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A transmissible cancer shifts from emergence to endemism in Tasmanian devils


The emergence of a devastating transmissible facial cancer among Tasmanian devils over the past few decades has caused substantial concern for their future because these animals are already threatened by a regional distribution and other stressors. Little is known about the overall history and trajectory of this disease. Patton et al. used an epidemiological phylodynamic approach to reveal the pattern of disease emergence and spread. They found that low Tasmanian devil densities appear to be contributing to slower disease growth and spread, which is good news for Tasmanian devil persistence and suggests that care should be taken when considering options for increasing devil populations. Science , this issue p. [eabb9772][1] ### INTRODUCTION Emerging infectious diseases pose one of the greatest threats to human health and biodiversity. Phylodynamics is an effective tool for inferring epidemiological parameters to guide intervention strategies, particularly for human viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, phylodynamic analysis has historically been limited to the study of rapidly evolving viruses and, in rare cases, bacteria. Nonetheless, application of phylodynamics to nonviral pathogens has immense potential, such as for predicting disease spread and informing the management of wildlife diseases. We conducted a phylodynamics analysis of devil facial tumor disease (DFTD), a transmissible cancer that has spread across nearly the entire geographic range of Tasmanian devils and threatens the species with extinction. DFTD is transmitted as an allograft through biting during common social interactions, susceptibility is nearly universal, and case fatality rates approach 100%. The goals of our study were to (i) characterize the geographic spread of DFTD, (ii) identify whether there are different circulating tumor lineages, and (iii) quantify rates of transmission among lineages. ### RATIONALE In principle, phylodynamics should be readily extended to the study of slowly evolving pathogens with large genomes through careful interrogation of genes to identify those that are measurably evolving. By testing individual genes for a clocklike signal, these genes may then be used for phylodynamic analysis. We demonstrate this proof of concept in DFTD. ### RESULTS We screened >11,000 genes across the DFTD genome, identifying 28 that exhibited a strong, clocklike signal, and performed the first phylodynamic analysis of a genome larger than a bacterium. We demonstrate here, contrary to field observations, that DFTD spread omnidirectionally throughout the epizootic, leaving little signal of geographic structuring of tumor lineages across Tasmania. Despite predictions of devil extinction, we found that the effective reproduction number ( R E), a summary of the rate at which disease spreads, has declined precipitously after the initial epidemic spread of DFTD. Specifically, R E peaked at a high of ~3.5 shortly after the discovery of DFTD in 1996 and is now ~1 in both extant tumor lineages. This is consistent with a shift from emergence to endemism. Except for a single gene, we found little evidence for convergent molecular evolution among tumor lineages. ### CONCLUSION We have demonstrated that phylodynamics can be applied to virtually any pathogen. In doing so, we show that through careful interrogation of the pathogen genome, a measurably evolving set of genes can be identified to characterize epidemiological dynamics of nonviral pathogens with large genomes. By applying this approach to DFTD, we have shown that the disease appears to be transitioning from emergence to endemism. Consistent with recent models, our inference that R E ~1 predicts that coexistence between devils and DFTD is a more likely outcome than devil extinction. Therefore, our findings present cautious optimism for the continued survival of the iconic Tasmanian devil but emphasize the need for evolutionarily informed conservation management to ensure their persistence. ![Figure][2] Tasmanian devils and their transmissible cancer. Healthy (top) and DFTD-infected (bottom) Tasmanian devils. Photos: David G. Hamilton (top), Alexandra K. Fraik (bottom). Emerging infectious diseases pose one of the greatest threats to human health and biodiversity. Phylodynamics is often used to infer epidemiological parameters essential for guiding intervention strategies for human viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). Here, we applied phylodynamics to elucidate the epidemiological dynamics of Tasmanian devil facial tumor disease (DFTD), a fatal, transmissible cancer with a genome thousands of times larger than that of any virus. Despite prior predictions of devil extinction, transmission rates have declined precipitously from ~3.5 secondary infections per infected individual to ~1 at present. Thus, DFTD appears to be transitioning from emergence to endemism, lending hope for the continued survival of the endangered Tasmanian devil. More generally, our study demonstrates a new phylodynamic analytical framework that can be applied to virtually any pathogen. [1]: /lookup/doi/10.1126/science.abb9772 [2]: pending:yes

