Michael Worobey

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Scholarly Contributions

Chapters

• Han, G., & Worobey, M. (2012). The origins and diversification of human immunodeficiency virus. In Global HIV/AIDS Medicine. Philadelphia: Elsevier.

Journals/Publications

• Yoon, J., Worobey, M., Quirk, G., Zhou, A., Khanthaphixay, B., Buchanan, B. C., & Liang, Y. (2023). Sensitive SARS-CoV-2 salivary antibody assays for clinical saline gargle samples using smartphone-based competitive particle immunoassay platforms. Biosensors and Bioelectronics, 229, 115221. doi:https://doi.org/10.1016/j.bios.2023.115221
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  Antibody assay for SARS-CoV-2 has become increasingly important to track latent and asymptomatic infections, check the individual's immune status, and confirm vaccine efficacy and durability. However, current SARS-CoV-2 antibody assays require invasive blood collection, requiring a remote laboratory and a trained phlebotomist. Direct detection of SARS-CoV-2 antibodies from clinical saline gargle samples has been considered challenging due to the smaller number of antibodies in such specimens and the high limit of detection of currently available rapid tests. This work demonstrates simple and non-invasive methods for detecting SARS-CoV-2 salivary antibodies. Competitive particle immunoassays were developed on a paper microfluidic chip using the receptor-binding domain (RBD) antigens on spike proteins. Using a smartphone, they were monitored by counting the captured fluorescent particles or evaluating the capillary flow velocities. The limit of detection (LOD), cross-binding between alpha- and omicron-strains, and the effect of angiotensin-converting enzyme 2 (ACE2) presence were investigated. LODs were 1–5 ng/mL in both 10% and 1% saliva. Clinical saline gargle samples were assayed using both methods, showing a statistical difference between virus-negative and virus-positive samples, although the assays targeted antibodies. Only a small number of virus-positive samples were antibody-negative. The high assay sensitivity detected a small number of antibodies developed even during the early phase of infections. Overall, this work demonstrates the ability to detect SARS-CoV-2 salivary IgG antibodies on simple, cost-effective, portable platforms towards mitigating SARS-CoV-2 and potentially other respiratory viruses.
• Yoon, J., Worobey, M., Nikolich-Zugich, J., Quirk, G., Uhrlaub, J. L., Eades, C., Nguyen, B. T., Baker, J., Sosnowski, K., Breshears, L. E., Kaarj, K., & Akarapipad, P. (2022). Smartphone-based sensitive detection of SARS-CoV-2 from saline gargle samples via flow profile analysis on a paper microfluidic chip. Biosensors and Bioelectronics, 207, 114192. doi:https://doi.org/10.1016/j.bios.2022.114192
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  Respiratory viruses, especially coronaviruses, have resulted in worldwide pandemics in the past couple of decades. Saliva-based paper microfluidic assays represent an opportunity for noninvasive and rapid screening, yet both the sample matrix and test method come with unique challenges. In this work, we demonstrated the rapid and sensitive detection of SARS-CoV-2 from saliva samples, which could be simpler and more comfortable for patients than existing methods. Furthermore, we systematically investigated the components of saliva samples that affected assay performance. Using only a smartphone, an antibody-conjugated particle suspension, and a paper microfluidic chip, we made the assay user-friendly with minimal processing. Unlike the previously established flow rate assays that depended solely on the flow rate or distance, this unique assay analyzes the flow profile to determine infection status. Particle-target immunoagglutination changed the surface tension and subsequently the capillary flow velocity profile. A smartphone camera automatically measured the flow profile using a Python script, which was not affected by ambient light variations. The limit of detection (LOD) was 1 fg/μL SARS-CoV-2 from 1% saliva samples and 10 fg/μL from simulated saline gargle samples (15% saliva and 0.9% saline). This method was highly specific as demonstrated using influenza A/H1N1. The sample-to-answer assay time was
• Yoon, J., Worobey, M., Nikolich-Zugich, J., Uhrlaub, J. L., Quirk, G., Kaarj, K., Sosnowski, K., Akarapipad, P., Nguyen, B. T., & Breshears, L. E. (2022). Sensitive, smartphone-based SARS-CoV-2 detection from clinical saline gargle samples. PNAS Nexus, pgac028. doi:https://doi.org/10.1093/pnasnexus/pgac028
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  Saliva specimens have drawn interest for diagnosing respiratory viral infections due to their ease of collection and decreased risk to healthcare providers. However, rapid and sensitive immunoassays have not yet been satisfactorily demonstrated for such specimens due to their viscosity and low viral loads. Using paper microfluidic chips and a smartphone-based fluorescence microscope, we developed a highly sensitive, low-cost immunofluorescence particulometric SARS-CoV-2 assay from clinical saline gargle samples. We demonstrated the limit of detection of 10 ag/μL. With easy-to-collect saline gargle samples, our clinical sensitivity, specificity, and accuracy were 100%, 86%, and 93%, respectively, for n = 27 human subjects with n = 13 RT-qPCR positives.
• Han, G., & Worobey, M. -. (2015). A Primitive Endogenous Lentivirus in a Colugo: Insights into the Early Evolution of Lentiviruses. Molecular Biology and Evolution, 32(1), 211-215.
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  Lentiviruses infect a wide range of mammal species. Much remains unknown about their deep history and host distribution. Here, we report the discovery of an endogenous lentivirus within the genome of the Sunda flying lemur (Galeopterus variegatus) (which we designate “Galeopterus variegatus endogenous lentivirus” [GvaELV]). We estimate the GvaELV genome invasion to have occurred more than 14 Ma, supporting an ancient origin of the lentivirus clade and an ancient lentiviral infection in colugo. Phylogenetic analyses show that GvaELV is a sister group of all previously known lentiviruses. The GvaELV genome appears to possess some primitive genomic features of a lentivirus, encoding not only a trans-activator of transcription (tat) gene but also two additional putative accessory genes that share no discernible similarity with other lentiviral accessory genes. The discovery of GvaELV provides novel insights into the prehistory and host distribution of lentivirus.
• Han, G., & Worobey, M. -. (2015). Endogenous Viral Sequences from the Cape Golden Mole (Chrysochloris asiatica) Reveal the Presence of Foamy Viruses in All Major Placental Mammal Clades. PLoS One, 9(5), e97931.
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  Endogenous retroviruses provide important insights into the deep history of this viral lineage. Endogenous foamy viruses are thought to be very rare and only a few cases have been identified to date. Here we report a novel endogenous foamy virus (CaEFV) within the genome of the Cape golden mole (Chrysochloris asiatica). The identification of CaEFV reveals the presence of foamy virus in the placental mammal superorder Afrotheria. Phylogenetic analyses place CaEFV basal to other foamy viruses of Eutherian origin, suggesting an ancient codivergence between foamy virus and placental mammals. These findings have implications for understanding the long-term evolution, diversity, and biology of retroviruses.
• Suzuki, T. A., & Worobey, M. (2014). Geographical variation of human gut microbial composition. Biology Letters, 10(2), 20131037.
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  Abstract: Althoughwe knowthere is considerable variation in gut microbial composition within host species, little is known about how this variation is shaped and why such variation exists. In humans, obesity is associated with the relative abundance of two dominant bacterial phyla: an increase in the proportion of Firmicutes and a decrease in the proportion of Bacteroidetes. As there is evidence that humans have adapted to colder climates by increasing their body mass (e.g. Bergmann's rule), we tested whether Firmicutes increase and Bacteroidetes decrease with latitude, using 1020 healthy individuals drawn from 23 populations and six published studies.We found a positive correlation between Firmicutes and latitude and a negative correlation between Bacteroidetes and latitude. The overall pattern appears robust to sex, age and bacterial detection methods. Comparisons between African Americans and native Africans and between European Americans and native Europeans suggest no evidence of host genotype explaining the observed patterns. The variation of gut microbial composition described here is consistentwith the pattern expected by Bergmann's rule. This surprising link between large-scale geography and human gut microbial composition merits further investigationl. © 2014 The Authors.
• Worobey, M., Han, G., & Rambaut, A. (2014). A synchronized global sweep of the internal genes of modern avian influenza virus. Nature, 3, 444-445.
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  Abstract: Zoonotic infectious diseases such as influenza continue to pose a grave threat to human health. However, the factors that mediate the emergence of RNA viruses such as influenza A virus (IAV) are still incompletely understood. Phylogenetic inference is crucial to reconstructing the origins and tracing the flow of IAV within and between hosts. Here we show that explicitly allowing IAV host lineages to have independent rates of molecular evolution is necessary for reliable phylogenetic inference of IAV and that methods that do not do so, including 'relaxed' molecular clock models, can be positively misleading. A phylogenomic analysis using a host-specific local clock model recovers extremely consistent evolutionary histories across all genomic segments and demonstrates that the equine H7N7 lineage is a sister clade to strains from birds-as well as those from humans, swine and the equine H3N8 lineage-sharing an ancestor with them in the mid to late 1800s. Moreover, major western and eastern hemisphere avian influenza lineages inferred for each gene coalesce in the late 1800s. On the basis of these phylogenies and the synchrony of these key nodes, we infer that the internal genes of avian influenza virus (AIV) underwent a global selective sweep beginning in the late 1800s, a process that continued throughout the twentieth century and up to the present. The resulting western hemispheric AIV lineage subsequently contributed most of the genomic segments to the 1918 pandemic virus and, independently, the 1963 equine H3N8 panzootic lineage. This approach provides a clear resolution of evolutionary patterns and processes in IAV, including the flow of viral genes and genomes within and between host lineages.
