on both linear and circular genome browsers. Evolutionary relationships among PaVE reference protein sequences can be analysed using multiple sequence alignments and phylogenetic trees. To assist in viral discovery, PaVE offers a typing tool; a simplified algorithm to determine whether a newly sequenced virus is novel. PaVE also now contains an image library containing gross clinical and histopathological images of papillomavirus infected lesions. Database URL: https://pave.niaid.nih.gov/.
• Van Doorslaer, K., Porter, S., McKinney, C., Stepp, W. H., & McBride, A. A. (2016). Novel recombinant papillomavirus genomes expressing selectable genes. Scientific reports, 6, 37782.
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  Papillomaviruses infect and replicate in keratinocytes, but viral proteins are initially expressed at low levels and there is no effective and quantitative method to determine the efficiency of infection on a cell-to-cell basis. Here we describe human papillomavirus (HPV) genomes that express marker proteins (antibiotic resistance genes and Green Fluorescent Protein), and can be used to elucidate early stages in HPV infection of primary keratinocytes. To generate these recombinant genomes, the late region of the oncogenic HPV18 genome was replaced by CpG free marker genes. Insertion of these exogenous genes did not affect early replication, and had only minimal effects on early viral transcription. When introduced into primary keratinocytes, the recombinant marker genomes gave rise to drug-resistant keratinocyte colonies and cell lines, which maintained the extrachromosomal recombinant genome long-term. Furthermore, the HPV18 "marker" genomes could be packaged into viral particles (quasivirions) and used to infect primary human keratinocytes in culture. This resulted in the outgrowth of drug-resistant keratinocyte colonies containing replicating HPV18 genomes. In summary, we describe HPV18 marker genomes that can be used to quantitatively investigate many aspects of the viral life cycle.
• Harrison, J. S., Jacobs, T. M., Houlihan, K., Van Doorslaer, K., & Kuhlman, B. (2015). Data in support of UbSRD: The Ubiquitin Structural Relational Database. Data in brief, 5, 605-15.
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  This article provides information to support the database article titled "UbSRD: The Ubiquitin Structural Relational Database" (Harrison et al., 2015) [1] . The ubiquitin-like homology fold (UBL) represents a large family that encompasses both post-translational modifications, like ubiquitin (UBQ) and SUMO, and functional domains on many biologically important proteins like Parkin, UHRF1 (ubiquitin-like with PDB and RING finger domains-1), and Usp7 (ubiquitin-specific protease-7) (Zhang et al., 2015; Rothbart et al., 2013; Burroughs et al., 2012; Wauer et al., 2015) [2], [3], [4], [5]. The UBL domain can participate in several unique protein-protein interactions (PPI) since protein adducts can be attached to and removed from amino groups of lysine side chains and the N-terminus of proteins. Given the biological significance of UBL domains, many have been characterized with high-resolution techniques, and for UBQ and SUMO, many protein complexes have been characterized. We identified all the UBL domains in the PDB and created a relational database called UbSRD (Ubiquitin Structural Relational Database) by using structural analysis tools in the Rosetta (Leaver et al., 2013; O'Meara et al., 2015; Leaver-fay et al., 2011) [1], [6], [7], [8]. Querying UbSRD permitted us to report many quantitative properties of UBQ and SUMO recognition at different types interfaces (noncovalent: NC, conjugated: CJ, and deubiquitanse: DB). In this data article, we report the average number of non-UBL neighbors, secondary structure of interacting motifs, and the type of inter-molecular hydrogen bonds for each residue of UBQ and SUMO. Additionally, we used PROMALS3D to generate a multiple sequence alignment used to construct a phylogram for the entire set of UBLs (Pei and Grishin, 2014) [9]. The data described here will be generally useful to scientists studying the molecular basis for recognition of UBQ or SUMO.
• Jang, M. K., Anderson, D. E., van Doorslaer, K., & McBride, A. A. (2015). A proteomic approach to discover and compare interacting partners of papillomavirus E2 proteins from diverse phylogenetic groups. Proteomics, 15(12), 2038-50.
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  Papillomaviruses are a very successful group of viruses that replicate persistently in localized regions of the stratified epithelium of their specific host. Infection results in pathologies ranging from asymptomatic infection, benign warts, to malignant carcinomas. Despite this diversity, papillomavirus genomes are small (7-8 kbp) and contain at most eight genes. To sustain the complex papillomaviral life cycle, each viral protein has multiple functions and interacts with and manipulates a plethora of cellular proteins. In this study, we use tandem affinity purification and MS to identify host factors that interact with 11 different papillomavirus E2 proteins from diverse phylogenetic groups. The E2 proteins function in viral transcription and replication and correspondingly interact with host proteins involved in transcription, chromatin remodeling and modification, replication, and RNA processing.
• Van Doorslaer, K., DeSalle, R., Einstein, M. H., & Burk, R. D. (2015). Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche. PLoS pathogens, 11(6), e1004980.
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  In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential) is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution.
• Warren, C. J., Van Doorslaer, K., Pandey, A., Espinosa, J. M., & Pyeon, D. (2015). Role of the host restriction factor APOBEC3 on papillomavirus evolution. Virus evolution, 1(1).
