Integrated analysis of protein sequence and structure redefines viral diversity and the taxonomy of the Flaviviridae

Integrated analysis of protein sequence and structure redefines viral diversity and the taxonomy of the Flaviviridae | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Integrated analysis of protein sequence and structure redefines viral diversity and the taxonomy of the Flaviviridae Peter Simmonds , Anamarija Butković , Joe Grove , View ORCID Profile Richard Mayne , View ORCID Profile Jonathon C. O. Mifsud , Martin Beer , Jens Bukh , J. Felix Drexler , Amit Kapoor , Volker Lohmann , Donald B. Smith , Jack T. Stapleton , View ORCID Profile Nikos Vasilakis , Jens H. Kuhn doi: https://doi.org/10.1101/2025.01.17.632993 Peter Simmonds 1 Nuffield Department of Medicine, University of Oxford , Oxford, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: Peter.Simmonds{at}ndm.ox.ac.uk Anamarija Butković 2 Archaeal Virology Unit, Institut Pasteur, Université Paris Cité , CNRS UMR6047, Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Joe Grove 3 MRC- University of Glasgow Centre for Virus Research , Glasgow, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Richard Mayne 1 Nuffield Department of Medicine, University of Oxford , Oxford, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Richard Mayne Jonathon C. O. Mifsud 4 Sydney Institute for Infectious Diseases, School of Medical Sciences, The University of Sydney , Sydney, New South Wales, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jonathon C. O. Mifsud Martin Beer 5 Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jens Bukh 6 Copenhagen Hepatitis C Program(CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site J. Felix Drexler 7 Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Virology , Berlin, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Amit Kapoor 8 Center for Vaccines and Immunity, The Research Institute at Nationwide Children’s Hospital , Columbus, Ohio, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Volker Lohmann 9 Department of Infectious Diseases, Molecular Virology, Heidelberg University , Heidelberg, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Donald B. Smith 1 Nuffield Department of Medicine, University of Oxford , Oxford, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jack T. Stapleton 10 Departments of Internal Medicine, Microbiology and Immunology, University of Iowa and Iowa City VA Healthcare , Iowa City, Iowa, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nikos Vasilakis 11 Department of Pathology and Center for Vector-Borne and Zoonotic Diseases, University of Texas Medical Branch , Galveston, Texas, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nikos Vasilakis Jens H. Kuhn 12 Integrated Research Facility at Fort Detrick, Division of Clinical Research, National Institute of Allergy and Infectious Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: kuhnjens{at}mail.nih.gov Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract The Flaviviridae are a family of non-segmented positive-sense enveloped RNA viruses containing significant pathogens including hepatitis C virus and yellow fever virus. Recent large-scale metagenomic surveys have identified many diverse RNA viruses related to classical orthoflaviviruses and pestiviruses but quite different genome lengths and configurations, and with a hugely expanded host range that spans multiple animal phyla, including molluscs, cnidarians and stramenopiles,, and plants. Grouping of RNA-directed RNA polymerase (RdRP) hallmark gene sequences of flavivirus and ‘flavi-like’ viruses into four divergent clades and multiple lineages within them was congruent with helicase gene phylogeny, PPHMM profile comparisons, and comparison of RdRP protein structure predicted by AlphFold2. These results support their classification into the established order, Amarillovirales , in three families ( Flaviviridae, Pestiviridae , and Hepaciviridae ), and 14 genera. This taxonomic framework informed by RdRP hallmark gene evolutionary relationships provides a stable reference from which major genome re-organisational events can be understood. Introduction The Flaviviridae are a family of positive-sense RNA viruses that incorporates several major human and veterinary pathogens, including hepatitis C virus (HCV) and a wide range of often highly virulent arthropod-borne viruses, including yellow fever virus (YFV) and dengue viruses 1 . Historically, the mosquito-borne YFV became the founding member of a growing group of ‘arthropod-borne viruses’, a term introduced in the 1940s 2 , although serologically split into two groups, A (Sindbis virus and relatives) and B (YFV and relatives) 3 . The taxonomic term ‘arbovirus’ was used in 1971 in the first report of the what became the International Committee on Taxonomy of Viruses (ICTV), with Arbovirus Group B renamed as the genus Flavivirus and group A as Alphavirus in the family Togavivirdae. This family also included the non-arbovirus transmitted genera Rubivirus (human rubella virus) and Pestivirus (bovine viral diarrhea virus (BVDV) and relatives infecting bovids and suids) 4 . Subsequent analyses of the growing amount of molecular, morphological, and serological data for these viruses indicated that the Flavivirus and Pestivirus genera should be removed from Togaviridae and re-classified into a new family, Flaviviridae 5 , 6 , a group that subsequently expanded to incorporate a third genus, Hepacivirus , for HCV 7 and relatives in non-human primates, bats, and rodents (reviewed in 8 ). Finally, a fourth genus, Pegivirus , was added in 2012 for a range of apparently non-pathogenic RNA viruses infecting humans (human pegivirus [HPgV]) and a broad range non-human primates, bats, and other mammas 9 and one avian (goose) host 10 . Since then, there have been only minor changes to flavivirus classification, essentially limited to the expansion of the number of species assigned to the Pestivirus, Pegivirus and Hepacivirus genera 11 and the renaming of the genus Flavivirus to Orthoflavivirus 11 . Current members of the Flaviviridae have consistent genome organizations (positive-sense, strategies (synthesis of a single polyprotein with a conserved organization that is cleaved into structural proteins located at the N terminus and nonstructural