• Worobey, M., Han, G., & Rambaut, A. (2014). The genesis and pathogenesis of the 1918 influenza pandemic. Proceedings of the National Academy of Sciences of the United States of America, 111(22), 8107–8112.
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  The source, timing, and geographical origin of the 1918–1920 pandemic influenza A virus have remained tenaciously obscure for nearly a century, as have the reasons for its unusual severity among young adults. Here, we reconstruct the origins of the pandemic virus and the classic swine influenza and (postpandemic) seasonal H1N1 lineages using a host-specific molecular clock approach that is demonstrably more accurate than previous methods. Our results suggest that the 1918 pandemic virus originated shortly before 1918 when a human H1 virus, which we infer emerged before ∼1907, acquired avian N1 neuraminidase and internal protein genes. We find that the resulting pandemic virus jumped directly to swine but was likely displaced in humans by ∼1922 by a reassortant with an antigenically distinct H1 HA. Hence, although the swine lineage was a direct descendent of the pandemic virus, the post-1918 seasonal H1N1 lineage evidently was not, at least for HA. These findings help resolve several seemingly disparate observations from 20th century influenza epidemiology, seroarcheology, and immunology. The phylogenetic results, combined with these other lines of evidence, suggest that the high mortality in 1918 among adults aged ∼20 to ∼40 y may have been due primarily to their childhood exposure to a doubly heterosubtypic putative H3N8 virus, which we estimate circulated from ∼1889–1900. All other age groups (except immunologically naive infants) were likely partially protected by childhood exposure to N1 and/or H1-related antigens. Similar processes may underlie age-specific mortality differences between seasonal H1N1 vs. H3N2 and human H5N1 vs. H7N9 infections.
• Chamberland, A., Diabaté, S., Sylla, M., Anagounou, S., Geraldo, N., Zannou, D. M., Labbé, A., Worobey, M., Alary, M., & Tremblay, C. (2012). Transmission of HIV-1 drug resistance in Benin could jeopardise future treatment options. Sexually Transmitted Infections, 88(3), 179-183.
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  PMID: 22158948;Abstract: Objectives As access to antiretrovirals (ARV) increases in developing countries, the identification of optimal therapeutic regimens and prevention strategies requires the identification of resistance pathways in non-B subtypes as well as the surveillance of drug mutation resistance (SDMR) including the trafficking of viral strains between high-risk groups such as commercial sex workers (CSW) and the general population (GP). In this study, the authors evaluated the rate of primary resistance mutations and the epidemiological link between isolates from GP and CSW from Bénin. Methods Plasma samples were obtained from 129 HIV- 1-infected treatment-nave individuals. Drug resistance mutations were identified using SDMR list and compared with other resistance algorithms. Results No nucleoside reverse transcriptase inhibitor resistance mutations were found. Four patients had nonnucleoside reverse transcriptase inhibitor resistance (K103N, G190A). One patient exhibited protease inhibitors resistance mutation, F53Y. Using the SDMR list, the authors obtained a rate of 3.9% of primary resistance. Nevertheless, the authors observed several mutations not on SDMR list but included in others resistance database, taking those mutations into account, the authors obtained a rate of 15.5%. Conclusions Although our results show a low rate of SDMR, this algorithm may underestimate resistance mutations that may impact treatment options in developing countries. Primary resistance rates were similar in CSW and in the GP. Our phylogenetic analysis confirmed the genetic exchange between groups.
• Han, G., & Worobey, M. (2012). An endogenous foamy virus in the aye-aye (Daubentonia madagascariensis). Journal of Virology, 86(14), 7696-7698.
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  PMID: 22573860;PMCID: PMC3416287;Abstract: We report the discovery and analysis of an endogenous foamy virus (PSFVaye) within the genome of the aye-aye (Daubentonia madagascariensis), a strepsirrhine primate from Madagascar. Phylogenetic analyses indicate that PSFVaye is divergent from all known simian foamy viruses, suggesting an association between foamy viruses and primates since the haplorrhine-strepsirrhine split. The discovery of PSFVaye indicates that primate foamy virus might be more broadly distributed than previously thought. © 2012, American Society for Microbiology.
• Han, G., & Worobey, M. (2012). Endogenous lentiviral elements in the weasel family (mustelidae). Molecular Biology and Evolution, 29(10), 2905-2908.
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  PMID: 22522310;PMCID: PMC3457773;Abstract: Endogenous retroviruses provide molecular fossils for studying the ancient evolutionary history of retroviruses. Here, we report our independent discovery and analysis of endogenous lentiviral insertions (Mustelidae endogenous lentivirus [MELV]) within the genomes of weasel family (Mustelidae). Genome-scale screening identified MELV elements in the domestic ferret (Mustela putorius furo) genome (MELVmpf). MELVmpf exhibits a typical lentiviral genomic organization. Phylogenetic analyses position MELVmpf basal to either primate lentiviruses or feline immunodeficiency virus. Moreover, we verified the presence of MELV insertions in the genomes of several species of the Lutrinae and Mustelinae subfamilies but not the Martinae subfamily, suggesting that the invasion of MELV into the Mustelidae genomes likely took place between 8.8 and 11.8 Ma. The discovery of MELV in weasel genomes extends the host range of lentiviruses to the Caniformia (order Carnivora) and provides important insights into the prehistoric diversity of lentiviruses. © 2012 The Author.
• Worobey, M. -., & Han, G. (2012). An endogenous foamy-like viral element in the coelacanth genome. PLoS pathogens, 8(6).
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  Little is known about the origin and long-term evolutionary mode of retroviruses. Retroviruses can integrate into their hosts' genomes, providing a molecular fossil record for studying their deep history. Here we report the discovery of an endogenous foamy virus-like element, which we designate 'coelacanth endogenous foamy-like virus' (CoeEFV), within the genome of the coelacanth (Latimeria chalumnae). Phylogenetic analyses place CoeEFV basal to all known foamy viruses, strongly suggesting an ancient ocean origin of this major retroviral lineage, which had previously been known to infect only land mammals. The discovery of CoeEFV reveals the presence of foamy-like viruses in species outside the Mammalia. We show that foamy-like viruses have likely codiverged with their vertebrate hosts for more than 407 million years and underwent an evolutionary transition from water to land with their vertebrate hosts. These findings suggest an ancient marine origin of retroviruses and have important implications in understanding foamy virus biology.
• Yingying, L., Ndjango, J., Learn, G. H., Ramirez, M. A., Keele, B. F., Bibollet-Ruche, F., Liu, W., Easlick, J. L., Decker, J. M., Rudicell, R. S., Inogwabini, B., Ahuka-Mundeke, S., Leendertz, F. H., Reynolds, V., Muller, M. N., Chancellor, R. L., Rundus, A. S., Simmons, N., Worobey, M., , Shaw, G. M., et al. (2012). Eastern chimpanzees, but not bonobos, represent a simian immunodeficiency virus reservoir. Journal of Virology, 86(19), 10776-10791.
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  PMID: 22837215;PMCID: PMC3457319;Abstract: Chimpanzees in west central Africa (Pan troglodytes troglodytes) are endemically infected with simian immunodeficiency viruses (SIVcpzPtt) that have crossed the species barrier to humans and gorillas on at least five occasions, generating pandemic and nonpandemic forms of human immunodeficiency virus type 1 (HIV-1) as well as gorilla SIV (SIVgor). Chimpanzees in east Africa (Pan troglodytes schweinfurthii) are also infected with SIVcpz; however, their viruses (SIVcpzPts) have never been found in humans. To examine whether this is due to a paucity of natural infections, we used noninvasive methods to screen wild-living eastern chimpanzees in the Democratic Republic of the Congo (DRC), Uganda, and Rwanda. We also screened bonobos (Pan paniscus) in the DRC, a species not previously tested for SIV in the wild. Fecal samples (n=3,108) were collected at 50 field sites, tested for species and subspecies origin, and screened for SIVcpz antibodies and nucleic acids. Of 2,565 samples from eastern chimpanzees, 323 were antibody positive and 92 contained viral RNA. The antibody-positive samples represented 76 individuals from 19 field sites, all sampled north of the Congo River in an area spanning 250,000 km2. In this region, SIVcpzPts was common and widespread, with seven field sites exhibiting infection rates of 30% or greater. The overall prevalence of SIVcpzPts infection was 13.4% (95% confidence interval, 10.7% to 16.5%). In contrast, none of the 543 bonobo samples from six sites was antibody positive. All newly identified SIVcpzPts strains clustered in strict accordance to their subspecies origin; however, they exhibited considerable genetic diversity, especially in protein domains known to be under strong host selection pressure. Thus, the absence of SIVcpzPts zoonoses cannot be explained by an insufficient primate reservoir. Instead, greater adaptive hurdles may have prevented the successful colonization of humans by P. t. schweinfurthii viruses. © 2012, American Society for Microbiology.
• Bjork, A., Liu, W., Wertheim, J. O., Hahn, B. H., & Worobey, M. (2011). Evolutionary history of chimpanzees inferred from complete mitochondrial genomes. Molecular Biology and Evolution, 28(1), 615-623.
