Virus TaxonomyThe ICTV Report on Virus Classification and Taxon Nomenclature

Flaviviridae Chapter

Flaviviridae

Rebecca Rico-Hesse, Tatjana Avsic-Zupanc, Bradley Blitvich, Jens Bukh, Van-Mai Cao-Lormeau, Allison Imrie, Amit Kapoor, Laura DKramer, Brett D Lindenbach, Peter Simmonds, Donald B. Smith and Pedro Fernando da Costa Vasconcelos

Edited by Nick J. Knowles and Stuart G. Siddell

Corresponding author: Rebecca Rico-Hesse (rebecca.rico-hesse@bcm.edu)

Posted January 2017, updated February 2019

PDF created: October 2020

Citation

A summary of this ICTV Report chapter has been published as an ICTV Virus Taxonomy Profile article in the Journal of General Virology, andshould be cited when referencing this online chapter as follows:

Simmonds, P., Becher, B., Bukh, J., Gould, E.A., Meyers, G., Monath, T., Muerhoff, S., Pletnev, A., Rico-Hesse, R., Smith, D.B., Stapleton,J.T., and ICTV Report Consortium. 2017, ICTV Virus Taxonomy Profile: Flaviviridae, Journal of General Virology, 98:2–3.

Summary

The Flaviviridae is a family of small enveloped viruses with positive-sense RNA genomes of approximately 9.0 –13 kb (Table 1.Flaviviridae).Most infect mammals and birds, and many are host-specific and pathogenic, such as hepatitis C virus (HCV) in the genus Hepacivirus. Mostmembers of the genus Flavivirus are arthropod-borne, and many are important human and veterinary pathogens (e.g., yellow fever virus,dengue virus, West Nile virus).

Table 1.Flaviviridae. Characteristics of members of the family Flaviviridae.

Characteristic Description

Typicalmember yellow fever virus-17D (X03700), species Yellow fever virus, genus Flavivirus

Virion Enveloped, 40–60 nm virions with a single core protein (except for genus Pegivirus) and 2 or 3 envelope glycoproteins

Genome 9.0–13 kb of positive-sense, non-segmented RNA

Replication Cytoplasmic, in membrane vesicles derived from the endoplasmic reticulum (ER); assembled virions bud into the lumen ofthe ER and are secreted through the vesicle transport pathway

Translation Directly from genomic RNA containing a type I cap (genus Flavivirus) or an internal ribosome entry site (other genera)

Host range Mammals (all genera); most members of genus Flavivirus are arthropod-borne

Taxonomy Four genera containing 89 species

Flavivirus. Most members of this genus, which includes 53 species, are arthropod-borne viruses, with distinct groups infecting mosquitoes orticks. Mammals and birds are the usual primary hosts, in which infections range from asymptomatic to severe or fatal haemorrhagic fever orneurological disease. Important human pathogens include yellow fever virus, dengue virus, Zika virus, Japanese encephalitis virus, West Nilevirus and tick-borne encephalitis virus. Other members cause economically important diseases in domestic or wild animals. Additional virusesinfect only arthropods or only mammals (e.g., Tamana bat virus).

Pestivirus. Pestiviruses infect pigs and ruminants, including cattle, sheep, goats and wild ruminants, and are transmitted through contact withinfected secretions (respiratory droplets, urine or faeces). Infections may be subclinical or cause enteric, haemorrhagic or wasting diseases,including the economically important bovine viral diarrhoea virus and classical swine fever virus. Additional pestiviruses of unknownpathogenicity infect bats and rats.

Hepacivirus. This genus includes hepatitis C virus (HCV), a major human pathogen causing chronic liver disease, including cirrhosis andcancer. Other viruses in the genus are of unknown pathogenicity and infect horses, rodents, bats, cows and primates. Infections are typically

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persistent and target the liver.

Pegivirus. Members of the genus Pegivirus are associated with persistent infections of a wide range of mammalian species. They have notbeen clearly associated with disease.

Virion

Morphology

Virions are 40–60 nm in diameter, spherical in shape with a lipid envelope. The capsid is comprised of a single protein and the envelopecontains two or three virus-encoded membrane proteins. Specific descriptions of members of the four individual genera are given in thecorresponding genus pages.

Physicochemical and physical properties

The virion Mr, buoyant density, sedimentation coefficient and other physicochemical properties differ among the members of the genera andare described separately in the corresponding genus pages.