proteins at the C terminus) ( Fig. 1 ), and a primarily mammalian host range (in case of orthoflaviviruses, also arthropods) 1 . In addition to the RdRP gene, flaviviruses are homologous in their superfamily 2 helicase (NS3) and serine protease domain sequences 12 . Download figure Open in new tab Fig. 1. Organization of example genomes in each lineage of flaviviruses and ‘flavi-like’ viruses. Genome diagrams for the example viruses listed in Table 1 drawn to scale (lower scale bar) and main functional domains identified by InterProScan browser v. 103 ( https://www.ebi.ac.uk/interpro/search/sequence/ ) 91 View this table: View inline View popup Table 1. Listing of clades and lineages of flavivirus and ‘flavi-like’ viruses and suggested revised taxonomy However, within flaviviruses, there are also some major differences, including possession of structurally distinct capsid proteins (and the apparent lack of a capsid in pegiviruses), packaging mechanisms, polyprotein translation strategy that may be 5’-cap-dependent (orthoflaviviruses) or driven through an internal ribosomal entry site (IRES) in other flaviviruses ( Fig. 1 ), and two disparate fusion glycoprotein systems. ( Fig. 1 ). Indeed, other than the serine protease, helicase and RdRP, no nonstructural protein genes are homologous across viruses of all four genera 11 . The more recent application of high throughput sequencing technologies to detect and genetically characterize viruses in a much wider range of potential hosts has transformed our understanding of flaviviral diversity and abundance 13 . Analyses particularly of arthropods and more recently fish and other aquatic life have created a case for a major expansion in viruses assigned to the existing genera, such as a range of novel insect-only (non-vectored) flaviviruses 14 – 19 and the expansion of species assignments to the Pestivirus , Pegivirus , and Hepacivirus genera that infect primates, other mammals, and birds (reviewed in 8 , 20 – 23 ). More perplexing has been the discovery and characterization of a huge number of flavi-like viruses 24 , so-called because of their phylogenetic grouping of RdRP gene sequences with those of classified members of the Flaviviridae , but often possessing quite different genome organisations, genome lengths and host ranges from currently classified flaviviruses. For example, the so-called ‘large-genome flaviviruses (LGFs)’ 13 , 25 , 26 have genomes significantly longer than currently classified flaviviruses, the longest to date being maximus pesti-like virus’s genome of ≈40 kb 27 . Jīngmén tick virus (JMTV) 28 and related jingmenviruses have multipartite genomes with four or more separate segments 29 – 32 . In addition, the majority of ‘flavi-like’ viruses have been discovered outside the primarily mammalian and vector host range of classified flaviviruses, being distributed across the animal kingdom, from poriferans (sponges) 27 , cnidarians (jellies) 33 , mollusks (squid) 34 , arthropods (insects 25 , 31 , 35 ; diplurans 31 ; scorpions 31 ; crustaceans 33 , 34 , 36 ), nematodes 37 , platyhelminths 38 , 39 ) to echinoderms (sea cucumbers) 40 , hemichordates (acorn worms) 33 , cartilaginous and bony fish 13 , 33 , 41 – 44 , amphibians (frogs) 33 , 45 , and reptiles 43 , 43 , 43 . Although only a few are known so far, ‘flavi-like’ viruses have also been discovered in stramenopiles (diatoms and oomycotes) 46 , 47 and, remarkably, in angiosperm plants 30 , 48 , 49 . These discoveries beg the question of how to classify these viruses and how to organize the current order Amarillovirales to best reflect their evolutionary relationships 13 , 50 . For example, by phylogenetic analysis of RdRP domain sequences, JMTV and other segmented ‘flavi-like’ viruses typically group closer to members of the Orthoflavivirus genus than to flaviviruses of other genera, but JMTV’s putative assignment to this genus conflicts with what had been previously considered family-defining characteristics across the Flaviviridae family, i.e. , a monopartite genome. The genomic diversity of ‘flavi-like’ viruses and expanded host ranges that now encompasses multiple kingdoms of eukaryotes profoundly questions how a virus family such as the Flaviviridae can be best defined 24 . The future development of a robust taxonomy framework for flaviviruses, and indeed other virus families with similarly expanded genetic and genome organisational complexities, clearly requires a radically different set of criteria than those that have guided their current classification. In this proposed update to the classification of flaviviruses, we have assigned primacy to the RdRP (hallmark) gene phylogeny using previously established principles for a genomics-based taxonomy of viruses 51 , and that assignments should be based on the most evolutionarily conserved gene within virus groups 52 – 54 . At present, most viruses are assigned into six realms that are divided based on their separate origins and possession of shared orthologous gene(s) 55 – 57 . Almost all RNA viruses, including flaviviruses, have been assigned to the realm Riboviria based on possession of evolutionarily related RdRPs that are distinct from those of cellular polymerases, substantiating their likely single origin. As the hallmark gene for ribovirians, its phylogenetic relationships therefore serve to best delineate the evolutionary history of RNA viruses. Re-casting virus taxonomy through evolutionary relationships of hallmark genes 51 has furthermore enabled the development of a hierarchical higher-rank classification of RNA viruses, in which the family Flaviviridae has been assigned as a (single) member of the order, Amarillovirales , in the class Flasuviricetes , phylum Kitrinoviricota , and kingdom Orthornavirae . This approach also acknowledges that the deeper evolution of all viruses may be punctuated by major genome reorganisations associated with changes in host range or ecologies. Examples of modular evolution in flaviviruses include multiple IRES exchanges within and between flaviviruses and viruses of the family, Picornavirusae 58 , 59 , and of acquisition of glycoproteins associated with commitment to vertebrate hosts 60 . Collectively, these create different evolutionary histories and phylogenetic relationships between different genome regions. While these evolutionary events