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  PMID: 20802239;PMCID: PMC3108604;Abstract: Investigations into the evolutionary history of the common chimpanzee, Pan troglodytes, have produced inconsistent results due to differences in the types of molecular data considered, the model assumptions employed, and the quantity and geographical range of samples used. We amplified and sequenced 24 complete P. troglodytes mitochondrial genomes from fecal samples collected at multiple study sites throughout sub-Saharan Africa. Using a "relaxed molecular clock," fossil calibrations, and 12 additional complete primate mitochondrial genomes, we analyzed the pattern and timing of primate diversification in a Bayesian framework. Our results support the recognition of four chimpanzee subspecies. Within P. troglodytes, we report a mean (95% highest posterior density [HPD]) time since most recent common ancestor (tMRCA) of 1.026 (0.811-1.263) Ma for the four proposed subspecies, with two major lineages. One of these lineages (tMRCA = 0.510 [0.387-0.650] Ma) contains P. t. verus (tMRCA = 0.155 [0.101-0.213] Ma) and P. t. ellioti (formerly P. t. vellerosus; tMRCA = 0.157 [0.102-0.215] Ma), both of which are monophyletic. The other major lineage contains P. t. schweinfurthii (tMRCA = 0.111 [0.077-0.146] Ma), a monophyletic clade nested within the P. t. troglodytes lineage (tMRCA = 0.380 [0.296-0.476] Ma). We utilized two analysis techniques that may be of widespread interest. First, we implemented a Yule speciation prior across the entire primate tree with separate coalescent priors on each of the chimpanzee subspecies. The validity of this approach was confirmed by estimates based on more traditional techniques. We also suggest that accurate tMRCA estimates from large computationally difficult sequence alignments may be obtained by implementing our novel method of bootstrapping smaller randomly subsampled alignments. © 2010 The Author.
• Han, G., & Worobey, M. (2011). Homologous recombination in negative sense RNA viruses. Viruses, 3(8), 1358-1373.
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  PMID: 21994784;PMCID: PMC3185808;Abstract: Recombination is an important process that influences biological evolution at many different levels. More and more homologous recombination events have been reported among negative sense RNA viruses recently. While sporadic authentic examples indicate that homologous recombination does occur, recombination seems to be generally rare or even absent in most negative sense RNA viruses, and most of the homologous recombination events reported in the literature were likely generated artificially due to lab contamination or inappropriate bioinformatics methods. Homologous recombination in negative sense RNA viruses should be reported with caution in the future, and only after stringent quality control efforts. Moreover, co-infection experiments should be performed to confirm whether recombination can occur. © 2011 by the authors; licensee MDPI, Basel, Switzerland.
• Ochman, H., Worobey, M., Kuo, C., Ndjango, J. N., Peeters, M., Hahn, B. H., & Hugenholtz, P. (2010). Evolutionary relationships of wild hominids recapitulated by gut microbial communities. PLoS Biology, 8(11).
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  PMID: 21103409;PMCID: PMC2982803;Abstract: Multiple factors over the lifetime of an individual, including diet, geography, and physiologic state, will influence the microbial communities within the primate gut. To determine the source of variation in the composition of the microbiota within and among species, we investigated the distal gut microbial communities harbored by great apes, as present in fecal samples recovered within their native ranges. We found that the branching order of host-species phylogenies based on the composition of these microbial communities is completely congruent with the known relationships of the hosts. Although the gut is initially and continuously seeded by bacteria that are acquired from external sources, we establish that over evolutionary timescales, the composition of the gut microbiota among great ape species is phylogenetically conserved and has diverged in a manner consistent with vertical inheritance. © 2010 Ochman et al.
• Wertheim, J. O., Worobey, M. -., Sanderson, M. J., & Bjork, A. (2010). Relaxed Molecular Clocks, the Bias-Variance Trade-off, and the Quality of Phylogenetic Inference. Systematic Biology, 59, 1-8.
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  Because a constant rate of DNA sequence evolution cannot be assumed to be ubiquitous, relaxed molecular clock inference models have proven useful when estimating rates and divergence dates. Furthermore, it has been recently suggested that using relaxed molecular clocks may provide superior accuracy and precision in phylogenetic inference compared with traditional time-free methods that do not incorporate a molecular clock. We perform a simulation study to determine if assuming a relaxed molecular clock does indeed improve the quality of phylogenetic inference. We analyze sequence data simulated under various rate distributions using relaxed-clocks, strict-clocks, and time-free Bayesian phylogenetic inference models. Our results indicate that no difference exists in the quality of phylogenetic inference between assuming a relaxed molecular clock and making no assumption about the clock-likeness of sequence evolution. This pattern is likely due to the bias–variance trade-off inherent in this type of phylogenetic inference. We also compared the quality of inference between Bayesian and maximum likelihood time-free inference models and found them to be qualitatively similar.
• Worobey, M. (2010). Island biogeography reveals the deep history of SIV (Science (1487)). Science, 330(6009), 1318-.
• Worobey, M., Telfer, P., Souquière, S., Hunter, M., Coleman, C. A., Metzger, M. J., Reed, P., Makuwa, M., Hearn, G., Honarvar, S., Roques, P., Apetrei, C., Kazanji, M., & Marx, P. A. (2010). Island biogeography reveals the deep history of SIV. Science, 329(5998), 1487-.
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  PMID: 20847261;
• J., G., Vijaykrishna, D., Bahl, J., Lycett, S. J., Worobey, M., Pybus, O. G., Ma, S. K., Cheung, C. L., Raghwani, J., Bhatt, S., S., J., Guan, Y., & Rambaut, A. (2009). Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza a epidemic. Nature, 459(7250), 1122-1125.
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  PMID: 19516283;Abstract: In March and early April 2009, a new swine-origin influenza A (HlNl) virus (S-OIV) emerged in Mexico and the United States'. During the first few weeks of surveillance, the virus spread worldwide to 30 countries (as of May 11 ) by human-to-human transmission, causing the World Health Organization to raise its pandemic alert to level 5 of 6. This virus has the potential to develop into the first influenza pandemic of the twenty-first century. Here we use evolutionary analysis to estimate the timescale of the origins and the early development of the S-OIV epidemic. We show that it was derived from several viruses circulating in swine, and that the initial transmission to humans occurred several months before recognition of the outbreak. A phylogenetic estimate of the gaps in genetic surveillance indicates a long period of unsampled ancestry before the S-OIV outbreak, suggesting that, the reassortment of swine lineages may have occurred years before emergence in humans, and that the multiple genetic ancestry of S-OIV is not indicative of an artificial origin. Furthermore, the unsampled history of the epidemic means that the nature and location of the genetically closest swine viruses reveal little about the immediate origin of the epidemic, despite the fact that we included a panel of closely related and previously unpublished swine influenza isolates. Our results highlight the need for systematic surveillance of influenza in swine, and provide evidence that the mixing of new genetic elements in swine can result in the emergence of viruses with pandemic potential in humans2. ©2009 Macmillan Publishers Limited. All rights reserved.
• Wertheim, J. O., & Worobey, M. (2009). Dating the age of the SIV lineages that gave rise to HIV-1 and HIV-2. PLoS Computational Biology, 5(5).
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  PMID: 19412344;PMCID: PMC2669881;Abstract: Great strides have been made in understanding the evolutionary history of simian immunodeficiency virus (SIV) and the zoonoses that gave rise to HIV-1 and HIV-2. What remains unknown is how long these SIVs had been circulating in nonhuman primates before the transmissions to humans. Here, we use relaxed molecular clock dating techniques to estimate the time of most recent common ancestor for the SIVs infecting chimpanzees and sooty mangabeys, the reservoirs of HIV-1 and HIV-2, respectively. The date of the most recent common ancestor of SIV in chimpanzees is estimated to be 1492 (1266-1685), and the date in sooty mangabeys is estimated to be 1809 (1729-1875). Notably, we demonstrate that SIV sequences sampled from sooty mangabeys possess sufficient clock-like signal to calibrate a molecular clock; despite the differences in host biology and viral dynamics, the rate of evolution of SIV in sooty mangabeys is indistinguishable from that of its human counterpart, HIV-2. We also estimate the ages of the HIV-2 human-to-human transmissible lineages and provide the first age estimate for HIV-1 group N at 1963 (1948-1977). Comparisons between the SIV most recent common ancestor dates and those of the HIV lineages suggest a difference on the order of only hundreds of years. Our results suggest either that SIV is a surprisingly young lentiviral lineage or that SIV and, perhaps, HIV dating estimates are seriously compromised by unaccounted-for biases. © 2009 Wertheim, Worobey.
• Wertheim, J. O., & Worobey, M. (2009). Relaxed selection and the evolution of RNA virus mucin-like pathogenicity factors. Journal of Virology, 83(9), 4690-4694.
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  PMID: 19224988;PMCID: PMC2668480;Abstract: Mucin-like regions contribute to pathogenicity in a variety of negative-stranded RNA viruses. These regions are characterized by a preponderance of O-linked glycosylation. They evolve exceptionally rapidly yet maintain their function as pathogenicity factors. Two hypotheses have been proposed to explain this evolutionary conundrum of phenotypic stability in the face of extreme genetic divergence: strong positive selection and relaxation of purifying selection. We determined the strength and direction of selection codon by codon across genes containing these regions and found that purifying selection is relaxed over the mucin-like regions relative to the genes in which they are found. This suggests that so long as these regions maintain sufficient O-linked glycosylation, they are free to evolve rapidly without loss of function as pathogenicity factors. Copyright © 2009, American Society for Microbiology. All Rights Reserved.