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  More than 270 different types of papillomaviruses have been discovered in a wide array of animal species. Despite the great diversity of papillomaviruses, little is known about the evolutionary processes that drive host tropism and the emergence of oncogenic genotypes. Although host defense mechanisms have evolved to interfere with various aspects of a virus life cycle, viruses have also coevolved copious strategies to avoid host antiviral restriction. Our and other studies have shown that the cytidine deaminase APOBEC3 family members edit HPV genomes and restrict virus infectivity. Thus, we hypothesized that host restriction by APOBEC3 served as selective pressure during papillomavirus evolution. To test this hypothesis, we analyzed the relative abundance of all dinucleotide sequences in full-length genomes of 274 papillomavirus types documented in the Papillomavirus Episteme database (PaVE). Here, we report that TC dinucleotides, the preferred target sequence of several human APOBEC3 proteins (hA3A, hA3B, hA3F, and hA3H), are highly depleted in papillomavirus genomes. Given that HPV infection is highly tissue-specific, the expression levels of APOBEC3 family members were analyzed. The basal expression levels of all APOBEC3 isoforms, excluding hA3B, are significantly higher in mucosal skin compared with cutaneous skin. Interestingly, we reveal that Alphapapillomaviruses (alpha-PVs), a majority of which infects anogenital mucosa, display the most dramatic reduction in TC dinucleotide content. Computer modeling and reconstruction of ancestral alpha-PV genomes suggest that TC depletion occurred after the alpha-PVs diverged from their most recent common ancestor. In addition, we found that TC depletion in alpha-PVs is greatly affected by protein coding potential. Taken together, our results suggest that PVs replicating in tissues with high APOBEC3 levels may have evolved to evade restriction by selecting for variants that contain reduced APOBEC3 target sites in their genomes.
• Harari, A., Wood, C. E., Van Doorslaer, K., Chen, Z., Domaingue, M. C., Elmore, D., Koenig, P., Wagner, J. D., Jennings, R. N., & Burk, R. D. (2013). Condylomatous genital lesions in cynomolgus macaques from Mauritius. Toxicologic pathology, 41(6), 893-901.
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  Genital condyloma-like lesions were observed on male and female cynomolgus macaque monkeys (Macaca fascicularis) originating from the island of Mauritius. Cytobrush and/or biopsy samples were obtained from lesions of 57 affected macaques. Primary histologic features included eosinophilic, neutrophilic, and lymphoplasmacytic penile and vulvar inflammation, epidermal hyperplasia with acanthosis, and increased collagenous stroma. Polymerase chain reaction-based assays to amplify viral DNA revealed the presence of macaque lymphocryptovirus (LCV) DNA but not papillomavirus or poxvirus DNA. Subsequent DNA analyses of 3 genomic regions of LCV identified isolates associated with lesions in 19/25 (76%) biopsies and 19/57 (33%) cytology samples. Variable immunolabeling for proteins related to the human LCV Epstein Barr Virus was observed within intralesional plasma cells, stromal cells, and epithelial cells. Further work is needed to characterize the epidemiologic features of these lesions and their association with LCV infection in Mauritian-origin macaques.
• Van Doorslaer, K. (2013). Evolution of the papillomaviridae. Virology, 445(1-2), 11-20.
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  Viruses belonging to the Papillomaviridae family have been isolated from a variety of mammals, birds and non-avian reptiles. It is likely that most, if not all, amniotes carry a broad array of viral types. To date, the complete genomic sequence of more than 240 distinct viral types has been characterized at the nucleotide level. The analysis of this sequence information has begun to shed light on the evolutionary history of this important virus family. The available data suggests that many different evolutionary mechanisms have influenced the papillomavirus phylogenetic tree. Increasing evidence supports that the ancestral papillomavirus initially specialized to infect different ecological niches on the host. This episode of niche sorting was followed by extensive episodes of co-speciation with the host. This review attempts to summarize our current understanding of the papillomavirus evolution.
• Van Doorslaer, K., Tan, Q., Xirasagar, S., Bandaru, S., Gopalan, V., Mohamoud, Y., Huyen, Y., & McBride, A. A. (2013). The Papillomavirus Episteme: a central resource for papillomavirus sequence data and analysis. Nucleic acids research, 41(Database issue), D571-8.
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  The goal of the Papillomavirus Episteme (PaVE) is to provide an integrated resource for the analysis of papillomavirus (PV) genome sequences and related information. The PaVE is a freely accessible, web-based tool (http://pave.niaid.nih.gov) created around a relational database, which enables storage, analysis and exchange of sequence information. From a design perspective, the PaVE adopts an Open Source software approach and stresses the integration and reuse of existing tools. Reference PV genome sequences have been extracted from publicly available databases and reannotated using a custom-created tool. To date, the PaVE contains 241 annotated PV genomes, 2245 genes and regions, 2004 protein sequences and 47 protein structures, which users can explore, analyze or download. The PaVE provides scientists with the data and tools needed to accelerate scientific progress for the study and treatment of diseases caused by PVs.
• Machado, F. S., Rodriguez, N. E., Adesse, D., Garzoni, L. R., Esper, L., Lisanti, M. P., Burk, R. D., Albanese, C., Van Doorslaer, K., Weiss, L. M., Nagajyothi, F., Nosanchuk, J. D., Wilson, M. E., & Tanowitz, H. B. (2012). Recent developments in the interactions between caveolin and pathogens. Advances in experimental medicine and biology, 729, 65-82.
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  The role of caveolin and caveolae in the pathogenesis of infection has only recently been appreciated. In this chapter, we have highlighted some important new data on the role of caveolin in infections due to bacteria, viruses and fungi but with particular emphasis on the protozoan parasites Leishmania spp., Trypanosoma cruzi and Toxoplasma gondii. This is a continuing area of research and the final chapter has not been written on this topic.