can be represented in the form of reticulated trees or networks, the strictly hierarchical classification of viruses required by the ICTV (and indeed of other biological taxonomies) requires primacy to be assigned to hallmark genes in the creation of coherent taxonomy frameworks. Accordingly, we present re-classification of flaviviruses and ‘flavi-like’ viruses grounded on varied analyses of RdRP hallmark gene relationships. This provides a robust, well-supported and coherent taxonomic framework that can serve as a scaffold for future expansion of this group of viruses. Moreover, our multi-modal approach may provide a template for re-examining other viral taxonomies, as we seek to organize the huge viral diversity revealed by meta-genomics. Results To establish a new taxonomic framework for classified flaviviruses and unclassified ‘flavi-like’ viruses, we generated a dataset of representative sequences from currently classified members of the family Flaviviridae (viruses of 52, 19, 14, and 11 species in four genera, Orthoflavivirus , Pestivirus , Hepacivirus and Pegivirus , respectively; 1 ) and a comprehensive sample of coding-complete sequences of currently unclassified ‘flavi-like’ viruses 60 (listed in Table S1; Suppl. Data). This dataset included representatives from groups of segmented ‘flavi-like’ viruses, divergent ‘pesti-like’ viruses, many with extended genomes, many additional ‘hepaci-’ and ‘pegi-like’ viruses primarily found in marine vertebrates, plant-infecting ‘koshoviruses’ and a selection of ‘flavi-like’ viruses recovered from environmental samples such as diatom colony-associated virus (DCAV). Genome regions encoding the enzymatic domains of the RdRP and helicase genes were extracted for alignment and analysis. RNA-directed RNA polymerase domain (NS5/NS5B) amino-acid sequence phylogenies differentiate flaviviruses and ‘flavi-like’ viruses into four highly supported main clades Our initial analysis focused on translated flavivirus and ‘flavi-like’ RdRP domain sequences. Using the distantly related tombusviruses as an outgroup, the RdRP domain phylogeny of our sequence set, calculated with IQ-TREE, resulted in four main bootstrap-supported clades (I–IV), each containing a number of bootstrap-supported lineages (labelled as Ia-l, IIm-s, IIIt-w anticlockwise around the tree) ( Fig. 2 ). Viruses of the four currently established flavivirus genera were distributed in three of the four clades, with hepaciviruses and pegiviruses clustering together in and largely defining clade III. In this phylogeny, ‘jingmenviruses’, ‘tamanaviruses’, and numerous ‘insect-specific flaviviruses (ISFs)’ cluster with orthoflaviviruses in clade I. The viruses that were previously loosely defined as ‘LGFs’ join pestiviruses in clade II, and include diverse viruses distributed across multiple lineages, eg . Bólè tick virus (a potentially tick-vectored pathogen of mammals) 26 in clade IIq and plant-infecting ‘koshoviruses’ 30 , 48 , 49 , in clade IIs ( Fig. 2 ; Table S1). Download figure Open in new tab Fig. 2. RNA-directed RNA polymerase domain (RdRP) amino-acid sequence phylogenies differentiate flaviviruses and ‘flavi-like’ viruses into four highly supported main clades. Maximum likelihood tree of aligned flavivirus and ‘flavi-like’ RNA-directed RNA polymerase (RdRP) domain amino-acid sequences, estimated using the LG+F+R10 model using IQ-TREE with tombusvirus sequences as an outgroup. Bootstrap support values for the main branches of the tree are shown in red if ≥70%. Already classified flaviviruses are shown in color-filled circles. The main clades are numbered I–IV and lineages are labeled with lower-case letters. The component sequences within each clade are provided in a fully annotated tree (RdRP_tree.NWK (File Sq; Suppl. Data) and listed in Table S1 (Suppl. data). To investigate the robustness of the clade and lineage groupings, the IQ-TREE-based maximum-likelihood phylogeny generated for Fig. 2 was compared with trees generated through a temporal reconstruction using the Bayesian evolutionary analysis by sampling trees cross-platform program (BEAST) and using the protein distance-based unweighted pair group method with arithmetic mean (UPGMA) phylogeny method ( Fig. 3 ). BEAST and UPGMA results reproduced clades I–IV with bootstrap support comparable to the original maximum likelihood analysis ( Figs. 2 , 3A ). However, in the time-rooted BEAST tree, Tombusviridae became an inlier ( Fig. 3C ), in marked contrast to its clearly defined outgroup positions in the IQ-TREE and UPGMA trees. The grouping of individual sequences within lineages a–w defined by IQ-TREE analysis were almost entirely reproduced in the BEAST and UPGMA trees, with the exception of eight sequences that did not group with their lineages in the UPGMA tree ( Fig. 3B ). Despite these, the clade and lineage assignments were relatively robust to different evolutionary reconstruction methods. Download figure Open in new tab Fig. 3. RNA-directed RNA polymerase domain (NS5/NS5B) amino-acid sequence phylogenies differentiate flaviviruses and ‘flavi-like’ viruses into four highly supported main clades Phylogenetic trees constructed by likelihood (A, B) and distance-based (C) methods using flavivirus and ‘flavi-like’ RNA-directed RNA polymerase (RdRP) domain amino-acid sequences. Clades (I–IV) and lineages (a–w) labelled in each tree are based on those in Fig. 2 . Tentative threshold levels of divergence separating clades and lineages are shown as red dotted lines in BEAST and UPGMA trees. An alternative threshold corresponding to the assignment of lineages Ia-Id to a common lineage is shown in a blue dotted line. Abbreviations: BP, before present; BEAST, Bayesian evolutionary analysis by sampling trees cross-platform program; JTT, Jones-Taylor-Thornton matrix; ML, maximum likelihood; UPGMA, unweighted pair group method with arithmetic mean. These RdRP phylogenies provide an excellent framework for the taxonomic reorganization of the Flaviviridae . Nonetheless, we recognized that phylogenetic inference over these very large genetic distances is challenging and therefore sought to corroborate (or, indeed, refute) these apparent taxonomic groupings with alternative and complementary approaches. Helicase domain (NS3) amino-acid sequence phylogeny supports partition of flaviviruses and ‘flavi-like’ viruses into four main clades While there is evidence for modular exchange of structural or assessor proteins among flaviviruses of the four established current genera, for instance acquisition of glycoproteins 60 , 61 , it is less clear whether sequences encoding the core replication module of these viruses (including serine protease, helicase and RdRP) evolve as a unit or are similarly subject to genome exchange and rearrangements. To investigate this, and to complement our RdRP analyses, we deduced flavivirus and ‘flavi-like’ helicase domain amino acid sequences, aligned them, performed IQ-TREE analysis, and compared the result to the RdRP IQ-TREE tree using a tanglegram ( Fig. 4 ). Phylogenetic groupings of viruses of the four current genera were highly concordant, with only minor differences in branching order within genera. The positions of ‘flavi-like’ viruses grouping with orthoflaviviruses were generally concordant between regions, but with some exceptions. For instance, there was a change in the topology of the deeper branches underlying the ‘LGF’, pestivirus and hepaci/pegivirus groupings, creating paraphyletic groups not observed in the RdRP tree. This result is not necessarily surprising because the helicase domain is shorter and more divergent than the RdRP domain and hence likely reflects lower resolution of relationships rather than indicating genome reorganization between the two domains. Other minor exceptions included Wēnl⍰ng moray eel hepacivirus (WMEHV) moving from an outlier position in clade III (lineage IIIt) into Hepacivirus in the helicase tree, a finding not incompatible with potential sequence errors in the deposited RdRP region sequence. Bólè tick virus 4 (BoTV4) fell within the IIq lineage in the RdRP domain tree, but as an outlier to pestiviruses in the helicase domain tree. Finally, ‘flavi-like’ viruses from environmental samples (clade IV in the RdRP tree) were located within the pestivirus-‘LGF’ branches in the helicase domain tree. Download figure Open in new tab Fig. 4. Helicase domain (NS3) amino-acid sequence phylogeny supports partition of flaviviruses and ‘flavi-like’ viruses into four main clades Tanglegram of flavivirus and ‘flavi-like’ virus helicase and RNA-directed RNA polymerase (RdRP) domain sequences constructed from ML phylogenetic trees generated by IQ-TREE trees, with established flavivirus genera colored as in Figs. 2 and 3 , and segmented ‘flavi-like’ viruses (lineages k and l in red). BoTV4, Bólè tick virus 4; LGFs, large genome flaviviruses, WMEHV, Wēnl⍰ng moray eel hepacivirus. A copy of the figure with the branches individually labelled is provided as Fig. S1. Structure-based comparisons of RNA-directed RNA polymerase (NS5/NS5B) recapitulate the sequence-based phylogeny The function of any given protein is primarily a feature of its three-dimensional structure. Consequently, protein structure is fundamentally more conserved than the underlying protein sequence. The advent of accurate protein structure prediction through machine-learning (e.g., AlphaFold2) is enabling surveys of protein form and function at enormous scales 62 , 63 ; and has driven the development of new high-throughput structure comparisons tools (e.g., Foldseek) that enable structure-guided inference of deep evolutionary relationships 64 , 65 . In a recent investigation of glycoproteins, we systematically applied protein structure prediction to the Flaviviridae 60 , generating thousands of structures spanning the complete polyproteins of all viruses represented in the RdRP phylogeny ( Fig. 2 ; Table S1, Suppl. Data). Drawing on this dataset, we generated complete NS5/NS5b RdRP domain structure predictions for each virus (see Methods). After filtering for prediction confidence and length, we analysed 400 RdRP structures using FoldTree 65 to produce a structure-guided tree based on the local distance difference test (lddt) structural similarity metric. This structure-based approach does not explicitly consider protein sequence and, therefore, represents an independent recapitulation of the sequence-based phylogeny ( Fig. 2 ). The topology of the structure-based tree was remarkably similar to that of the sequence-based tree and supports the existence of the same four major clades ( Fig. 5A ). Moreover, most of the lineages represented in the sequence phylogeny were consistent in their position and composition. The only exceptions are lineages Ia, which in the structure-based tree formed a basal branch from lineage Ic, and lineages Ii and IIIv which moved subtly in relation to their neighboring clades. Clades Ij, IIn, and IIIt were lost from the structure-based tree due to filtering of lower confidence structural predictions. Major clade IV, containing ‘flavi-like’ viruses from environmental samples, also shifted position slightly, branching between clade II and the Tombusviridae outgroup in the structure-based tree. However, this divergent and basal taxon will likely remain difficult to place by any method without further discovery of similar viruses. Download figure Open in new tab Download figure Open in new tab Fig. 5. RNA-directed RNA polymerase domain (NS5/NS5B) structural comparison supports partition of flaviviruses and ‘flavi-like’ viruses into four main clades A) Structure-based tree of 400 flaviviruses and ‘flavi-like’ viruses’ RdRP domains, derived from a lddt distance matrix (calculated by FoldTree 65 , powered by Foldseek 64 ), scale bar indicates lddt distance (which is approximate to the inverse of the pairwise lddt score). Main clades and lineages are labelled as in Figure 2 . B) Examples of aligned RdRP domain structures, color-coded as stated in the label, lineage identifiers (e.g. b vs w) indicate the position of the compared structures on the tree. lddt values represent structural similarity, with values of 1 being perfectly aligned identical structures. BVDV, bovine viral diarrhea virus. Example structural alignments of clade representatives corroborate their distribution on the tree ( Fig. 5B ). Dengue virus 1 and HCV genotype 1 were at opposite ends of the tree (clade I and III) and their respective RdRPs align with a relatively low lddt score (0.47), whereas pairings within clade I, II and III give higher scores: 0.67, 0.61 and 0.74 respectively (note that an lddt score of 1.0 represents perfect alignment