• Worobey, M., Wertheim, J. O., & Worobey, M. -. (2009). Dating the age of the SIV lineages that gave rise to HIV-1 and HIV-2. PLoS computational biology, 5(5).
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  Great strides have been made in understanding the evolutionary history of simian immunodeficiency virus (SIV) and the zoonoses that gave rise to HIV-1 and HIV-2. What remains unknown is how long these SIVs had been circulating in non-human primates before the transmissions to humans. Here, we use relaxed molecular clock dating techniques to estimate the time of most recent common ancestor for the SIVs infecting chimpanzees and sooty mangabeys, the reservoirs of HIV-1 and HIV-2, respectively. The date of the most recent common ancestor of SIV in chimpanzees is estimated to be 1492 (1266-1685), and the date in sooty mangabeys is estimated to be 1809 (1729-1875). Notably, we demonstrate that SIV sequences sampled from sooty mangabeys possess sufficient clock-like signal to calibrate a molecular clock; despite the differences in host biology and viral dynamics, the rate of evolution of SIV in sooty mangabeys is indistinguishable from that of its human counterpart, HIV-2. We also estimate the ages of the HIV-2 human-to-human transmissible lineages and provide the first age estimate for HIV-1 group N at 1963 (1948-1977). Comparisons between the SIV most recent common ancestor dates and those of the HIV lineages suggest a difference on the order of only hundreds of years. Our results suggest either that SIV is a surprisingly young lentiviral lineage or that SIV and, perhaps, HIV dating estimates are seriously compromised by unaccounted-for biases.
• Worobey, M., Wertheim, J. O., & Worobey, M. -. (2009). Relaxed selection and the evolution of RNA virus mucin-like pathogenicity factors. Journal of virology, 83(9).
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  Mucin-like regions contribute to pathogenicity in a variety of negative-stranded RNA viruses. These regions are characterized by a preponderance of O-linked glycosylation. They evolve exceptionally rapidly yet maintain their function as pathogenicity factors. Two hypotheses have been proposed to explain this evolutionary conundrum of phenotypic stability in the face of extreme genetic divergence: strong positive selection and relaxation of purifying selection. We determined the strength and direction of selection codon by codon across genes containing these regions and found that purifying selection is relaxed over the mucin-like regions relative to the genes in which they are found. This suggests that so long as these regions maintain sufficient O-linked glycosylation, they are free to evolve rapidly without loss of function as pathogenicity factors.
• Liu, W., Worobey, M., Yingying, L. i., Keele, B. F., Bibollet-Ruche, F., Guo, Y., Goepfert, P. A., Santiago, M. L., Ndjango, J. N., Neel, C., Clifford, S. L., Sanz, C., Kamenya, S., Wilson, M. L., Pusey, A. E., Gross-Camp, N., Boesch, C., Smith, V., Zamma, K., , Huffman, M. A., et al. (2008). Molecular ecology and natural history of Simian foamy virus infection in wild-living chimpanzees. PLoS Pathogens, 4(7).
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  PMID: 18604273;PMCID: PMC2435277;Abstract: Identifying microbial pathogens with zoonotic potential in wild-living primates can be important to human health, as evidenced by human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) and Ebola virus. Simian foamy viruses (SFVs) are ancient retroviruses that infect Old and New World monkeys and apes. Although not known to cause disease, these viruses are of public health interest because they have the potential to infect humans and thus provide a more general indication of zoonotic exposure risks. Surprisingly, no information exists concerning the prevalence, geographic distribution, and genetic diversity of SFVs in wild-living monkeys and apes. Here, we report the first comprehensive survey of SFVcpz infection in free-ranging chimpanzees (Pan troglodytes) using newly developed, fecal-based assays. Chimpanzee fecal samples (n = 724) were collected at 25 field sites throughout equatorial Africa and tested for SFVcpzspecific antibodies (n = 706) or viral nucleic acids (n = 392). SFVcpz infection was documented at all field sites, with prevalence rates ranging from 44% to 100%. In two habituated communities, adult chimpanzees had significantly higher SFVcpz infection rates than infants and juveniles, indicating predominantly horizontal rather than vertical transmission routes. Some chimpanzees were co-infected with simian immunodeficiency virus (SIVcpz); however, there was no evidence that SFVcpz and SIVcpz were epidemiologically linked. SFVcpz nucleic acids were recovered from 177 fecal samples, all of which contained SFVcpz RNA and not DNA. Phylogenetic analysis of partial gag (616 bp), pol-RT (717 bp), and pol-IN (425 bp) sequences identified a diverse group of viruses, which could be subdivided into four distinct SFVcpz lineages according to their chimpanzee subspecies of origin. Within these lineages, there was evidence of frequent superinfection and viral recombination. One chimpanzee was infected by a foamy virus from a Cercopithecus monkey species, indicating cross-species transmission of SFVs in the wild. These data indicate that SFVcpz (i) is widely distributed among all chimpanzee subspecies; (ii) is shed in fecal samples as viral RNA; (iii) is transmitted predominantly by horizontal routes; (iv) is prone to superinfection and recombination; (v) has co-evolved with its natural host; and (vi) represents a sensitive marker of population structure that may be useful for chimpanzee taxonomy and conservation strategies.
• Worobey, M. (2008). Phylogenetic evidence against evolutionary stasis and natural abiotic reservoirs of influenza A virus. Journal of Virology, 82(7), 3769-3774.
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  PMID: 18234791;PMCID: PMC2268457;Abstract: Zhang et al. (G. Zhang, D. Shoham, D. Gilichinsky, S. Davydov, J. D. Castello, and S. O. Rogers, J. Virol. 80:12229-12235, 2006) have claimed to have recovered influenza A virus RNA from Siberian lake ice, postulating that ice might represent an important abiotic reservoir for the persistence and reemergence of this medically important pathogen. A rigorous phylogenetic analysis of these influenza A virus hemagglutinin gene sequences, however, indicates that they originated from a laboratory reference strain derived from the earliest human influenza A virus isolate, WS/33. Contrary to Zhang et al.'s assertions that the Siberian "ice viruses" are most closely related either to avian influenza virus or to human influenza virus strains from Asia from the 1960s (Zhang et al., J. Virol. 81:2538 [erratum], 2007), they are clearly contaminants from the WS/33 positive control used in their laboratory. There is thus no credible evidence that environmental ice acts as a biologically relevant reservoir for influenza viruses. Several additional cases with findings that seem at odds with the biology of influenza virus, including modern-looking avian influenza virus RNA sequences from an archival goose specimen collected in 1917 (T. G. Fanning, R. D. Siemens, A. H. Reid, T. A. Janczewski, J. Dean, and J. K. Taubenberger, J. Virol. 76:7860-7862, 2002), can also be explained by laboratory contamination or other experimental errors. Many putative examples of evolutionary stasis in influenza A virus appear to be due to laboratory artifacts. Copyright © 2008, American Society for Microbiology. All Rights Reserved.
• Worobey, M. (2008). The Origins and Diversification of HIV. Global HIV/AIDS Medicine, 13-21.
• Worobey, M., & Worobey, M. -. (2008). Phylogenetic evidence against evolutionary stasis and natural abiotic reservoirs of influenza A virus. Journal of virology, 82(7).
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  Zhang et al. (G. Zhang, D. Shoham, D. Gilichinsky, S. Davydov, J. D. Castello, and S. O. Rogers, J. Virol. 80:12229-12235, 2006) have claimed to have recovered influenza A virus RNA from Siberian lake ice, postulating that ice might represent an important abiotic reservoir for the persistence and reemergence of this medically important pathogen. A rigorous phylogenetic analysis of these influenza A virus hemagglutinin gene sequences, however, indicates that they originated from a laboratory reference strain derived from the earliest human influenza A virus isolate, WS/33. Contrary to Zhang et al.'s assertions that the Siberian "ice viruses" are most closely related either to avian influenza virus or to human influenza virus strains from Asia from the 1960s (Zhang et al., J. Virol. 81:2538 [erratum], 2007), they are clearly contaminants from the WS/33 positive control used in their laboratory. There is thus no credible evidence that environmental ice acts as a biologically relevant reservoir for influenza viruses. Several additional cases with findings that seem at odds with the biology of influenza virus, including modern-looking avian influenza virus RNA sequences from an archival goose specimen collected in 1917 (T. G. Fanning, R. D. Slemons, A. H. Reid, T. A. Janczewski, J. Dean, and J. K. Taubenberger, J. Virol. 76:7860-7862, 2002), can also be explained by laboratory contamination or other experimental errors. Many putative examples of evolutionary stasis in influenza A virus appear to be due to laboratory artifacts.
• Worobey, M., Gemmel, M., Teuwen, D. E., Haselkorn, T., Kunstman, K., Bunce, M., Muyembe, J., Kabongo, J. M., Kalengayi, R. M., Marck, E. V., Thomas, M., & Wolinsky, S. M. (2008). Direct evidence of extensive diversity of HIV-1 in Kinshasa by 1960. Nature, 455(7213), 661-664.