• Van Doorslaer, K., & Burk, R. D. (2012). Association between hTERT activation by HPV E6 proteins and oncogenic risk. Virology, 433(1), 216-9.
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  Expression of activated telomerase and subversion of the p16/pRb pathway is sufficient and essential for the in vitro immortalization of primary keratinocytes. Most cancers-including cervical carcinoma-over-express hTERT, the catalytic domain of the telomerase complex. Only a limited set of viruses within the Alphapapillomavirus genus are oncogenic. The viral functions responsible for this distinction are not well understood. The human papillomavirus type 16 E6 protein activates the hTERT promoter. We used a luciferase-based assay to test the ability of 29 viral types, representing all current species within the Alphapapillomavirus genus, to activate the hTERT promoter. We show that oncogenic types specifically activate the hTERT promoter, while non-oncogenic types do not. Statistical analysis supports the notion that activation of the hTERT promoter is uniquely associated with oncogenic types, independent of evolutionary relationships. This finding begins to shed light on the viral phenotypes correlated with oncogenic potential.
• Smith, B., Chen, Z., Reimers, L., van Doorslaer, K., Schiffman, M., Desalle, R., Herrero, R., Yu, K., Wacholder, S., Wang, T., & Burk, R. D. (2011). Sequence imputation of HPV16 genomes for genetic association studies. PloS one, 6(6), e21375.
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  Human Papillomavirus type 16 (HPV16) causes over half of all cervical cancer and some HPV16 variants are more oncogenic than others. The genetic basis for the extraordinary oncogenic properties of HPV16 compared to other HPVs is unknown. In addition, we neither know which nucleotides vary across and within HPV types and lineages, nor which of the single nucleotide polymorphisms (SNPs) determine oncogenicity.
• Van Doorslaer, K., Bernard, H. U., Chen, Z., de Villiers, E. M., zur Hausen, H., & Burk, R. D. (2011). Papillomaviruses: evolution, Linnaean taxonomy and current nomenclature. Trends in microbiology, 19(2), 49-50; author reply 50-1.
• Wood, C. E., Tannehill-Gregg, S. H., Chen, Z., Doorslaer, K. v., Nelson, D. R., Cline, J. M., & Burk, R. D. (2011). Novel betapapillomavirus associated with hand and foot papillomas in a cynomolgus macaque. Veterinary pathology, 48(3), 731-6.
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  Betapapillomavirus is a genus of papillomaviruses (PVs) commonly found in human skin and associated with both benign and malignant skin lesions. Only 2 previous beta-PVs have been fully characterized in nonhuman species. This report describes a novel beta-PV, named Macaca fascicularis PV type 2 (MfPV2), isolated from exophytic skin papillomas on the hands and feet of a 2-year-old male cynomolgus monkey (M. fascicularis). On histology the papillomas were composed of diffusely thickened epidermis with superficial foci of cytomegaly, cytoplasmic pallor, marginalized chromatin, and rare eosinophilic intranuclear inclusion bodies. Positive immunostaining for p16 and the proliferation marker Ki67 was present multifocally within affected epidermis, most prominently within basal-type cells. Complete sequence identity (100%) was noted between PV genomes fully sequenced from hand and foot lesions. The MfPV2 genome was 7632 base pairs in length and included putative open reading frames (ORFs) for E1, E2, E4, E6, E7, L1, and L2 genes, similar to other PVs. The closest relatives to MfPV2 based on the L1 ORF sequence were all beta-PVs. These included human PV (HPV) 9, HPV115, HPV76, HPV75, and MfPV1 (60-70% pairwise identity for all), the latter of which was also isolated from hand and foot papillomas in a cynomolgus macaque. Phylogenetic analysis placed MfPV2 in a new species group (beta-6), distinct from HPVs (beta-1 to beta-5) and MfPV1 (beta-1). These findings characterize a new nonhuman beta-PV and provide additional support for the idea that tissue tropism among ancestral primate PVs developed prior to divergence of certain Old World primate lineages.
• Bernard, H. U., Burk, R. D., Chen, Z., van Doorslaer, K., zur Hausen, H., & de Villiers, E. M. (2010). Classification of papillomaviruses (PVs) based on 189 PV types and proposal of taxonomic amendments. Virology, 401(1), 70-9.
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  We present an expansion of the classification of the family Papillomaviridae, which now contains 29 genera formed by 189 papillomavirus (PV) types isolated from humans (120 types), non-human mammals, birds and reptiles (64, 3 and 2 types, respectively). To accommodate the number of PV genera exceeding the Greek alphabet, the prefix "dyo" is used, continuing after the Omega-PVs with Dyodelta-PVs. The current set of human PVs is contained within five genera, whereas mammalian, avian and reptile PVs are contained within 20, 3 and 1 genera, respectively. We propose standardizations to the names of a number of animal PVs. As prerequisite for a coherent nomenclature of animal PVs, we propose founding a reference center for animal PVs. We discuss that based on emerging species concepts derived from genome sequences, PV types could be promoted to the taxonomic level of species, but we do not recommend implementing this change at the current time.
• Connolly, E. C., Van Doorslaer, K., Rogler, L. E., & Rogler, C. E. (2010). Overexpression of miR-21 promotes an in vitro metastatic phenotype by targeting the tumor suppressor RHOB. Molecular cancer research : MCR, 8(5), 691-700.