of identical structures). Thus, inferring evolutionary relatedness through structure-only analysis corroborates sequence-based approaches and is highly supportive of the organization of flaviviruses and ‘flavi-like’ viruses into four main clades. Alignment-free hidden Markov model homology analysis supports partition of flaviviruses and ‘flavi-like’ viruses into four main clades Genome Relationships Applied to Virus Taxonomy (GRAViTy) is a non-supervised, alignment free method to assess the relatedness of virus genome sequences though calculation of protein profile hidden Markov model (PPHMM) homologies and through metrics of genome organization such as the order and orientation of genes 66 . We performed GRAViTy analysis using our flavivirus and ‘flavi-like’ virus dataset (Table S1) for phylogeny and RdRP structure comparisons; genomes of segmented ‘jingmenviruses’ were concatenated in order of segment length from short-to-long. Results were remarkably concordant with those determined by RdRP and helicase domain phylogenies ( Figs. 2 – 4 ), with bootstrap-supported segregation of the same sequences into three main clades I–III ( Fig. 6A ). ‘Flavi-like’ viruses from environmental samples (formed an outlier position as clade IV. Download figure Open in new tab Download figure Open in new tab Fig. 6. Alignment-free hidden Markov model homology analysis supports partition of flaviviruses and ‘flavi-like’ viruses into one order and four family rank clades A)GRAViTy Jaccard distances calculated for classified flaviviruses and ‘flavi-like viruses and a representative member of each established RNA virus family in ribovirian kingdom Orthornavirae (n=135), showing approximate demarcation thresholds for orders and families (dashed vertical red lines). Bootstrap support values (10 iterations) are shown in red if ≥70%. A copy of the figure with the branches individually labelled is provided as Fig. S3. B) Heatmap and dendrogram depicting relationships among classified flaviviruses and ‘flavi-like’ viruses. Clades I–IV identified in the RdRP phylogeny ( Fig. 2 ) were added to equivalent branches in dendrogram. Bootstrap support values (10 iterations) for deeper branches are shown in red if ≥70%. A copy of the figure with the branches individually labelled is provided as Fig. S2.s To depict the wider inter-relationships of classified flaviviruses and ‘flavi-like’ viruses, we compared their sequences with sequences representing viruses of all other established ribovirian families. Flaviviruses and ‘flavi-like’ viruses were monophyletic in the GRAViTy dendrogram, supporting their assignment to a common higher taxonomic rank, the established order Amarillovirales ( Fig. 6B ). The Jaccard distances calculated by GRAViTy do not provide a precise quantitative estimate of evolutionary distance or thresholds for taxonomic assignments. However, ribovirian families differ from each other in the distance range of 0.7–0.85, whereas viruses within families typically are associated with Jaccard distances of approximately 0.9 ( Fig. 6B ). Although very general, the distances between clades I–IV were within the range of between-family distances elsewhere in the dendrogram, whereas their combined grouping occurs at the level of order assignments for other virus families assigned to orders (e.g., Nidovirales and Picornavirales ). Taxonomic groupings correlate with genome properties and host range Within clade I, lineage Ib contains the currently classified members of the Orthoflavirus genus, as well as a high number of currently unclassified primarily insect-specific flaviviruses (ISFs). Lineage Ic is similarly populated by ISFs, including the current unclassified cell fusing agent virus (CFAV), consistent with a previous proposed assignment to a new genus within the Flaviviridae 67 , 64 , 66 . The similarly divergent Tamana bat virus (TABV) falls in the very diverse lineage If, that also contains ‘flavi-like’ viruses infecting lumpfish ( Cyclopterus_lumpus ) and the pygmy squid ( Xipholeptos notoides ). The segmented JMTV and Guaico Culex virus (GCuV) and their relatives have been assigned to lineages Ik and Il, adjacent to lineage Ij containing the non-segmented infectious precocity virus. Extending the host range of this clade were Cnidaria flavivirus and Harrimaniidae flavivirus infecting basal metazoa such as jellies and acorn worms 33 in lineage Ia. Despite the evident lineage (and host) diversity, clade I is clearly monophyletic with a bootstrap-supported long branch separating members from other clades. Clade II similarly sub-divides into a number of bootstrap-supported lineages IIm – IIs, with currently classified pestiviruses clustering exclusively in lineage IIo along with currently unassigned more divergent viruses exclusively infecting vertebrates, as does lineage IIn (in fish). Viruses assigned to other lineages within clade II are arthropod-hosted with the striking exception of the plant-infecting Apis flavivirus, carrot flavi-like virus 1, Gentian Kobu-sho-associated virus, Coptis virus 1 and Sonchus virus 1 that form a sub-group, termed ‘koshoviruses’, within lineage IIs 48 , 49 , 68 . Arthropod and plant-infecting members of clade II frequently showed substantially longer genomes than the vertebrate-infecting members of lineages IIn and IIo (ranges 11038–11450 and 11555–15154 respectively) compared with lineage IIm (20432–22622), IIq (13599–18696), IIr (14731–26314) and IIs (18749–27708). Members of clade III were all non-segmented, possessed similar genome lengths (8294–12290) and formed a well-defined separate grouping from other flaviviruses. In contrast to the wide host range of clade I and II, all clade III members had presumed or demonstrated vertebrate hosts, spanning a wide range of mammals, birds, reptiles, and bony and cartilaginous fish. Two lineages, IIIu and IIIw, contained currently classified members of the Pegivirus and Hepacivirus genera along with a range of more divergent viruses with a greater host range beyond mammals and birds. Genomic relationships between flaviviruses and ‘flavi-like’ viruses provides a basis for their classification The reproducible phylogenetic relationships between flaviviruses and ‘flavi-like’ viruses using different tree construction methods in the RdRP domain sequences ( Fig. 2 & 3 ) were recapitulated by protein