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  PMID: 18833279;PMCID: PMC3682493;Abstract: Human immunodeficiency virus type 1 (HIV-1) sequences that pre-date the recognition of AIDS are critical to defining the time of origin and the timescale of virus evolution. A viral sequence from 1959 (ZR59) is the oldest known HIV-1 infection. Other historically documented sequences, important calibration points to convert evolutionary distance into time, are lacking, however; ZR59 is the only one sampled before 1976. Here we report the amplification and characterization of viral sequences from a Bouin's-fixed paraffin-embedded lymph node biopsy specimen obtained in 1960 from an adult female in Léopoldville, Belgian Congo (now Kinshasa, Democratic Republic of the Congo (DRC)), and we use them to conduct the first comparative evolutionary genetic study of early pre-AIDS epidemic HIV-1 group M viruses. Phylogenetic analyses position this viral sequence (DRC60) closest to the ancestral node of subtype A (excluding A2). Relaxed molecular clock analyses incorporating DRC60 and ZR59 date the most recent common ancestor of the M group to near the beginning of the twentieth century. The sizeable genetic distance between DRC60 and ZR59 directly demonstrates that diversification of HIV-1 in west-central Africa occurred long before the recognized AIDS pandemic. The recovery of viral gene sequences from decades-old paraffin-embedded tissues opens the door to a detailed palaeovirological investigation of the evolutionary history of HIV-1 that is not accessible by other methods. ©2008 Macmillan Publishers Limited. All rights reserved.
• Worobey, M., Pitchenik, A. E., Thomas, M., Wlasiuk, G., & Rambaut, A. (2008). Reply to Pape et al.: The phylogeography of HIV-1 group M subtype B. Proceedings of the National Academy of Sciences of the United States of America, 105(12), E16.
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  PMID: 18337514;PMCID: PMC2290816;
• Cooper, M. A., Adam, R. D., Worobey, M., & Sterling, C. R. (2007). Population Genetics Provides Evidence for Recombination in Giardia. Current Biology, 17(22), 1984-1988.
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  PMID: 17980591;Abstract: Giardia lamblia (syn. Giardia intestinalis, Giardia duodenalis) is an enteric protozoan parasite with two nuclei, and it might be one of the earliest branching eukaryotes [1]. However, the discovery of at least rudimentary forms of certain features, such as Golgi and mitochondria, has refuted the proposal that its emergence from the eukaryotic lineage predated the development of certain eukaryotic features. The recent recognition of many of the genes known to be required for meiosis in the genome has also cast doubt on the idea that Giardia is primitively asexual, but so far there has been no direct evidence of sexual reproduction in Giardia, and population data have suggested clonal reproduction. We did a multilocus sequence evaluation of the genotype A2 reference strain, JH, and five genotype A2 isolates from a highly endemic area in Peru. Loci from different chromosomes yielded significantly different phylogenetic trees, indicating that they do not share the same evolutionary history; within individual loci, tests for recombination yielded significant statistical support for meiotic recombination. These observations provide genetic data supportive of sexual reproduction in Giardia. © 2007 Elsevier Ltd. All rights reserved.
• Gilbert, M. T., Djurhuus, D., Melchior, L., Lynnerup, N., Worobey, M., Wilson, A. S., Andreasen, C., & Dissing, J. (2007). Erratum: mtDNA from hair and nail clarifies the genetic relationship of the 15^th century Qilakitsoq Inuit mummies (American Journal of Physical Anthropology (2007) 133, (847-853)). American Journal of Physical Anthropology, 133(4), 1174-.
• Gilbert, M. T., Sanchez, J. J., Haselkorn, T., Jewell, L. D., Lucas, S. B., Marck, E. V., Børsting, C., Morling, N., & Worobey, M. (2007). Multiplex PCR with minisequencing as an effective high-throughput SNP typing method for formalin-fixed tissue. Electrophoresis, 28(14), 2361-2367.
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  PMID: 17578837;Abstract: Extensive collections of formalin-fixed paraffin-embedded (FFPE) tissues exist that could be exploited for genetic analyses in order to provide important insights into the genetic basis of disease or host/pathogen cointeractions. We report here an evaluation of a 44 SNP multiplex genotyping method, multiplex PCR with minisequencing (MPMS), on 92 DNA extractions performed on six archival FFPE samples of variable DNA quality, which date between 9 and 25 years old. On the three extracts with highest quality, we found the assay efficiency to be near 100%. However, the efficiency of the lowest quality extracts varied significantly. In this study, we demonstrate that although direct measures of DNA concentration in the extracts provide no useful information with regard to subsequent MPMS success, the success of the assay can be determined to some degree a priori, through initial screening of the DNA quality using a simple quantitative real-time PCR (qPCR) assay for nuclear DNA, and/or an assay of the maximum PCR amplifiable size of nuclear DNA. MPMS promises to be of significant use in future genetic studies on FFPE material. It provides a streamlined approach for retrieving a large amount of genetic information using simple, single reactions and minute amounts of archival tissue/DNA. In the light of this evidence, we suggest that the systematic screening of FFPE collections may in the future provide valuable insights into the past. © 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
• Thomas, M., Djurhuus, D., Melchior, L., Lynnerup, N., Worobey, M., Wilson, A. S., Andreasen, C., & Dissing, J. (2007). mtDNA from hair and nail clarifies the genetic relationship of the 15th century Qilakitsoq Inuit mummies. American Journal of Physical Anthropology, 133(2), 847-853.
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  PMID: 17427925;Abstract: The 15th century Inuit mummies excavated at Qilakitsoq in Greenland in 1978 were exceptionally well preserved and represent the largest find of naturally mummified specimens from the Arctic. The estimated ages of the individuals, their distribution between two adjacent graves, the results of tissue typing, and incomplete STR results led researchers to conclude that the eight mummies formed two distinct family groups: A grandmother (I/5), two daughters (I/3, I/4), and their two children (I/1, I/2) in one grave, and two sisters (II/6, II/8) and a daughter (II/7) of one of them in the other. Using mtDNA from hair and nail, we have reanalyzed the mummies. The results allowed the unambiguous assignment of each of the mummies to one of three mtDNA haplogroups: A2b (I/5); A2a (I/2, I/3, II/6, II/8); A2a-311 (I/1, I/4, II/7), excluded some of the previous relations, and pointed to new ones. I/5 is not the grandmother/mother of the individuals in Grave I, and she is not maternally related to any of the seven other mummies; I/3 and I/4 are not sisters and II/7 is neither the daughter of II/6 nor of II/8. However, I/1 may be the child of either I/4 or II/7 and these two may be sisters. I/2 may be the son of I/3, who may be the daughter of either II/6 or II/8, and these two may be sisters. The observation of haplogroups A2a and A2b amongst the 550-year-old Inuit puts a lower limit on the age of the two lineages in Greenland. ©2007 Wiley-Liss, Inc.
• Thomas, M., Haselkorn, T., Bunce, M., Sanchez, J. J., Lucas, S. B., Jewell, L. D., Marck, E. v., & Worobey, M. (2007). The Isolation of Nucleic Acids from Fixed, Paraffin-Embedded Tissues-Which Methods Are Useful When?. PLoS ONE, 2(6).
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  PMID: 17579711;PMCID: PMC1888728;Abstract: Museums and pathology collections around the world represent an archive of genetic material to study populations and diseases. For preservation purposes, a large portion of these collections has been fixed in formalin-containing solutions, a treatment that results in cross-linking of biomolecules. Cross-linking not only complicates isolation of nucleic acid but also introduces polymerase "blocks" during PCR. A wide variety of methods exists for the recovery of DNA and RNA from archival tissues, and although a number of previous studies have qualitatively compared the relative merits of the different techniques, very few have undertaken wide scale quantitative comparisons. To help address this issue, we have undertaken a study that investigates the quality of nucleic acids recovered from a test panel of fixed specimens that have been manipulated following a number of the published protocols. These include methods of pre-treating the samples prior to extraction, extraction and nucleic acid purification methods themselves, and a post-extraction enzymatic repair technique. We find that although many of the published methods have distinct positive effects on some characteristics of the nucleic acids, the benefits often come at a cost. In addition, a number of the previously published techniques appear to have no effect at all. Our findings recommend that the extraction methodology adopted should be chosen carefully. Here we provide a quick reference table that can be used to determine appropriate protocols for particular aims. © 2007 Gilbert et al.
• Thomas, M., Rambaut, A., Wlasiuk, G., Spira, T. J., Pitchenik, A. E., & Worobey, M. (2007). The emergence of HIV/AIDS in the Americas and beyond. Proceedings of the National Academy of Sciences of the United States of America, 104(47), 18566-18570.
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  PMID: 17978186;PMCID: PMC2141817;Abstract: HIV-1 group M subtype B was the first HIV discovered and is the predominant variant of AIDS virus in most countries outside of sub-Saharan Africa. However, the circumstances of its origin and emergence remain unresolved. Here we propose a geographic sequence and time line for the origin of subtype B and the emergence of pandemic HIV/AIDS out of Africa. Using HIV-1 gene sequences recovered from archival samples from some of the earliest known Haitian AIDS patients, we find that subtype B likely moved from Africa to Haiti in or around 1966 (1962-1970) and then spread there for some years before successfully dispersing elsewhere. A "pandemic" clade, encompassing the vast majority of non-Haitian subtype B infections in the United States and elsewhere around the world, subsequently emerged after a single migration of the virus out of Haiti in or around 1969 (1966-1972). Haiti appears to have the oldest HIV/AIDS epidemic outside sub-Saharan Africa and the most genetically diverse subtype B epidemic, which might present challenges for HIV-1 vaccine design and testing. The emergence of the pandemic variant of subtype B was an important turning point in the history of AIDS, but its spread was likely driven by ecological rather than evolutionary factors. Our results suggest that HIV-1 circulated cryptically in the United States for ≈12 years before the recognition of AIDS in 1981. © 2007 by The National Academy of Sciences of the USA.