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  Metastasis is a multistep process that involves the deregulation of oncogenes and tumor suppressors beyond changes required for primary tumor formation. RHOB is known to have tumor suppressor activity, and its knockdown is associated with more aggressive tumors as well as changes in cell shape, migration, and adhesion. This study shows that oncogenic microRNA, miR-21, represses RHOB expression by directly targeting the 3' untranslated region. Loss of miR-21 is associated with an elevation of RHOB in hepatocellular carcinoma cell lines Huh-7 and HepG2 and in the metastatic breast cancer cell line MDA-MB-231. Using in vitro models of distinct stages of metastasis, we showed that loss of miR-21 also causes a reduction in migration, invasion, and cell elongation. The reduction in migration and cell elongation can be mimicked by overexpression of RHOB. Furthermore, changes in miR-21 expression lead to alterations in matrix metalloproteinase-9 activity. Therefore, we conclude that miR-21 promotes multiple components of the metastatic phenotype in vitro by regulating several important tumor suppressors, including RHOB.
• Fu, L., Van Doorslaer, K., Chen, Z., Ristriani, T., Masson, M., Travé, G., & Burk, R. D. (2010). Degradation of p53 by human Alphapapillomavirus E6 proteins shows a stronger correlation with phylogeny than oncogenicity. PloS one, 5(9).
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  Human Papillomavirus (HPV) E6 induced p53 degradation is thought to be an essential activity by which high-risk human Alphapapillomaviruses (alpha-HPVs) contribute to cervical cancer development. However, most of our understanding is derived from the comparison of HPV16 and HPV11. These two viruses are relatively distinct viruses, making the extrapolation of these results difficult. In the present study, we expand the tested strains (types) to include members of all known HPV species groups within the Alphapapillomavirus genus.
• Safaeian, M., van Doorslaer, K., Schiffman, M., Chen, Z., Rodriguez, A. C., Herrero, R., Hildesheim, A., & Burk, R. D. (2010). Lack of heterogeneity of HPV16 E7 sequence compared with HPV31 and HPV73 may be related to its unique carcinogenic properties. Archives of virology, 155(3), 367-70.
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  To assess the role of human papillomavirus virus (HPV) genetics in cervical lesions, we sequenced the E7 gene of HPV16, 31, or 73 from singly infected women who (1) cleared the infection quickly, (2) had type-specific persistent infection, or (3) progressed to CIN2 or worse lesions. Four of the 296 HPV16 E7 nucleotides were variable, compared with 7 of 296 for HPV31 E7 and 4 of 296 for HPV73 E7. While most of the polymorphisms in HPV31 and -73 resulted in non-synonymous amino acid changes, the polymorphisms in the HPV16 E7 resulted in synonymous changes. The lack of heterogeneity of HPV16 E7 suggests high evolutionary purifying selection that might be related to the unique carcinogenicity of HPV16.
• Van Doorslaer, K., & Burk, R. D. (2010). Evolution of human papillomavirus carcinogenicity. Advances in virus research, 77, 41-62.
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  Members of the Alphapapillomavirus genus are the causative agent for virtually all cases of cervical cancer. However, strains (commonly referred to as types) within this genus span the entire range of pathogenicity from highly carcinogenic (e.g., HPV16, odds ratio = 281.9, responsible for 50% of all cervical cancers), moderately carcinogenic (e.g., HPV31) to not carcinogenic (e.g., HPV71). The persistent expression of the viral oncoproteins (E6 and E7) from HPV16 has been shown to be necessary and sufficient to transform primary human keratinocytes in vitro. A plethora of functions have been described for both oncoproteins, and through functional comparisons between HPV16 and HPV6, a subset of these functions have been suggested to be oncogenic. However, extrapolating functional differences from these comparisons is unlikely to tease apart the fine details. In this review, we argue that a thorough understanding of the molecular mechanisms differentiating oncogenic from nononcogenic types should be obtained by performing functional assays in an evolutionary and epidemiological framework. We continue by interpreting some recent results using this paradigm and end by suggesting directions for future inquiries.
• Van Doorslaer, K., Reimers, L. L., Studentsov, Y. Y., Einstein, M. H., & Burk, R. D. (2010). Serological response to an HPV16 E7 based therapeutic vaccine in women with high-grade cervical dysplasia. Gynecologic oncology, 116(2), 208-12.
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  Infection with oncogenic human papillomaviruses has been linked to the development of cervical neoplasia and cancer. The exclusive expression of E7, a viral oncogene, in infected cells makes this protein an ideal target for immunotherapy. We recently reported on the results of a trial in women with cervical carcinoma-in-situ using HspE7, a protein vaccine consisting of full length HPV16 E7 linked to a heat shock protein from M. bovis. The stimulating effects of HspE7 on specific cytotoxic T lymphocytes have been demonstrated in vitro and in (pre-)clinical trials. The induction of a B-cell response by HspE7 and its association with clinical outcome is unknown, and is the purpose of this study.
• Burk, R. D., Chen, Z., & Van Doorslaer, K. (2009). Human papillomaviruses: genetic basis of carcinogenicity. Public health genomics, 12(5-6), 281-90.