The emerging plasticity of SARS-CoV-2


Viruses evolve as a result of mutation (misincorporations, insertions or deletions, and recombination) and natural selection for favorable traits such as more efficient viral replication, transmission, and evasion of host defenses. Newly selected traits may be linked in unpredictable ways and raise concern that virus spread and evolution could result in greater virulence (disease severity). The limited diversity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reported during 2020, ascribed to the 3′-5′ exonuclease proofreading function of nonstructural protein 14 (nsp14), led to the view that vaccines based on a single sequence of the viral spike (S) protein, which mediates host cell entry, would likely generate immune protection to all circulating variants ([ 1 ][1]). However, variants of SARS-CoV-2 with mutations in S have emerged around the world, posing potential challenges for vaccination and antibody-based therapies. The continued spread of SARS-CoV-2 creates the opportunity for accumulation of additional consequential mutations in S and throughout the viral genome. Although SARS-CoV-2 shares high sequence homology with SARS-CoV, which caused the 2002–2004 SARS outbreak, the coronavirus family is diverse in both sequence and in host receptor preference. For example, SARS-CoV-2 and a “common cold” human coronavirus, HCoV-NL63, both recognize angiotensin-converting enzyme 2 (ACE2) as the host cell receptor, but SARS-CoV-2 and HCoV-NL63 belong to different coronavirus genera and have major sequence and structural differences in the receptor-binding domain (RBD) of S, sharing <30% sequence homology ([ 2 ][2]). This diversity in S indicates that coronaviruses have broad potential to tolerate changes in both sequence and structure without substantial loss of function. This may partially explain why coronaviruses can undergo zoonotic transmission and suggests that the full evolutionary potential of SARS-CoV-2 has yet to be revealed. The S protein comprises two subunits: S1, which contains the RBD, and S2, which mediates virus–host cell fusion. Antibody-neutralizing epitopes are scattered throughout S but are mostly concentrated within the RBD. Despite the potential for plasticity, after nearly a year of spread (from December 2019) to >100 million people, there was limited evidence for evolution of SARS-CoV-2 S. The only notable evolutionary event was the D614G (Asp614→Gly) substitution in S1, which increases ACE2 affinity, leading to higher infectivity and transmissibility. Viral sequences deposited in public databases were mostly obtained from the upper respiratory tract during acute infection, before major immune responses have occurred. Such sequences might not capture the effect of within-host immune selection on viral diversification. Extensive intrahost evolution of SARS-CoV-2 has been reported in at least five individuals with protracted infection because of immune impairment from therapy for hematologic malignancies or autoimmunity ([ 3 ][3]–[ 7 ][4]). They had active SARS-CoV-2 infection for an average of 115 days before clearing the infection or succumbing to COVID-19. Each patient also had at least one convalescent plasma (CP) treatment (intravenous transfusion of blood plasma from a donor who has recovered from COVID-19) and/or monoclonal antibody therapy. Some of these individuals were shedding high titers of SARS-CoV-2 at the time of discharge from hospital or before death, indicating the potential for transmission. SARS-CoV-2 variants from two of these patients had up to fivefold reduction in neutralization sensitivity to CP ([ 3 ][3], [ 7 ][4]). Although these are case studies in immunocompromised individuals, they raise concern because the deletions of amino acids 69 to 70 (Δ69–70), Δ141–144, or Δ242–248 in S1 were observed in four out of the five infections ([ 3 ][3], [ 5 ][5]–[ 7 ][4]); the N501T (Asn501→Thr) or N501Y (Asn501→Tyr) mutations were seen in two out of the five ([ 5 ][5], [ 6 ][6]); and the E484K (Glu484→Lys) and Q493K (Gln493→Lys) mutations in the RBD of one infection also arose in antibody-resistant viruses after in vitro selection. These reports preceded the detection of three major circulating variants—B.1.1.7, B.1.351, and P.1—which all contain at least eight single, nonsynonymous nucleotide changes, including E484K, N501Y, and/or K417N (Lys417→Asn) in the ACE2 interface of the RBD (shown in the illustration). There are also various deletions in the amino (N)-terminal domain (NTD) of S1 in B.1.1.7 and B.1.351 (see the figure). Although most of the mutations in these variants were observed in a minor fraction of SARS-CoV-2 sequences during the first year of the pandemic, including K417N, E484K, and N501Y, there is no evidence to suggest that these variants were created through sequential addition of each substitution during interhost transmission. Because only a few SARS-CoV-2 mutations were in circulation during most of 2020, it is likely that the three major variants are the result of selective pressures and adaptation of the virus during prolonged individual infections and subsequent transmission. All the case reports of individuals with extensive intrahost SARS-CoV-2 evolution indicated that they had been treated with suboptimal neutralizing antibodies (that is, the CP treatment did not neutralize the entire virus population). Whether or not antibody therapy played a role, it is likely that the same variants or variants containing new mutations will continue to emerge in different geographic locations as the result of intrahost selection and subsequent transmission. Indeed, other variants have been reported with multiple mutations in S1, including the lineages B.1.526 (detected in New York) and B.1.429 (which originated in California) containing a substitution in the RBD that is distinct from other variants; and B.1.525 and A.23.1 that are thought to have originated in Nigeria and Uganda, respectively ([ 8 ][7]) (see the figure). The individual phenotypic effects of the mutations in S1 are incompletely understood, but some initial clues are emerging. Substitution at position Asn501 with Thr or Phe increases affinity for ACE2 binding ([ 9 ][8]), and Tyr501 increases infectivity and virulence in a mouse model ([ 10 ][9]). Some circulating variants may have reduced sensitivity to neutralizing antibodies that bind to the RBD directly (attributed to triple substitutions of key amino acids in the RBD at the ACE2-binding interface: Lys417, Glu484, and Asn501) or to the NTD (conformational changes in the NTD are required for ACE2 attachment). More studies to correlate viral genotype and phenotype are needed. It is possible that mutations that reduce neutralizing antibody binding, such as E484K, may require compensatory mutations that restore infectivity, such as N501Y. There appears to be convergent association of mutations such as the triple RBD mutation (Lys417, Glu484, and Asn501) that evolved in two distinct lineages (B.1.351 and P.1). Moreover, E484K was also recently detected with N501Y in the B.1.1.7 lineage ([ 11 ][10]). Δ69–70 in S1 doubled the infectivity of SARS-CoV-2 pseudovirus, implying that the deletion may have been required to compensate for a mutation, D796H (Asp796→His), that reduced antibody neutralization sensitivity at a cost to viral fitness ([ 7 ][4]). The role of compensatory mutations is also supported by the emerging B.1.525 lineage that has both E484K (reduction in antibody sensitivity) and Δ69–70 (compensatory increase in infectivity). ![Figure][11] Mutations and deletions in the spike protein Currently, B.1.1.7, B.1.351, and P.1 are the major circulating variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); others are emerging. The spike S1 subunit contains an amino (N)-terminal domain (NTD) and receptor-binding domain (RBD), which mediate host receptor recognition and contain epitopes for antibody binding. Deletions and substitutions in S1 can affect transmissibility (Tr), vaccine efficacy (Ef), and virulence (Vi). Additional mutations that define the variants can be tracked at ([ 8 ][7]). SP, signal peptide. GRAPHIC: N. CARY/ SCIENCE It is not yet known whether the complex mutational patterns observed in SARS-CoV-2 variants are linked on the same viral genome or represent mixtures of different variants within the same patient. Studies evaluating the linkage of these mutations in individual SARS-CoV-2 genomes using single-genome amplification and sequencing, as has been used to characterize genetic diversity of HIV-1 