structure-guided analysis ( Fig. 5 ). RdRP is evidently co-evolving with the helicase gene ( Fig. 4 ), suggesting that the essential replicase of flaviviruses and ‘flavi-like’ viruses traces a single coherent evolutionary history. This is also reflected in the relationships identified by an alignment-free method for analyzing whole genome sequences ( Fig. 6 ). Therefore, we are confident that the foundational RdRP phylogeny in Figure 2 provides a robust framework for a genomics-based re-classification of flaviviruses. These analyses based on clade and lineage relationships provide a framework that might map clades and lineages onto families and genera. However, there are quite variable branch lengths within lineages ( Fig. 2 ) and differences in thresholds that split lineages in BEAST and UPGMA tree across clades I, II and III ( Fig. 3 ). These observations indicate that a purely cladistic classification may not conform to specific sequence divergence thresholds that are often used elsewhere in virus taxonomy. Indeed, formal comparison of mean amino acid sequence identities between and within lineages in the three clades showed considerable variation (Fig. S4; Suppl. Data), particularly in clade I where sequence identities between lineages Ia, Ib, Ic, and Id were all much greater than within-group values of other lineages (notably If, Ih, Ii and Ij, and lineage n in clade II). For classification purposes, it might be considered that these four lineages were assigned as a group equivalent to those formed in other lineages; this would create a higher threshold in the UPGMA and BEAST trees (blue dotted line in Figs. 3B and 3C ) that is more consistent with between lineage thresholds in clades II and III. With this caveat, we can make some tentative proposals for the re-classification of flaviviruses that accommodates the large number of additional ‘flavi-like’ viruses described since the last classification of the family 1 ( Table 1 ). The following findings were considered in the design of a new flavivirus taxonomy: Flavivirus and ‘flavi-like’ viruses form a monophyletic group in comparison with all other members of the Riboviria , enabling their assignment to a single taxonomic rank. Although not precise, divergence among flaviviruses at this rank was comparable to that of members of virus orders in GRAViTy analysis ( Fig. 5B ). We therefore propose that all flavivirus and ‘flavi-like’ viruses can be assigned to the established order Amarillovirales . The level of sequence divergence among members of clades I–IV was comparable to inter-family distances of other RNA viruses on GRAViTy analysis, and we therefore suggest assignment of members of lineages I, II, and III to the new families. We provisionally name these Flaviviridae , ‘ Pestiviridae ’, and ‘ Hepaciviridae ’, reflecting the historical key virus members of these families. While a separate, bootstrap-supported lineage IV was consistently observed, its members are highly divergent genetically and derive from environmental samples. Where hosts are suspected, these are extremely diverse and require further verification. We therefore do not suggesting creating a family for lineage IV at this time. All three suggested families contain a number of bootstrap-supported clades of sequences, often with distinct genome organisations, lengths and host tropisms. We suggest that the clade assignments can be used for genus demarcation in certain circumstances: Clades Ib, IIo, IIIu, and IIIw contain currently classified flaviviruses and should be assigned in the revised taxonomy. We suggest that the current genus names Orthoflavivirus , Pegivirus , and Hepacivirus are retained, with Pestivirus modified to ‘ Orthopestivirus ’ (because of the proposed Pestiviridae family name) and Hepacivirus modified to ‘ Orthohepacivirus ’ (because of the proposed Hepaciviridae family name) to maintain continuity with established nomenclature while acknowledging the greatly expanded diversity of viruses assigned to each. Criteria for designating further genera include: Bootstrap-supported groupings consistent with multiple methods A minimum of three members so that the extent of grouping can be assessed Priority should be given to clades containing previously described and well-characterised viruses, such as cell fusing agent virus (CFAV) and Tamana bat virus (TABV). On this basis, we propose the creation and naming of a total of 10 additional genera in addition to the four currently assigned ( Table 1 ). An alternative classification within clade I would assign members of lineages Ia, Ib, Ic, and Id to the same genus to provide greater comparability with genetic divergence thresholds between genera elsewhere. The name Orthoflavivirus could then be applied to a greatly expanded range of viruses including CFAV, while its component lineages could be assigned as the subgenera ‘ Euflavivirus ’ (true flaviviruses), ‘ Crangovirus ’, and iFusivirus ’. Discussion The RdRP domain is considered the “hallmark gene” for the classification of RNA viruses, primarily because comparison of RdRP sequences provided evidence for their monophyletic origin separate from all known cellular polymerases 56 , 69 . Using the RdRP phylogeny as the reference point, the evolutionary history of flaviviruses and ‘flavi-like’ viruses has evidently been punctuated by multiple genome re-organizations, expansions, exchange of structural sequence modules, and genome segmentation 13 , 50 , 60 . The continuous discovery of ‘flavi-like’ viruses in highly divergent hosts means that it is no longer possible to use the criteria of genome length, coding strategy and host range that had been used for taxonomic placement of currently classified flaviviruses. While the evolutionary history of these virus could be more completely and better represented as a multi-dimensional network or reticulate tree, the requirement of all biological taxonomies for a hierarchical classification necessitates selection of a common marker gene present in all clades. Thus, the confirmed grouping of already classified flaviviruses and ‘flavi-like’ viruses into established order Amarillovirales and their suggested assignment to three families and 14 genera ( Table 1 ) is based on the phylogeny of RdRP. Other genome regions exhibit much more horizontal gene transfer, resulting in distinct evolutionary