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  Persistent infection by specific oncogenic human papillomaviruses (HPVs) is established as the necessary cause of cervix cancer. DNA sequence differences between HPV genomes determine whether an HPV has the potential to cause cancer. Of the more than 100 HPV genotypes characterized at the genetic level, at least 15 are associated, to varying degrees, with cervical cancer. Classification based on nucleotide similarity places nearly all HPVs that infect the cervicovaginal area within the alpha-PV genus. Within this genus, phylogenetic trees inferred from the entire viral genome cluster all cancer-causing types together, suggesting the existence of a common ancestor for the oncogenic HPVs. However, in separate trees built from the early open reading frames (ORFs; i.e. E1, E2, E6, E7) or the late ORFs (i.e. L1, L2), the carcinogenic potential sorts with the early region of the genome, but not the late region. Thus, genetic differences within the early region specify the pathogenic potential of alpha-HPV infections. Since the HPV genomes are monophyletic and sites are highly correlated across the genome, diagnosis of oncogenic types and non-oncogenic types can be accomplished using any region across the genome. Here we review our current understanding of the evolutionary history of the oncogenic HPVs, in particular, we focus on the importance of viral genome heterogeneity and discuss the genetic basis for the oncogenic phenotype in some but not all alpha-PVs.
• Chen, Z., van Doorslaer, K., DeSalle, R., Wood, C. E., Kaplan, J. R., Wagner, J. D., & Burk, R. D. (2009). Genomic diversity and interspecies host infection of alpha12 Macaca fascicularis papillomaviruses (MfPVs). Virology, 393(2), 304-10.
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  Alpha human papillomaviruses (HPVs) are among the most common sexually transmitted agents of which a subset causes cervical neoplasia and cancer in humans. Alpha-PVs have also been identified in non-human primates although few studies have systematically characterized such types. We cloned and characterized 10 distinct types of PVs from exfoliated cervicovaginal cells from different populations of female cynomolgus macaques (Macaca fascicularis) originating from China and Indonesia. These include 5 novel genotypes and 5 previously identified genotypes found in rhesus (Macaca mulatta) (RhPV-1, RhPV-a, RhPV-b and RhPV-d) and cynomolgus macaques (MfPV-a). Type-specific primers were designed to amplify the complete PV genomes using an overlapping PCR method. Four MfPVs were associated with cervical intraepithelial neoplasia (CIN). The most prevalent virus type was MfPV-3 (formerly RhPV-d), which was identified in 60% of animals with CIN. In addition, the complete genomes of variants of MfPV-3 and RhPV-1 were characterized. These variants are 97.1% and 97.7% similar across the L1 nucleotide sequences with the prototype genomes, respectively. Sequence comparisons and phylogenetic analyses indicate that these novel MfPVs cluster together within the alpha12 PV species closely related to the alpha9 (e.g., HPV16) and alpha11 species (e.g., HPV34), and all share a most recent common ancestor. Our data expand the molecular diversity of non-human primate PVs and suggest a recent expansion of alpha-PV species groups. Moreover, identification of an overlapping set of MfPVs in rhesus and cynomolgus macaques indicates that non-human primate alpha-PVs might not be strictly species-specific and may represent past interspecies infection.
• Herbst, L. H., Lenz, J., Van Doorslaer, K., Chen, Z., Stacy, B. A., Wellehan, J. F., Manire, C. A., & Burk, R. D. (2009). Genomic characterization of two novel reptilian papillomaviruses, Chelonia mydas papillomavirus 1 and Caretta caretta papillomavirus 1. Virology, 383(1), 131-5.
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  In this paper we describe the characterization of the genomes of two sea turtle papillomaviruses, Chelonia mydas PV (CmPV-1) and Caretta caretta PV (CcPV-1). The isolation and sequencing of the first non-avian reptilian PVs extend the evolutionary history of PVs to include all amniotes. PVs have now been described in mammals, birds and non-avian reptiles. The chelonian PVs form a distinct clade most closely related to the avian PVs. Unlike the avian PVs, both chelonian PVs have canonical E6 and E7 ORFs, indicating that these genes were present in the common ancestor to mammalian and non-mammalian amniote PVs. Rates of evolution among the non-mammalian PVs were generally slower than those estimated for mammalian PVs, perhaps due to lower metabolic rates among the ectothermic reptiles.
• Heslin, D. J., Murcia, P., Arnaud, F., Van Doorslaer, K., Palmarini, M., & Lenz, J. (2009). A single amino acid substitution in a segment of the CA protein within Gag that has similarity to human immunodeficiency virus type 1 blocks infectivity of a human endogenous retrovirus K provirus in the human genome. Journal of virology, 83(2), 1105-14.
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  Human endogenous retrovirus K (HERV-K) is the most intact retrovirus in the human genome. However, no single HERV-K provirus in the human genome today appears to be infectious. Since the Gag protein is the central component for the production of retrovirus particles, we investigated the abilities of Gag from two HERV-K proviruses to support production of virus-like particles and viral infectivity. HERV-K113 has full-length open reading frames for all viral proteins, while HERV-K101 has a full-length gag open reading frame and is expressed in human male germ cell tumors. The Gag of HERV-K101 allowed production of viral particles and infectivity, although at lower levels than observed with a consensus sequence Gag. Thus, including HERV-K109, at least two HERV-K proviruses in human genome today have functional Gag proteins. In contrast, HERV-K113 Gag supported only very low levels of particle production, and no infectivity was detectable due to a single amino acid substitution (I516M) near the extreme C terminus of the CA protein within Gag. The sequence of this portion of HERV-K CA showed similarities to that of human immunodeficiency virus type 1 and other primate immunodeficiency viruses. The extreme C terminus of CA may be a general determinant of retrovirus particle production. In addition, precise mapping of the defects in HERV-K proviruses as was done here identifies the key polymorphisms that need to be analyzed to assess the possible existence of infectious HERV-K alleles within the human population.
• Joh, J., Hopper, K., Van Doorslaer, K., Sundberg, J. P., Jenson, A. B., & Ghim, S. J. (2009). Macaca fascicularis papillomavirus type 1: a non-human primate betapapillomavirus causing rapidly progressive hand and foot papillomatosis. The Journal of general virology, 90(Pt 4), 987-994.