and other viruses, are needed to accurately assess the infectivity and phenotype of individual variants. A case report of intrahost SARS-CoV-2 evolution showed that SARS-CoV-2 can evolve multiple distinct lineages within the same individual ([ 6 ][6]). Several studies suggest that the major circulating variants have reduced neutralizing sensitivity to CP and plasma from recently vaccinated individuals. For example, CP from individuals who were infected with the B.1 lineage (D614G-containing SARS-CoV-2) had varying reductions in neutralizing activity against live virus isolates of the B.1.351 lineage. Additionally, vaccine-elicited antibodies have reduced neutralization of pseudovirus containing the triple mutation in S1 (K417N, E484K, and N501Y) of the P.1 and B.1.351 variants ([ 12 ][12]). Pseudovirus bearing the deletions and mutations found in the B.1.1.7 lineage also showed reduced neutralization sensitivity, but titers of antibody were sufficient for complete neutralization of B.1.1.7 in sera from 40 individuals vaccinated with BNT162b2 (Pfizer/BioNTech) ([ 13 ][13]). Continued phenotypic assessments of emerging, rapidly spreading variants, including those with nonsynonymous mutations in S1 (NTD and RBD) and S2, to neutralization by CP and postvaccination sera should be a high priority to monitor possible effects on vaccine efficacy. Phase 3 trials of SARS-CoV-2 vaccines derived from a single S sequence have shown them to be highly effective in preventing infection with the initial SARS-CoV-2 variants, including those with the D614G mutation ([ 14 ][14], [ 15 ][15]). More recent data suggest that certain vaccines are less protective against the B.1.351 variant, although additional studies are needed. Studies showing reduced antibody sensitivities against new variants do not inherently prove that a vaccine is less effective. In addition to effector B cells (which produce neutralizing antibodies), there are numerous additional vaccine-induced responses of the innate and adaptive immune systems that may protect against infection and further viral immune escape. Conversely, there are uncharacterized mutations outside of S that could facilitate SARS-CoV-2 immune evasion. The growing evidence for the emergence of immune escape mutations in protracted SARS-CoV-2 infection and for multiple, rapidly spreading variants should raise broad concern and action. Reducing the spread of SARS-CoV-2 is most likely to prevent further selection of immune escape variants. This will require a coordinated and comprehensive global vaccination and prevention strategy. Partial roll-out and incomplete immunization of individuals leading to suboptimal titers of neutralizing antibody could promote selection of escape variants that negatively affect vaccine efficacy. Increased genotypic and phenotypic testing capacities are essential worldwide to detect and characterize circulating SARS-CoV-2 variants that may emerge from selection by natural or vaccine-mediated immune responses. Infections that occur among vaccinated individuals should be aggressively evaluated for the mechanisms of breakthrough. The explosive, global spread of SARS-CoV-2 and the devastation it has wreaked is a stark warning of the potential for new variants to further complicate pandemic control. Vaccine manufacturers are now testing potential booster vaccines against circulating SARS-CoV-2 variants, and more broadly active monoclonal antibodies are in development for therapy. Such proactive approaches are likely to be needed to ensure pandemic control and elimination. 1. [↵][16]1. B. Dearlove et al ., Proc. Natl. Acad. Sci. U.S.A. 117, 23652 (2020). [OpenUrl][17][Abstract/FREE Full Text][18] 2. [↵][19]1. N. M. A. Okba et al ., Emerg. Infect. Dis. 26, 1478 (2020). [OpenUrl][20][PubMed][21] 3. [↵][22]1. V. A. Avanzato et al ., Cell 183, 1901 (2020). [OpenUrl][23][PubMed][21] 4. 1. J. H. Baang et al ., J. Infect. Dis. 223, 23 (2021). [OpenUrl][24] 5. [↵][25]1. B. Choi et al ., N. Engl. J. Med. 383, 2291 (2020). [OpenUrl][26][PubMed][21] 6. [↵][27]1. M. K. Hensley et al ., Clin. Infect. Dis. 10.1093/cid/ciab072 (2021). 7. [↵][28]1. S. A. Kemp et al ., Nature (2021). 10.1038/s41586-021-03291-y 8. [↵][29]1. A. H. O'Toole et al ., International Lineage Report. 2021, PANGO lineages, 9. [↵][30]1. T. N. Starr et al ., Cell 182, 1295 (2020). [OpenUrl][31] 10. [↵][32]1. H. Gu et al ., Science 369, 1603 (2020). [OpenUrl][33][Abstract/FREE Full Text][34] 11. [↵][35]Public Health England, Investigation of novel SARS-CoV-2 variant: Variant of Concern 202012/01 Technical Briefing 5 (2021); . 