histories that may even originate from cellular life (e.g., E rns , found in the pestivirus and ‘LGF’, is of bacterial origin 60 ); clearly, organization based on these genomic regions would confound taxonomic classification. We observed a primary division into four clades, all of which are bootstrap-supported by each of the methods we used. The established genus assignments were replicated in these analyses, but there is a much closer relationship of hepaciviruses and pegiviruses than between or to the other genera. Indeed, hepaciviruses and pegiviruses cluster in one main clade (III), ‘LGFs’ cluster with pestiviruses in another (II), whereas ‘jingmenviruses’, orthoflaviviruses, and ‘tamanaviruses’ cluster in another (I). There is no pre-defined level or evolutionary distance range in the RdRP phylogeny (or of hallmark genes elsewhere in the ICTV taxonomy) that dictate family rank assignments. However, a threshold Jaccard distance level of 0.8–0.85 drawn through the amarilloviral grouping reproduces family rank assignments elsewhere in realm Riboviria ( Fig. 6B ). Consequently, splitting the current family Flaviviridae into three families is consistent with degrees of relatedness between and within other classified RdRP-encoding RNA virus families. This split results in the removal of genera Hepacivirus , Pegivirus , and Pestivirus from family Flaviviridae , which would be restricted to genus Orthoflavivirus and novel genera for ‘flavi-like’ viruses. The suggested family ‘ Pestiviridae ’ would absorb genus Pestivirus but also includes the multiple and highly divergent clades of ‘LGFs’. Mammalian pestiviruses possess a type IV IRES 70 , 71 and major envelope proteins that are structurally homologous to those of hepaciviruses and pegiviruses 60 . However, unclassified pesti-like viruses of spiders (lineage IIm) may use cap-dependent translation and encode envelope proteins structurally unrelated to those of the originally assigned mammalian pestiviruses 60 . ‘LGFs’ are far more diverse genetically in the RdRP sequence than those of viruses in the other suggested families. However, lowering the family rank assignment threshold would create up to five or more new families of ‘pesti-like’ viruses, a step we consider inappropriate at least until these viruses are better characterized. The remainder of flaviviruses and ‘flavi-like’ viruses group in a deep and phylogenetically well-defined third family, ‘ Hepaciviridae ’. The depth of grouping provides no support for the formation of separate families for hepaciviruses and pegiviruses. The addition of recently described ‘hepaci-like’ or ‘pegi-like’ viruses infecting fish has greatly expanded the genetic diversity of both groups and blurs the originally clear distinction between them. Generally, however, the apparent absence of a capsid-encoding sequence in pegiviruses, indicative of a likely radically different virion structure (or conceivably undetected segmentation of the pegivirus genome), differentiates pegiviruses from hepaciviruses at least for now. The analysis provides the basis for assignments of new genera within each of the three new families ( Table 1 ), where they are supported into genetically well-defined groups and have some commonality in phenotypic properties, such as host range, and of genome organisation. The proposed additional ten taxa, or alternatively into eight genera and three subgenera, represent obvious candidates for classification by these criteria. However, additional assignments may be made in the future pending collection of further characterised ‘flavi-like’ viruses in ongoing clinical, veterinary and entomology screens, and in metagenomic data from the widening range of invertebrate species. Overall, we believe we have made a robust case for an evolutionarily-based reclassification of flaviviruses using an approach that puts primacy on genetic relationships of the hallmark RdRP gene. The seeming propensity of flaviviruses and potentially other RNA viruses to undergo radical changes in genome organisation such as segmentation, changes to translation mechanisms and exchanges of structural gene modules indeed reinforces the need to base classification and inference on evolutionary histories of the most stable elements within the genome. The use of protein structure relationships of RdRP and potentially other replication-associated enzymes provides an exciting new method to determine deeper evolutionary histories of RNA viruses beyond the level of family and order analysed in the current study. Methods Genome sequences A comprehensive set of coding-complete genome sequences representing the 97 currently established flavivirus species 11 , supplemented with ‘flavi-like’ viruses with analyzed previously 60 was used as the basic set for analysis and putative taxon assignments (Table S1; File S1). Genome regions encoding the RNA-directed RNA polymerase (RdRP) and helicase domains were extracted for amino-acid sequence deduction, alignment, and analysis. Sequence alignment and phylogenetic analysis Alignment and trimming of RdRP domain amino-acids sequences with TrimAI 72 was performed using multiple methods and conservation thresholds as previously described 60 . The phylogeny of sequences in the final alignment was reconstructed by the maximum likelihood-based IQ-TREE program version 1.6.12 73 with an empirically determined optimal model, Lascuel + F + 10 rate categories (LG+F+R10) selected based on the minimum Bayesian information criteria (BIC) score 74 . Robustness of branching was estimated by bootstrap resampling (1,000 replicates) 75 . An unrooted tree with bootstrap support values shown for the main clades and lineages was plotted using MEGA7.0 76 . The RdRP domain amino-acid sequence dataset was analyzed in parallel by the unweighted pair group method with arithmetic mean (UPGMA) as implemented in MEGA7.0, using Jones-Taylor-Thornton (JTT) matrix protein distances and 100 bootstrap replicates. A temporal reconstruction of amarilloviral evolution was performed using BEAST version 10.05 using dated sequences based on sample date (or International Nucleotide Sequence Database Collaboration [INSDC] submission date if this information was not annotated; n=143), using uniform rate, constant population size, and BLOSUM protein distances as priors. A maximum likelihood