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  Papillomaviruses (PVs) are a group of small, non-enveloped DNA viruses that cause mucosal or cutaneous neoplasia in a variety of animals. Whilst most papillomas will regress spontaneously, some may persist or undergo malignant transformation. In this study, aggressive, persistent and extensive warts were observed on the hands and feet of a cynomolgus macaque (Macaca fascicularis). The presence of PV in the wart biopsies was identified by immunohistochemistry and PCR amplification of PV DNA. The genomic DNA of this PV was cloned and sequenced, and the PV was designated M. fascicularis papillomavirus type 1 (MfPV-1). Its genome was 7588 bp in length and the organization of its putative open reading frames (E1, E2, E6, E7, L1, L2 and E4) was similar to that of other PVs. MfPV-1 had a short non-coding region (NCR) of 412 bp. Molecular analysis of MfPV-1 genomic DNA classified it into the genus Betapapillomavirus, to which all epidermodysplasia verruciformis (EV)-type PVs belong. Diseases caused by PVs of the genus Betapapillomavirus are usually associated with natural or iatrogenic immunosuppression. The genomic characterization performed in this study showed that MfPV-1 clustered within the genus Betapapillomavirus and also contained EV-type-specific motifs in its NCR. Further characterization of this virus and its host interactions may allow us to develop a non-human primate model for human betapapillomaviruses, a genus populated by human PV types causing EV.
• Van Doorslaer, K., Sidi, A. O., Zanier, K., Rybin, V., Deryckère, F., Rector, A., Burk, R. D., Lienau, E. K., van Ranst, M., & Travé, G. (2009). Identification of unusual E6 and E7 proteins within avian papillomaviruses: cellular localization, biophysical characterization, and phylogenetic analysis. Journal of virology, 83(17), 8759-70.
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  Papillomaviruses (PVs) are a large family of small DNA viruses infecting mammals, reptiles, and birds. PV infection induces cell proliferation that may lead to the formation of orogenital or skin tumors. PV-induced cell proliferation has been related mainly to the expression of two small oncoproteins, E6 and E7. In mammalian PVs, E6 contains two 70-residue zinc-binding repeats, whereas E7 consists of a natively unfolded N-terminal region followed by a zinc-binding domain which folds as an obligate homodimer. Here, we show that both the novel francolin bird PV Francolinus leucoscepus PV type 1 (FlPV-1) and the chaffinch bird PV Fringilla coelebs PV contain unusual E6 and E7 proteins. The avian E7 proteins contain an extended unfolded N terminus and a zinc-binding domain of reduced size, whereas the avian E6 proteins consist of a single zinc-binding domain. A comparable single-domain E6 protein may have existed in a common ancestor of mammalian and avian PVs. Mammalian E6 C-terminal domains are phylogenetically related to those of single-domain avian E6, whereas mammalian E6 N-terminal domains seem to have emerged by duplication and subsequently diverged from the original ancestral domain. In avian and mammalian cells, both FlPV-1 E6 and FlPV-1 E7 were evenly expressed in the cytoplasm and the nucleus. Finally, samples of full-length FlPV-1 E6 and the FlPV-1 E7 C-terminal zinc-binding domain were prepared for biophysical analysis. Both constructs were highly soluble and well folded, according to nuclear magnetic resonance spectroscopy measurements.
• Rector, A., Stevens, H., Lacave, G., Lemey, P., Mostmans, S., Salbany, A., Vos, M., Van Doorslaer, K., Ghim, S. J., Rehtanz, M., Bossart, G. D., Jenson, A. B., & Van Ranst, M. (2008). Genomic characterization of novel dolphin papillomaviruses provides indications for recombination within the Papillomaviridae. Virology, 378(1), 151-61.
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  Phylogenetic analysis of novel dolphin (Tursiops truncatus) papillomavirus sequences, TtPV1, -2, and -3, indicates that the early and late protein coding regions of their genomes differ in evolutionary history. Sliding window bootscan analysis showed a significant a change in phylogenetic clustering, in which the grouped sequences of TtPV1 and -3 move from a cluster with the Phocoena spinipinnis PsPV1 in the early region to a cluster with TtPV2 in the late region. This provides indications for a possible recombination event near the end of E2/beginning of L2. A second possible recombination site could be located near the end of L1, in the upstream regulatory region. Selection analysis by using maximum likelihood models of codon substitutions ruled out the possibility of intense selective pressure, acting asymmetrically on the viral genomes, as an alternative explanation for the observed difference in evolutionary history between the early and late genomic regions of these cetacean papillomaviruses.
• Van Doorslaer, K., Rector, A., Jenson, A. B., Sundberg, J. P., Van Ranst, M., & Ghim, S. J. (2007). Complete genomic characterization of a murine papillomavirus isolated from papillomatous lesions of a European harvest mouse (Micromys minutus). The Journal of general virology, 88(Pt 5), 1484-1488.
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  The papillomaviruses form a large group of species-specific pathogens that cause epithelial proliferations in a wide spectrum of animal hosts. Previous reports demonstrated a relatively high frequency of a variety of skin lesions in captive European harvest mice. The Micromys minutus papillomavirus (MmPV) was isolated from one of these lesions found on a captive European harvest mouse in a regional zoo in Chicago. In this study we present the entire genomic sequence of MmPV. The MmPV genome is organized into the seven classical papillomaviral open reading frames. Phylogenetic analysis places MmPV together with a papillomavirus (PV) isolated from a Syrian golden Hamster (HaOPV) in the genus Pipapillomavirus. The similar clustering pattern of the MmPV-HaOPV pair and their rodent hosts support the hypothesis of papillomaviral and host co-phylogenetic descent. The availability of the complete genomic sequence of a mouse PV should allow researchers to use MmPV as a model for PV carcinogenesis.