12. [↵][36]1. Z. Wang et al ., Nature 10.1038/s41586-021-03324-6 (2021). 13. [↵][37]1. A. Muik et al ., Science 371, eabg6105 (2021). [OpenUrl][38] 14. [↵][39]1. F. P. Polack et al ., N. Engl. J. Med. 383, 2603 (2020). [OpenUrl][40][CrossRef][41][PubMed][21] 15. [↵][42]1. L. R. Baden et al ., N. Engl. J. Med. 384, 403 (2021). [OpenUrl][43][CrossRef][44][PubMed][21] Acknowledgments: We thank L. Pollini for assistance. J.W.M. is a consultant to Gilead Sciences and holds shares or share options in Co-Crystal Pharma, Inc., Abound Bio, Inc., and Infectious Disease Connect. 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Phylogenetic analysis of SARS-CoV-2 in Boston highlights the impact of superspreading events


One important characteristic of coronavirus epidemiology is the occurrence of superspreading events. These are marked by a disproportionate number of cases originating from often-times asymptomatic individuals. Using a rich sequence dataset from the early stages of the Boston outbreak, Lemieux et al. identified superspreading events in specific settings and analyzed them phylogenetically (see the Perspective by Alizon). Using ancestral trait inference, the authors identified several importation events, further investigated the context and contribution of particular superspreading events to the establishment of local and wider SARS-CoV-2 transmission, and used viral phylogenies to describe sustained transmission. Science , this issue p. [eabe3261][1]; see also p. [574][2] ### INTRODUCTION We used genomic epidemiology to investigate the introduction and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the Boston area across the first wave of the pandemic, from March through May 2020, including high-density sampling early in this period. Our analysis provides a window into the amplification of transmission in an urban setting, including the impact of superspreading events on local, national, and international spread. ### RATIONALE Superspreading is recognized as an important driver of SARS-CoV-2 transmission, but the determinants of superspreading—why apparently similar circumstances can lead to very different outcomes—are poorly understood. The broader impact of such events, both on local transmission and on the overall trajectory of the pandemic, can also be difficult to determine. Our dataset includes hundreds of cases that resulted from superspreading events with different epidemiological features, which allowed us to investigate the nature and effect of superspreading events in the first wave of the pandemic in the Boston area and to track their broader impact. ### RESULTS Our data suggest that there were more than 120 introductions of SARS-CoV-2 into the Boston area, but that only a few of these were responsible for most local transmission: 29% of the introductions accounted for 85% of the cases. At least some of this variation results from superspreading events amplifying some lineages and not others. Analysis of two superspreading events in our dataset illustrate how some introductions can be amplified by superspreading. One occurred in a skilled nursing facility, where multiple introductions of SARS-CoV-2 were detected in a short time period. Only one of these led to rapid and extensive spread within the facility, and significant mortality in this vulnerable population, but there was little onward transmission. A second superspreading event, at an international business conference, led to sustained community transmission, including outbreaks in homeless and other higher-risk communities, and was exported domestically and internationally, ultimately resulting in hundreds of thousands of cases. The two events also differed substantially in the genetic variation they generated, possibly suggesting varying transmission dynamics in superspreading events. Our results also show how genomic data can be used to support cluster investigations in real time—in this case, ruling out connections between contemporaneous cases at Massachusetts General Hospital, where nosocomial transmission was suspected. ### CONCLUSION Our results provide powerful evidence of the importance of superspreading events in shaping the course of this pandemic and illustrate how some introductions, when amplified under unfortunate circumstances, can have an outsized effect with devastating consequences that extend far beyond the initial events themselves. Our findings further highlight the close relationships between seemingly disconnected groups and populations during a pandemic: Viruses introduced at an international business conference seeded major outbreaks among individuals experiencing