• Vuissoz, A., Worobey, M., Odegaard, N., Bunce, M., Machado, C. A., Lynnerup, N., Peacock, E. E., & Thomas, M. (2007). The survival of PCR-amplifiable DNA in cow leather. Journal of Archaeological Science, 34(5), 823-829.
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  Abstract: We have investigated the survival of PCR-amplifiable mitochondrial and nuclear DNA in a small number of modern and medieval bovine leather samples. The results of this preliminary investigation demonstrate that, while no nuclear DNA can be PCR-amplified from any of the specimens, mitochondrial DNA can be amplified from all samples. To investigate this contrasting pattern of DNA survival further, we have vegetable-tanned cow skin in our own laboratory, and directly assayed the survival of PCR-amplifiable mitochondrial and nuclear DNA at each step of the process. The results indicate that nuclear DNA is reduced to sub-amplifiable levels as a result of the tanning baths, whereas amplifiable mitochondrial DNA survives the complete process. Our results suggest that old and archaeological bovine leather may represent a useful source of genetic information, although this information will most likely be limited to that which can be gained from mitochondrial DNA. © 2006 Elsevier Ltd. All rights reserved.
• Wertheim, J. O., & Worobey, M. (2007). A challenge to the ancient origin of SIVagm based on African green monkey mitochondrial genomes. PLoS Pathogens, 3(7), 0866-0873.
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  Abstract: While the circumstances surrounding the origin and spread of HIV are becoming clearer, the particulars of the origin of simian immunodeficiency virus (SIV) are still unknown. Specifically, the age of SIV, whether it is an ancient or recent infection, has not been resolved. Although many instances of cross-species transmission of SIV have been documented, the similarity between the African green monkey (AGM) and SIVagm phylogenies has long been held as suggestive of ancient codivergence between SIVs and their primate hosts. Here, we present well-resolved phylogenies based on full-length AGM mitochondrial genomes and seven previously published SIVagm genomes; these allowed us to perform the first rigorous phylogenetic test to our knowledge of the hypothesis that SIVagm codiverged with the AGMs. Using the Shimodaira-Hasegawa test, we show that the AGM mitochondrial genomes and SIVagm did not evolve along the same topology. Furthermore, we demonstrate that the SIVagm topology can be explained by a pattern of west-to-east transmission of the virus across existing AGM geographic ranges. Using a relaxed molecular clock, we also provide a date for the most recent common ancestor of the AGMs at approximately 3 million years ago. This study substantially weakens the theory of ancient SIV infection followed by codivergence with its primate hosts. © 2007 Wertheim and Worobey.
• Wertheim, J. O., & Worobey, M. (2007). A challenge to the ancient origin of SIVagm based on African green monkey mitochondrial genomes.. PLoS pathogens, 3(7), e95.
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  PMID: 17616975;PMCID: PMC1904472;Abstract: While the circumstances surrounding the origin and spread of HIV are becoming clearer, the particulars of the origin of simian immunodeficiency virus (SIV) are still unknown. Specifically, the age of SIV, whether it is an ancient or recent infection, has not been resolved. Although many instances of cross-species transmission of SIV have been documented, the similarity between the African green monkey (AGM) and SIVagm phylogenies has long been held as suggestive of ancient codivergence between SIVs and their primate hosts. Here, we present well-resolved phylogenies based on full-length AGM mitochondrial genomes and seven previously published SIVagm genomes; these allowed us to perform the first rigorous phylogenetic test to our knowledge of the hypothesis that SIVagm codiverged with the AGMs. Using the Shimodaira-Hasegawa test, we show that the AGM mitochondrial genomes and SIVagm did not evolve along the same topology. Furthermore, we demonstrate that the SIVagm topology can be explained by a pattern of west-to-east transmission of the virus across existing AGM geographic ranges. Using a relaxed molecular clock, we also provide a date for the most recent common ancestor of the AGMs at approximately 3 million years ago. This study substantially weakens the theory of ancient SIV infection followed by codivergence with its primate hosts.
• Wilson, A. S., Taylor, T., Ceruti, M. C., Chavez, J. A., Reinhard, J., Grimes, V., Meier-Augenstein, W., Cartmell, L., Stern, B., Richards, M. P., Worobey, M., Barnes, I., & Thomas, M. (2007). Stable isotope and DNA evidence for ritual sequences in Inca child sacrifice. Proceedings of the National Academy of Sciences of the United States of America, 104(42), 16456-16461.
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  PMID: 17923675;PMCID: PMC2034262;Abstract: Four recently discovered frozen child mummies from two of the highest peaks in the south central Andes now yield tantalizing evidence of the preparatory stages leading to Inca ritual killing as represented by the unique capacocha rite. Our interdisciplinary study examined hair from the mummies to obtain detailed genetic and diachronic isotopic information. This approach has allowed us to reconstruct aspects of individual identity and diet, make inferences concerning social background, and gain insight on the hitherto unknown processes by which victims were selected, elevated in social status, prepared for a high-altitude pilgrimage, and killed. Such direct information amplifies, yet also partly contrasts with, Spanish historical accounts. © 2007 by The National Academy of Sciences of the USA.
• Worobey, M., Bjork, A., & Wertheim, J. O. (2007). Point, counterpoint: The evolution of pathogenic viruses and their human hosts. Annual Review of Ecology, Evolution, and Systematics, 38, 515-540.
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  Abstract: Viral pathogens play a prominent role in human health owing to their ability to rapidly evolve creative new ways to exploit their hosts. As elegant and deceptive as many viral adaptations are, humans and their ancestors have repeatedly answered their call with equally impressive adaptations. Here we argue that the coevolutionary arms race between humans and their viral pathogens is one of the most important forces in human molecular evolution, past and present. With a focus on HIV-1 and other RNA viruses, we highlight recent developments in our understanding of the human innate and adaptive immune systems and how the selective pressures exerted by viruses have shaped the human genome. We also discuss how the antiviral function of cellular machinery like RNAi and APOBEC3G blur the lines between innate and adaptive immunity. The remarkable power of natural selection is revealed in each host-pathogen arms race examined. Copyright © 2007 by Annual Reviews. All rights reserved.
• Worobey, M., Gilbert, M. T., Haselkorn, T., Bunce, M., Sanchez, J. J., Lucas, S. B., Jewell, L. D., Van Marck, E., & Worobey, M. -. (2007). The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when?. PloS one, 2(6).
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  Museums and pathology collections around the world represent an archive of genetic material to study populations and diseases. For preservation purposes, a large portion of these collections has been fixed in formalin-containing solutions, a treatment that results in cross-linking of biomolecules. Cross-linking not only complicates isolation of nucleic acid but also introduces polymerase "blocks" during PCR. A wide variety of methods exists for the recovery of DNA and RNA from archival tissues, and although a number of previous studies have qualitatively compared the relative merits of the different techniques, very few have undertaken wide scale quantitative comparisons. To help address this issue, we have undertaken a study that investigates the quality of nucleic acids recovered from a test panel of fixed specimens that have been manipulated following a number of the published protocols. These include methods of pre-treating the samples prior to extraction, extraction and nucleic acid purification methods themselves, and a post-extraction enzymatic repair technique. We find that although many of the published methods have distinct positive effects on some characteristics of the nucleic acids, the benefits often come at a cost. In addition, a number of the previously published techniques appear to have no effect at all. Our findings recommend that the extraction methodology adopted should be chosen carefully. Here we provide a quick reference table that can be used to determine appropriate protocols for particular aims.
• Worobey, M., Gilbert, M. T., Moore, W., Melchior, L., & Worobey, M. -. (2007). DNA extraction from dry museum beetles without conferring external morphological damage. PloS one, 2(3).
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  A large number of dry-preserved insect specimens exist in collections around the world that might be useful for genetic analyses. However, until now, the recovery of nucleic acids from such specimens has involved at least the partial destruction of the specimen. This is clearly undesirable when dealing with rare species or otherwise important specimens, such as type specimens.
• Worobey, M., Gilbert, M. T., Rambaut, A., Wlasiuk, G., Spira, T. J., Pitchenik, A. E., & Worobey, M. -. (2007). The emergence of HIV/AIDS in the Americas and beyond. Proceedings of the National Academy of Sciences of the United States of America, 104(47).
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  HIV-1 group M subtype B was the first HIV discovered and is the predominant variant of AIDS virus in most countries outside of sub-Saharan Africa. However, the circumstances of its origin and emergence remain unresolved. Here we propose a geographic sequence and time line for the origin of subtype B and the emergence of pandemic HIV/AIDS out of Africa. Using HIV-1 gene sequences recovered from archival samples from some of the earliest known Haitian AIDS patients, we find that subtype B likely moved from Africa to Haiti in or around 1966 (1962-1970) and then spread there for some years before successfully dispersing elsewhere. A "pandemic" clade, encompassing the vast majority of non-Haitian subtype B infections in the United States and elsewhere around the world, subsequently emerged after a single migration of the virus out of Haiti in or around 1969 (1966-1972). Haiti appears to have the oldest HIV/AIDS epidemic outside sub-Saharan Africa and the most genetically diverse subtype B epidemic, which might present challenges for HIV-1 vaccine design and testing. The emergence of the pandemic variant of subtype B was an important turning point in the history of AIDS, but its spread was likely driven by ecological rather than evolutionary factors. Our results suggest that HIV-1 circulated cryptically in the United States for approximately 12 years before the recognition of AIDS in 1981.