• Rector, A., Mostmans, S., Van Doorslaer, K., McKnight, C. A., Maes, R. K., Wise, A. G., Kiupel, M., & Van Ranst, M. (2006). Genetic characterization of the first chiropteran papillomavirus, isolated from a basosquamous carcinoma in an Egyptian fruit bat: the Rousettus aegyptiacus papillomavirus type 1. Veterinary microbiology, 117(2-4), 267-75.
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  The complete genomic DNA of a novel papillomavirus (PV) was isolated from a basosquamous carcinoma on the wing of an Egyptian fruit bat (Rousettus aegyptiacus). Initial short sequences of the E1 and L1 genes of this virus were retrieved by PCR with degenerate papillomavirus-specific primers, and the entire R. aegyptiacus papillomavirus type 1 (RaPV-1) DNA was then amplified by long template PCR, cloned and sequenced with a transposon insertion method. The RaPV-1 genome counts 7970 basepairs and contains the typical papillomavirus open reading frames (ORF) (E1, E2, E4, E6, E7, L1 and L2). Based on a concatenated alignment of the E1, E2, L1 and L2 open reading frames of RaPV-1 and 46 other human and animal papillomavirus type species, a neighbor-joining phylogenetic tree was constructed. This phylogenetic analysis shows that RaPV-1 has a close-to-root position in the papillomavirus evolutionary tree. Since RaPV-1 is only distantly related to other papillomaviruses (with maximally 50% nucleotide sequence identity across the L1 open reading frame), it cannot be assigned to one of the existing papillomavirus genera and therefore represents the first member of a novel, as yet unnamed, close-to-root papillomavirus genus. This is the first time a papillomavirus has been isolated and characterized from a member of the Chiroptera order.
• Van Doorslaer, K., Rector, A., Vos, P., & Van Ranst, M. (2006). Genetic characterization of the Capra hircus papillomavirus: a novel close-to-root artiodactyl papillomavirus. Virus research, 118(1-2), 164-9.
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  The healthy skin of a seven-year-old female goat (Capra hircus) was sampled with a cotton tip swab. Total genomic DNA was extracted from the sample and subjected to multiply primed rolling-circle amplification (RCA). Restriction analysis showed that the complete genome of a papillomavirus was amplified, measuring approximately 7,600 bp in length. The Capra hircus papillomavirus (ChPV-1) genome was cloned from this rolling-circle amplification product, and the complete nucleotide sequence was determined. The ChPV-1 genome counts 7,542 bp, and contains the typical papillomaviral open reading frames (ORFs). Based on a concatenated alignment of the E1, E2, L1 and L2 open reading frames of ChPV-1 and 54 other animal and human papillomavirus types, a neighbor-joining phylogenetic tree was constructed. In this tree ChPV-1 clusters with BPV-3, -4 and -6. Pairwise nucleotide sequence alignments of the L1 open reading frame of ChPV-1 with its closest relatives showed less than 60% similarity, placing the ChPV-1 in a novel genus.
• Rector, A., Tachezy, R., Van Doorslaer, K., MacNamara, T., Burk, R. D., Sundberg, J. P., & Van Ranst, M. (2005). Isolation and cloning of a papillomavirus from a North American porcupine by using multiply primed rolling-circle amplification: the Erethizon dorsatum papillomavirus type 1. Virology, 331(2), 449-56.
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  The complete genome of a novel papillomavirus was isolated from a cutaneous papillomatous lesion of a North American porcupine (Erethizon dorsatum) using multiply primed rolling-circle amplification (RCA). The nucleotide sequence, genome organization, and phylogenetic position of the Erethizon dorsatum papillomavirus type 1 (EdPV-1) were determined. EdPV-1 is only distantly related to other benign cutaneous papillomavirus sequences and is the first member of the novel Sigma papillomavirus genus.
• Rector, A., Van Doorslaer, K., Bertelsen, M., Barker, I. K., Olberg, R. A., Lemey, P., Sundberg, J. P., & Van Ranst, M. (2005). Isolation and cloning of the raccoon (Procyon lotor) papillomavirus type 1 by using degenerate papillomavirus-specific primers. The Journal of general virology, 86(Pt 7), 2029-2033.
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  Partial sequences of a novel papillomavirus were amplified from a cutaneous lesion biopsy of a raccoon (Procyon lotor), by using PCR with degenerate papillomavirus-specific primers. The Procyon lotor papillomavirus type 1 (PlPV-1) DNA was amplified with long template PCR in two overlapping fragments, together encompassing the entire genome, and the complete PlPV-1 genomic sequence was determined. The PlPV-1 genome consists of 8170 bp, and contains the typical papillomaviral open reading frames, encoding five early proteins and two late capsid proteins. Besides the classical non-coding region (NCR1) between the end of L1 and the start of E6, PlPV-1 contains an additional non-coding region (NCR2) of 1065 bp between the early and late protein region, which has previously also been described for the canine oral papillomavirus (COPV) and the Felis domesticus papillomavirus (FdPV-1). Phylogenetic analysis places PlPV-1 together with COPV and FdPV-1 in a monophyletic branch which encompasses the Lambda papillomavirus genus.