phylogenetic tree of helicase-encoding sequences was generated similarly using IQ-TREE, with the (lowest BIC) substitution model. The alignment was obtained with MAFFT 77 and trimmed using TrimAI 72 and the gappyout option. RdRP and helicase domain trees were compared using Tanglegram 78 , 79 . Potyviruses, poxvirus, and DEAH helicase family sequences were used to root the tree. Genome Relatedness Applied to Virus Taxonomy (GRAViTy) Genome relatedness of flaviviruses and ‘flavi-like’ viruses to all currently classified ribovirians was performed using the GRAViTy version 2 implementation 66 of the original algorithm 80 . Analysis was performed in a single step, using the new classification function. Default parameters were used, except for initial translated open reading frame sequence clustering inflation (6.0), translated open reading frame alignment method (G-INS-I), and protein profile hidden Markov model (PPHMM) similarity cutoff hitscore. Taxonomic assignments were bootstrapped with 10 iterations, using the sumtrees method. RdRP structure comparisons We started with a previous dataset of Flaviviridae protein structure predictions ( https://zenodo.org/records/11092288 ) 60 . This covers all viruses examined in the current study, with their respective polyproteins sequences broken into sequential 300 residue blocks (overlapping by 100 residues) for protein structure prediction with ColabFold and ESMFold 63 , 81 , 82 . The fragmented nature of these structures was insufficient for accurate structural alignment, therefore we queried the dataset against experimental RdRP structure references using Foldseek 64 : orthoflavivirus PDB:5F3Z, pestivirus PDB:5YF6 and hepacivirus PDB: 1C2P) 83 – 85 , enabling us to extract continuous RdRP domain sequences for all viruses. Structures were predicted for these RdRP sequences using ColabFoldv1.5.5 82 , taking the highest confidence model from five predictions. These structures were filtered for average local distance difference test (lddt) prediction confidence using a semi-arbitrary cut-off of ≥80%. We also discarded structures below 400 residues in length, reasoning that heavily truncated structures may be misinformative. This yielded a final structure dataset for this study containing 400 flavivirus and ‘flavi-like’ virus RdRP structures. RdRP structures were analysed using FoldTree to produce a structure-guided tree based on the lddt structural similarity metric, which provides a measure of structural similarity whilst accommodating for some structural flexibility 86 . In short, FoldTree 65 performs an all-vs.-all structure comparison, driven by Foldseek 64 , to derive pairwise lddt values that are used to calculate a distance matrix and derive a Neighbour-Joining tree via QuickTree 87 . The tree was visualised and prepared for publication using iTOL 88 . Example structural models in Figure 5b were aligned for visualization using flexible FATCAT 89 , models were viewed and prepared for publication using UCSF ChimeraX 90 . Resource Availability Lead Contact Peter Simmonds Materials availability Not applicable, no biological materials were used in the study Data and code availability Databases, sequence alignments and raw sequence distance data provided in Suppl. Data. Correspondence and requests for materials should be addressed to PS / JCOM (RdRP sequence analysis), JG (RdRP structure analysis, RM (GRAViTy analysis) or AB (helicase analysis). Author contributions PS, JHK, and members of the original ICTV Study Group (MB, JFD, AK, VL, JS and NV) conceived the study. PS, AB, JG, RM, JCOM and JHK conceptualized the experimental section. PS, AB, JG, RM and JCOM performed analyses. All authors wrote/revised the manuscript and PS supervised the work. All authors read and approved the manuscript. Declaration of interests All authors declare no competing interests. Supplementary information The online version contains supplementary material comprising: Fig. S1. Helicase domain (NS3) amino-acid sequence phylogeny supports partition of flaviviruses and ‘flavi-like’ viruses into one order and four family level clades. Fully branch-labeled version of Fig 4 . Fig. S2. Alignment-free hidden Markov model homology analysis supports partition of flaviviruses and ‘flavi-like’ viruses into one order and four family level clades. Fully branch-labeled version of Fig 6A . Fig. S3 . Alignment-free hidden Markov model homology analysis supports partition of flaviviruses and ‘flavi-like’ viruses into four main clades. Fully branch-labeled version of Fig 6B .’ Fig. S4 . Mean pairwise amino acid sequence identities between lineages in clades I–III. File S1 - RdRP_tree.NWK File S2 - GRAViTyv2 Run parameters.json File S3 - RdRP_lddt_structure tree.NWK Acknowledgements The authors thank Anya Crane (Integrated Research Facility at Fort Detrick/National Institute of Allergy and Infectious Diseases/National Institutes of Health, Fort Detrick, Frederick, MD, USA) for critically editing the manuscript. This work was supported in part through a Laulima Government Solutions, LLC, prime contract with the National Institute of Allergy and Infectious Diseases (Contract No. HHSN272201800013C). J.H.K. performed this work as an employee of Tunnell Government Services (TGS), a subcontractor of Laulima Government Solutions, LLC, under Contract No. HHSN272201800013C. NV acknowledges partial support from the Centers for Research in Emerging Infectious Diseases (CREID) Coordinating Research on Emerging Arboviral Threats Encompassing the NEOtropics (CREATE-NEO) 1U01AI151807 grant by the National Institutes of Health (NIH). A.B. is supported by a postdoctoral fellowship from Foundation pour la Recherche Mèdicale (grant number SPF202110014092). JG was supported by a Wellcome Trust/Royal Society Sir Henry Dale Fellowship (107653/Z/15/Z) and MRC-University of Glasgow Centre for Virus Research core support from the Medical Research Council (MC_UU_00034/1). JTS was supported by Veterans Administration Merit Review BX000207 and VA SEQCure Network grants. be interpreted as necessarily representing the official policies, either expressed or implied, of the U.S. Department of Health and Human Services or of the institutions and companies affiliated with the authors, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. 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