homelessness; spread throughout the Boston area, including to other higher-risk communities; and were exported extensively to other domestic and international sites. They also illustrate an important reality: Although superspreading among vulnerable populations has a larger immediate impact on mortality, the cost to society is greater for superspreading events that involve younger, healthier, and more mobile populations because of the increased risk of subsequent transmission. This is relevant to ongoing efforts to control the spread of SARS-CoV-2, particularly if vaccines prove to be more effective at preventing disease than blocking transmission. ![Figure][3] Schematic outline of this genomic epidemiology study. Illustrated are the numerous introductions of SARS-CoV-2 into the Boston area; the minimal spread of most introductions; and the local, national, and international impact of the amplification of one introduction by a large superspreading event. Analysis of 772 complete severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from early in the Boston-area epidemic revealed numerous introductions of the virus, a small number of which led to most cases. The data revealed two superspreading events. One, in a skilled nursing facility, led to rapid transmission and significant mortality in this vulnerable population but little broader spread, whereas other introductions into the facility had little effect. The second, at an international business conference, produced sustained community transmission and was exported, resulting in extensive regional, national, and international spread. The two events also differed substantially in the genetic variation they generated, suggesting varying transmission dynamics in superspreading events. Our results show how genomic epidemiology can help to understand the link between individual clusters and wider community spread. [1]: /lookup/doi/10.1126/science.abe3261 [2]: /lookup/doi/10.1126/science.abg0100 [3]: pending:yes

Recurrent deletions in the SARS-CoV-2 spike glycoprotein drive antibody escape


Influenza viruses evade immunity initiated by previous infection, which explains recurrent influenza pandemics. Unlike the error-prone RNA-dependent RNA polymerase of influenza, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and related viruses contain polymerases with proofreading activity. However, proofreading cannot correct deletions, which during a long-term persistent infection could result in the generation of viruses showing alteration of entire stretches of amino acids and the structures they form. McCarthy et al. identified an evolutionary signature defined by prevalent and recurrent deletions in the spike protein of SARS-CoV-2 at four antigenic sites. Deletion variants show human-to-human transmission of viruses with altered antigenicity. Science , this issue p. [1139][1] Zoonotic pandemics, such as that caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can follow the spillover of animal viruses into highly susceptible human populations. The descendants of these viruses have adapted to the human host and evolved to evade immune pressure. Coronaviruses acquire substitutions more slowly than other RNA viruses. In the spike glycoprotein, we found that recurrent deletions overcome this slow substitution rate. Deletion variants arise in diverse genetic and geographic backgrounds, transmit efficiently, and are present in novel lineages, including those of current global concern. They frequently occupy recurrent deletion regions (RDRs), which map to defined antibody epitopes. Deletions in RDRs confer resistance to neutralizing antibodies. By altering stretches of amino acids, deletions appear to accelerate SARS-CoV-2 antigenic evolution and may, more generally, drive adaptive evolution. [1]: /lookup/doi/10.1126/science.abf6950

Metagenomic sequencing at the epicenter of the Nigeria 2018 Lassa fever outbreak


Lassa fever is a hemorrhagic viral disease endemic to West Africa. Usually, each year sees only a smattering of cases reported, but hospitalized patients risk a 15% chance of death. Responding to fears that a 10-fold surge in cases in Nigeria in 2018 signaled an incipient outbreak, Kafetzopoulou et al. performed metagenomic nanopore sequencing directly from samples from 120 patients (see the Perspective by Bhadelia). Results showed no strong evidence of a new strain emerging nor of person-to-person transmission; rather, rodent contamination was the main source. To prevent future escalation of this disease, we need to understand what triggers the irruption of rodents into human dwellings.