• Worobey, M., Gilbert, M. T., Sanchez, J. J., Haselkorn, T., Jewell, L. D., Lucas, S. B., Van Marck, E., Børsting, C., Morling, N., & Worobey, M. -. (2007). Multiplex PCR with minisequencing as an effective high-throughput SNP typing method for formalin-fixed tissue. Electrophoresis, 28(14).
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  Extensive collections of formalin-fixed paraffin-embedded (FFPE) tissues exist that could be exploited for genetic analyses in order to provide important insights into the genetic basis of disease or host/pathogen cointeractions. We report here an evaluation of a 44 SNP multiplex genotyping method, multiplex PCR with minisequencing (MPMS), on 92 DNA extractions performed on six archival FFPE samples of variable DNA quality, which date between 9 and 25 years old. On the three extracts with highest quality, we found the assay efficiency to be near 100%. However, the efficiency of the lowest quality extracts varied significantly. In this study, we demonstrate that although direct measures of DNA concentration in the extracts provide no useful information with regard to subsequent MPMS success, the success of the assay can be determined to some degree a priori, through initial screening of the DNA quality using a simple quantitative real-time PCR (qPCR) assay for nuclear DNA, and/or an assay of the maximum PCR amplifiable size of nuclear DNA. MPMS promises to be of significant use in future genetic studies on FFPE material. It provides a streamlined approach for retrieving a large amount of genetic information using simple, single reactions and minute amounts of archival tissue/DNA. In the light of this evidence, we suggest that the systematic screening of FFPE collections may in the future provide valuable insights into the past.
• Worobey, M., Wertheim, J. O., & Worobey, M. -. (2007). A challenge to the ancient origin of SIVagm based on African green monkey mitochondrial genomes. PLoS pathogens, 3(7).
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  While the circumstances surrounding the origin and spread of HIV are becoming clearer, the particulars of the origin of simian immunodeficiency virus (SIV) are still unknown. Specifically, the age of SIV, whether it is an ancient or recent infection, has not been resolved. Although many instances of cross-species transmission of SIV have been documented, the similarity between the African green monkey (AGM) and SIVagm phylogenies has long been held as suggestive of ancient codivergence between SIVs and their primate hosts. Here, we present well-resolved phylogenies based on full-length AGM mitochondrial genomes and seven previously published SIVagm genomes; these allowed us to perform the first rigorous phylogenetic test to our knowledge of the hypothesis that SIVagm codiverged with the AGMs. Using the Shimodaira-Hasegawa test, we show that the AGM mitochondrial genomes and SIVagm did not evolve along the same topology. Furthermore, we demonstrate that the SIVagm topology can be explained by a pattern of west-to-east transmission of the virus across existing AGM geographic ranges. Using a relaxed molecular clock, we also provide a date for the most recent common ancestor of the AGMs at approximately 3 million years ago. This study substantially weakens the theory of ancient SIV infection followed by codivergence with its primate hosts.
• Worobey, M. (2005). Anthrax and the art of war (against ascertainment bias). Heredity, 94(5), 459-460.
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  PMID: 15674386;
• Barnett, O. E., Worobey, M., Holmes, E. C., & Cooper, A. (2004). Detection of TT virus among chimpanzees in the wild using a noninvasive technique. Journal of Wildlife Diseases, 40(2), 230-237.
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  PMID: 15362822;Abstract: Zoonotic transmission and emergence of pathogens are serious threats to endangered populations of free-ranging primate species. Recent discovery of a nonpathogenic yet highly prevalent virus in human populations, TT virus (TTV), has prompted studies into the presence of this virus among captive individuals of other species of nonhuman primates. In this study, we screened captive primate species for TTV. In addition, we provide the first data on TTV infection in free-ranging primates by noninvasive screening of three chimpanzee (Pan troglodytes sweinfurthii) communities. Phylogenetic relationships between virus isolates and those previously reported from human populations, captive primates, and domesticated species are inferred. Our findings are discussed with respect to potential zoonotic events that may result from increased levels of human encroachment into wild habitats. © Wildlife Disease Association 2004.
• Damond, F., Worobey, M., Campa, P., Farfara, I., Colin, G., Matheron, S., Brun-Vézinet, F., Robertson, D. L., & Simon, F. (2004). Identification of a highly divergent HIV type 2 and proposal for a change in HIV type 2 classification. AIDS Research and Human Retroviruses, 20(6), 666-672.
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  PMID: 15242544;Abstract: We report the complete genome sequence of a highly divergent strain of human immunodeficiency virus type 2 (HIV-2), 96FR12034, identified in France from a patient of West African origin. This lineage, H, represents only the third definitive instance of a monkey-to-human transfer of SIVsm that has given rise to pathogenic HIV-2. As the different "subtypes" of HIV-2 are analogous to the different groups of HIV-1 we propose that HIV-2 subtypes henceforth by renamed groups in agreement with the HIV Nomenclature Committee. The single-strain lineages C to G and the 96FR12034 lineage identified here should be considered only as putative groups until related strains are identified that confirm circulation of these viruses in the human population.
• Lemey, P., Pybus, O. G., Rambaut, A., Drummond, A. J., Robertson, D. L., Roques, P., Worobey, M., & Vandamme, A. (2004). The molecular population genetics of HIV-1 group O. Genetics, 167(3), 1059-1068.
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  PMID: 15280223;PMCID: PMC1470933;Abstract: HIV-1 group O originated through cross-species transmission of SIV from chimpanzees to humans and has established a relatively low prevalence in Central Africa. Here, we infer the population genetics and epidemic history of HIV-1 group O from viral gene sequence data and evaluate the effect of variable evolutionary rates and recombination on our estimates. First, model selection tools were used to specify suitable evolutionary and coalescent models for HIV group O. Second, divergence times and population genetic parameters were estimated in a Bayesian framework using Markov chain Monte Carlo sampling, under both strict and relaxed molecular clock methods. Our results date the origin of the group O radiation to around 1920 (1890-1940), a time frame similar to that estimated for HIV-1 group M. However, group O infections, which remain almost wholly restricted to Cameroon, show a slower rate of exponential growth during the twentieth century, explaining their lower current prevalence. To explore the effect of recombination, the Bayesian framework is extended to incorporate multiple unlinked loci. Although recombination can bias estimates of the time to the most recent common ancestor, this effect does not appear to be important for HIV-1 group O. In addition, we show that evolutionary rate estimates for different HIV genes accurately reflect differential selective constraints along the HIV genome.
• Worobey, M., Santiago, M. L., Keele, B. F., Ndjango, J. N., Joy, J. B., Labama, B. L., Dhed'a, B. D., Rambaut, A., Sharp, P. M., Shaw, G. M., & Hahn, B. H. (2004). Contaminated polio vaccine theory refuted. Nature, 428(6985), 820-.
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  PMID: 15103367;
• P., R., Worobey, M., Visser, J. A., & Schuitemaker, H. (2003). Evolution of R5 and X4 human immunodeficiency virus type 1 gag sequences in vivo: Evidence for recombination. Virology, 314(1), 451-459.
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  PMID: 14517097;Abstract: Human immunodeficiency virus type 1 (HIV-1) infection is in general established by CCR5-utilizing (R5) virus variants, which persist throughout the course of infection. R5 HIV-1 variants evolve into CXCR4-utilizing (X4) HIV-1 variants in approximately half of the infected individuals. We have previously observed an ongoing genetic evolution with a continuous divergence of envelope gp120 sequences of coexisting R5 and X4 virus variants over time. Here, we studied evolution of gag p17 sequences in two patients who developed X4 variants in the course of infection. In contrast to the envelope gp120 sequences, gag p17 sequences of R5 and X4 virus populations intermingled in phylogenetic trees and did not diverge from each other over time. Statistical evaluation using the Shimodaira-Hasegawa test indicated that the different genomic regions evolved along different topologies, supporting the hypothesis of recombination. Therefore, our data imply that recombination between R5 and X4 HIV-1 variants occurs in vivo. © 2003 Elsevier Inc. All rights reserved.
• Walker, P. R., Worobey, M., Rambaut, A., Holmes, E. C., & Pybus, O. G. (2003). Sexual transmission of HIV in Africa. Nature, 422(6933), 679-.
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  PMID: 12700750;
• Worobey, M., Rambaut, A., Pybus, O. G., & Robertson, D. L. (2002). Questioning the evidence for genetic recombination in the 1918 "Spanish flu" virus.. Science, 296(5566), 211 discussion 211.
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  PMID: 11951002;
• Jenkins, G. M., Worobey, M., Woelk, C. H., & Holmes, E. C. (2001). Erratum: Evidence for the non-quasispecies evolution of RNA viruses (Molecular Biology and Evolution (2001) 18 (987)). Molecular Biology and Evolution, 18(8), 1610-.
• Jenkins, G. M., Worobey, M., Woelk, C. H., & Holmes, E. C. (2001). Nonquasispecies evidence for the evolution of rna viruses. Molecular Biology and Evolution, 18(6), 987-994.
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  PMID: 11371587;Abstract: The quasispecies model of RNA virus evolution differs from those formulated in conventional population genetics in that neutral mutations do not lead to genetic drift of the population, and natural selection acts on the mutant distribution as a whole rather than on individual variants. By computer simulation, we show that this model could be inappropriate for many RNA viruses because the neutral sequence space may be too large to allow the formation of a quasispecies distribution. This view is supported by our analysis of gene sequences from vesicular stomatitis virus, which is considered a prototype RNA virus quasispecies. Our results are relevant to the evolution of RNA systems in general.