Presentations

• Van Doorslaer, K. M. (2019, September). Papillomavirus evolution and immunity. LSU invited seminar. LSU: LSU.
• Van Doorslaer, K. M. (2019, july). Papillomaviruses in cancer. DNA tumor virus meeting.
• Van Doorslaer, K. M. (2018, August). Fish Associated Viruses Provide Insights into the Evolution of Viral Oncogenes. DNA Tumor Virus Meeting. Madison, WI.
• Van Doorslaer, K. M. (2018, August). Viral transcriptional enhancer cis elements are necessary for persistent HPV18 replication. DNA Tumor Virus Meeting. Madison, WI.
• Van Doorslaer, K. M. (2018, July). Fish Associated Viruses Provide Insights into the Evolution of Viral Oncogenes. American Society for Virology Annual Meeting.
• Van Doorslaer, K. M. (2018, July). The Papillomavirus Episteme. American Society for Virology Annual Meeting. College Park, MD.
• Van Doorslaer, K. M. (2018, July). Viral transcriptional enhancer cis elements are necessary for persistent HPV18 replication. American Society for Virology Annual Meeting. College Park, MD.
• Van Doorslaer, K. M. (2018, June). Fish associated viruses provide insights into the evolution of viral oncogenes. Virus Genomics and evolution. Cambridge, UK.
• Van Doorslaer, K. M. (2018, October). Non-mammalian viruses provide insights into the evolution of viral oncogenes. 32nd International Papillomavirus Conference. Sydney, Aus.
• Van Doorslaer, K. M. (2018, October). Proposing a new scheme for papillomavirus classification. 32nd International Papillomavirus Conference. Sydney Australia.
• Van Doorslaer, K. M. (2018, October). PuMA: Towards an automated annotation tool. 32nd International Papillomavirus Conference. Sydney, Australia.
• Van Doorslaer, K. M. (2017, April). Comparative Genomics as a Tool to Understand Papillomavirus Evolution and Disease. 56th Annual Meeting of the Arizona/Southern Nevada ASM Branch. Tucson, AZ: ASM local Branch.
• Van Doorslaer, K. M. (2017, April). Comparative Genomics as a Tool to Understand Papillomavirus Evolution and Disease. 56th Annual meeting of the Arizona/Southern Nevada ASM Branch. Tucson, AZ, USA: Arizona/Southern Nevada ASM Branch.
• Van Doorslaer, K. M. (2017, April). Papillomaviruses as tools to study evolution. MicroLunch, Cross Campus Microbiology Focus Group, University of Arizona. Tucson, AZ, USA: MicroLunch, Cross Campus Microbiology Focus Group, University of Arizona.
• Van Doorslaer, K. M. (2017, Feb). Papillomaviruses; replication and cancer. MARC Biomedical Research Colloquium. Tucson, AZ, USA: MARC Biomedical Research program, University of Arizona.
• Van Doorslaer, K. M. (2017, Feb). Papillomaviruses; replication and cancer. UA Microbiolgy Club meeting, University of Arizona. Tucson, AZ, USA: UA Microbiolgy Club, University of Arizona.
• Van Doorslaer, K. M. (2017, July). Genomic plasticity near the root of the papillomavirus evolutionary tree. International DNA tumor virus meeting. Birmingham, UK: International DNA tumor virus meeting.
• Van Doorslaer, K. M. (2017, March). Comparative Genomics as a Tool to Understand Papillomavirus Evolution and Disease. CIDD seminar (Penn State University). Hershey, PA, USA: Penn State University.
• Van Doorslaer, K. M. (2017, March). Use of comparative virology to understand papillomavirus oncogenicity. Frontiers in Immunobiology & Immunopathogenesis Symposium 2017. Tucson, AZ, USA: Dept. of Immunobiology, University of Arizona.
• Van Doorslaer, K. M. (2017, November). Evolutionary clues to papillomavirus oncogenicity. UA-ASU Virology Symposium. Phoenix, AZ, USA: University of Arizona and Arizona State University.
• Van Doorslaer, K. M. (2017, November). Papillomaviruses; challenges for cancer control. Arizona Wellbeing Commons - Cancer Prevention, Detection, Management and Treatment Division Meeting. Phoenix, AZ, USA: Arizona Wellbeing Commons.
• Van Doorslaer, K. M. (2016, Nov). Comparative Genomics as a Tool to Understand Papillomavirus Evolution and Disease. Cancer Biology Program Seminar, University of Arizona Cancer Center. Tucson, AZ, USA: Arizona Cancer Center.
• Van Doorslaer, K. M. (2016, Nov). In vitro tools to study the papillomavirus lifecycle. Head and Neck Cancer collaborative seminar, University of Arizona Cancer Center. Tucson, AZ, USA: Arizona Cancer Center.
• Van Doorslaer, K. M. (2016, Oct). Comparative Genomics as a Tool to Understand Papillomavirus Evolution and Disease. Inaugural CME collaborative seminar Arizona State University. Phoenix, AZ, USA: Arizona State University.
• Van Doorslaer, K. M. (2016, Oct). Comparative Genomics as a Tool to Understand Papillomavirus Evolution and Disease. MicroLunch, Cross Campus Microbiology Focus Group, University of Arizona. Tucson, AZ, USA: Arizona Cancer Center.

Poster Presentations

• Jackson, J., Jandova, J., King, K., & Van Doorslaer, K. M. (2017, Nov). Detecting papillomavirus recombination in vitro. ABRCMS. Phoenix, AZ: ASM.