• Worobey, M. (2001). A novel approach to detecting and measuring recombination: New insights into evolution in viruses, bacteria, and mitochondria. Molecular Biology and Evolution, 18(8), 1425-1434.
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  PMID: 11470833;Abstract: An accurate estimate of the extent of recombination is important whenever phylogenetic methods are applied to potentially recombining nucleotide sequences. Here, data sets from viruses, bacteria, and mitochondria were examined for deviations from clonality using a new approach for detecting and measuring recombination. The apparent rate heterogeneity (ARH) among sites in a sequence alignment can be inflated as an artifact of recombination. However, the composition of polymorphic sites will differ in a data set with recombination-generated ARH versus a clonal data set that exhibits the equivalent degree of rate heterogeneity. This is because recombinant data sets, encompassing regions of conflicting phylogenetic history, tend to yield "starlike" trees that are superficially similar to those inferred from clonal data sets with weak phylogenetic signal throughout. Specifically, a recombinant data set will be unexpectedly rich in conflicting phylogenetic information compared with clonally generated data sets supporting the same tree shape. Its value of q - Defined as the proportion of two-state parsimony-informative sites to all polymorphic sites - Will be greater than that expected for nonrecombinant data. The method proposed here, the informative-sites test, compares the value of q against a null distribution of values found using Monte Carlo-simulated data evolved under the null hypothesis of clonality. A significant excess of q indicates that the assumption of clonality is not valid and hence that the ARH in the data is at least partly an artifact of recombination. Investigations of the procedure using simulated sequences indicated that it can successfully detect and measure recombination and that it is unlikely to produce "false positives." Simulations also showed that for recombinant data, naïve use of maximum-likelihood models incorporating rate heterogeneity can lead to overestimation of the time to the most recent common ancestor. Application of the test to real data revealed for the first time that populations of viruses, like those of bacteria, can be brought close to complete linkage equilibrium by pervasive recombination. On the other hand, the test did not reject the hypothesis of clonality when applied to a data set from the coding region of human mitochondrial DNA, despite its high level of ARH and homoplasy.
• Worobey, M., & Holmes, E. C. (2001). Homologous recombination in GB virus C/hepatitis G virus. Molecular Biology and Evolution, 18(2), 254-261.
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  PMID: 11158384;Abstract: Analysis of 33 GB virus C/hepatitis G virus (GBV-C/HGV) full or nearly full genome sequences revealed several putative inter- and intrasubtype recombinants. The breakpoints of the recombinant regions were mapped using a maximum-likelihood method, and the statistical significance for each region was tested using Monte Carlo simulation. The results were highly significant and provided evidence for the existence of complex mosaic genomes showing as many as nine recombination events, with breakpoints in the 5′ UTR and in all of the coding regions except the short NS4b gene. Recombination was confirmed by separate phylogenetic analysis of the various recombinant regions and by Sawyer's runs test. Taken together, these findings demonstrate for the first time that recombination is common in natural populations of GBV-C and that it takes place both within and between subtypes. The wide-ranging implications of such nonclonal history for reconstructing the spread and timescale of GBV-C evolution are discussed.
• Worobey, M. (2000). Extensive homologous recombination among widely divergent TT viruses. Journal of Virology, 74(16), 7666-7670.
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  PMID: 10906223;PMCID: PMC112290;Abstract: Analyses of a collection of full-length TT virus genomes showed nearly half of them to be recombinant. The results were highly significant and revealed homologous recombination both within and among genotypes, often involving extremely divergent lineages. Recombination breakpoints were significantly more common in the noncoding region of the TT virus genome than in the coding region.
• Holmes, E. C., Worobey, M., & Rambaut, A. (1999). Phylogenetic evidence for recombination in dengue virus. Molecular Biology and Evolution, 16(3), 405-409.
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  PMID: 10331266;Abstract: A split decomposition analysis of dengue (DEN) virus gene sequences revealed extensive networked evolution, indicative of recombination, among DEN-1 strains but not within serotypes DEN-2, DEN-3, or DEN-4. Within DEN-1, two viruses sampled from South America in the last 10 years were identified as recombinants. To map the breakpoints and test their statistical support, we developed a novel maximum likelihood method. In both recombinants, the breakpoints were found to be in similar positions, within the fusion peptide of the envelope protein, demonstrating that a single recombination event occurred prior to the divergence of these two strains. This is the first report of recombination in natural populations of dengue virus.
• Worobey, M., & Holmes, E. C. (1999). Evolutionary aspects of recombination in RNA viruses. Journal of General Virology, 80(10), 2535-2543.
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  PMID: 10573145;
• Worobey, M., Rambaut, A., & Holmes, E. C. (1999). Widespread intra-serotype recombination in natural populations of dengue virus. Proceedings of the National Academy of Sciences of the United States of America, 96(13), 7352-7357.
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  PMID: 10377418;PMCID: PMC22089;Abstract: Diversity analysis of 71 published dengue virus gene sequences revealed several strains that appeared to be mosaics comprising gene regions with conflicting evolutionary histories. Subsequent maximum likelihood breakpoint estimation identified seven recombinants, including members of three of the four dengue virus serotypes, with breakpoints in the premembrane/membrane gene, the envelope gene, and at the junction of the envelope and first nonstructural genes. Many of the individual recombinants contain sequence representing separate genetic subtypes. The results were highly statistically significant and were confirmed by phylogenetic analysis of the regions of interest. These findings indicate that recombination may play a very significant role in shaping genetic diversity in dengue virus and, as such, have important implications for its biology and its control.
• Crespi, B. J., Carmean, D. A., Mound, L. A., Worobey, M., & Morris, D. (1998). Phylogenetics of Social Behavior in Australian Gall-Forming Thrips: Evidence from Mitochondrial DNA Sequence, Adult Morphology and Behavior, and Gall Morphology. Molecular Phylogenetics and Evolution, 9(1), 163-180.
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  PMID: 9479705;Abstract: Six species of Australian gall-forming thrips (Insecta: Thysanoptera) onAcaciaexhibit soldier castes, individuals with reduced wings and enlarged forelegs that defend their gall against interspecific invaders. We used data from two mitochondrial genes (cytochrome oxidase I and 16S rDNA), adult morphology and behavior, and gall morphology to infer a phylogeny forAcaciagall-forming thrips with and without soldiers, and we used this phylogeny to evaluate hypotheses concerning soldier evolution. Phylogenies inferred from each data set analyzed separately yielded large numbers of most-parsimonious trees and weak support for most nodes. However, when analyzed together the data sets complemented and reinforced one another in such a way as to yield a well-resolved phylogeny. Our phylogeny implies that soldiers originated once or twice early in the history of this clade, that soldiers were lost once or twice, and that soldiers evolved from winged dispersers rather than from nonsoldier within-gall reproductive offspring of foundresses. The phylogeny also provides evidence for long-term morphological stasis, an ancient split between eastern and western gall thrips species, and a high degree of conservatism in host-plant affiliations. © 1998 Academic Press.
• Crespi, B., & Worobey, M. (1998). Comparative analysis of gall morphology in Australian gall thrips: The evolution of extended phenotypes. Evolution, 52(6), 1686-1696.
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  Abstract: We used a combination of morphometric, phylogenetic, and life-history information to analyze the evolution and possible adaptive significance of gall morphology in a clade of 24 species of gall-inducing thrips (Insecta: Thysanoptera) on Australian Acacia trees. Principal components analysis revealed that galls varied in morphology along two main axes, spherical versus elongate (PC1) and general size (PC2). A high degree of conservation of gall shape on an independently derived phylogeny of the insects and the presence of nine species of Acacia each bearing two or three morphologically disparate gall forms induced by different thrips species indicate that interspecific variation in gall form is determined predominantly by the insects. Character optimization of PC1 on the phylogeny of gall thrips suggested that the ancestral gall form was a simple roll or curl. The diversification of gall form involved four main processes: (1) the convergent evolution of relatively spherical galls in two clades; (2) the evolution of small elongate and hemispherical galls in one clade; (3) the evolution of a lobed interior in a species with a spherical gall and multiple within-gall generations; and (4) the evolution of intraspecific gall polymorphism in a clade of apparent sibling species. Comparative analyses indicated that gall sphericity was associated with the presence of physogastry (foundress hyperfecundity) and that small elongate and hemispherical forms may be associated with the presence of multiple generations in a gall and, perhaps, with the presence of soldier castes. The evolution of a lobed interior in one species, which greatly increases inner surface area, coincided with the evolution of multiple generations. In the clade with intraspecific gall polymorphism in some species, patterns of intraspecific variation mirror patterns of interspecific variation within the clade as a whole. This is the first study to analyze the evolution of gall size and shape in a phylogenetic context and to investigate the life-history correlates of evolutionary changes in gall form. Taken together, our findings indicate that the main selective pressures driving the evolution of gall form in Australian gall thrips on Acacia involve inner surface area to volume relationships, which change in concert with foundress fecundity and the number of within-gall generations.

Presentations

• Yoon, J., Worobey, M., Baker, J., Nguyen, B. T., Sosnowski, K., Breshears, L. E., Akarapipad, P., & Kaarj, K. (2021, July). Rapid and sensitive detection of SARS-CoV-2 from human clinical samples using flow-based smartphone analysis on paper microfluidic chip. 31st Anniversary World Congress on Biosensors. Online: Elsevier.