Nucleic acid

Genomes are positive-sense ssRNA of approximately 9.2–11.0, 11.3–13.0, 8.9–10.5 and 8.9–11.3 kb for members of the genera Flavivirus,Pestivirus, Hepacivirus and Pegivirus, respectively. All members of the family lack a 3′-terminal poly(A) tract. Only the genomes of membersof the genus Flavivirus contain a 5′-terminal type I cap structure, the others possess an internal ribosomal entry site (IRES).

Proteins

Virions of members of the family have a single, small basic capsid (C) and two ( Flavivirus, Hepacivirus and Pegivirus) or three (Pestivirus)membrane-associated envelope proteins. Pegiviruses appear to lack a complete nucleocapsid protein gene. The nonstructural proteinscontain sequence motifs characteristic of a serine protease, RNA helicase and RNA-dependent RNA polymerase (RdRP) that are encoded atsimilar locations along the genome in all genera. Further details of specific functional properties are given in the corresponding sections of theindividual genera pages.

Lipids

Lipids present in virions are derived from host cell membranes and make up 17% of the total virion weight in the case of members of thegenus Flavivirus. The lipid content of pestiviruses, hepaciviruses and pegiviruses is unknown.

Carbohydrates

Virions contain carbohydrates in the form of glycolipids and glycoproteins.

Genome organization and replication

The genomic RNA of all members of the family has a similar organization and is the viral mRNA found in infected cells. It contains a singlelong open reading frame (ORF) flanked by 5′- and 3′-terminal non-coding regions (NCRs) that form specific secondary structures required forgenome replication and translation. Members of the genus Flavivirus, but not pestiviruses, hepaciviruses or pegiviruses produce a unique,subgenomic, small (300–500 nt) non-coding RNA that is derived from the 3′-NCR of genomic RNA (Lin et al., 2004) that is essential for virusreplication in cells and modulates pathogenicity in animals. Translation-initiation of genomic RNA is cap-dependent for members of the genusFlavivirus, whereas IRES elements are present in viruses of the other genera. Viral proteins are synthesized as part of a polyprotein that is co-and post-translationally cleaved by viral and cellular proteases. The structural proteins are contained in the N-proximal portion of thispolyprotein and the nonstructural proteins in the remainder. The latter include a serine protease, an RNA helicase and the RdRP. Genomereplication occurs in the cytoplasm in association with modified cellular membranes via the synthesis of genome-length negative-strandintermediates. Virion assembly, including acquisition of a glycoprotein-containing lipid envelope, occurs by budding through intracellularmembranes. Viral particles are transported in cytoplasmic vesicles through the secretory pathway before they are released by exocytosis, asshown for members of the genus Flavivirus and assumed for members of the other genera. In addition, release of infectious RNA viaexosomes has recently been demonstrated (Ramakrishnaiah et al., 2013).

Biology

The biological properties of viruses in the four genera exhibit different characteristics and are described in the corresponding sections of thegenus pages.

Antigenicity

The viruses of different genera are antigenically unrelated, but serological cross-reactivity exists among members within each genus.

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Derivation of names

Flavi: from Latin flavus, “yellow”.

Pesti: from Latin pestis, “plague”.

Hepaci: from Greek hepar, hepatos, “liver” and identifying letter from hepatitis C virus

Pegi: from persistent, and the original names of the GB viruses and hepatitis G, deriving from the initials of the original source, the surgeon“GB”

Relationships within the family

Phylogenetic relationships of amino acid sequences in a conserved domain of the RdRP show clustering of members of the Flaviviridae intothe four currently assigned genera, although there is a closer phylogenetic relationship between members of the Hepacivirus and Pegivirusgenera than between others (Figure 1.Flaviviridae). Another exception is the outlier position of Tamana bat virus, currently listed as a potentialmember of the Flavivirus genus, but sufficiently distinct to potentially merit assignment into a new genus, should further related viruses befound in the future.

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Figure 1.Flaviviridae. Phylogeny of conserved amino acid sequences in the RdRP (NS5 or NS5B) of members of the family Flaviviridae.Partial gene sequences between positions 8,040–8,897 (numbered using positions in the HCV sequence, AF011751) from representativeisolates of each species and from several related unclassified viruses were aligned as inferred amino acid sequences using MUSCLE(Edgar 2004) and verified by the presence of aligned motifs. An unrooted phylogenetic tree was constructed from the sequence alignmentby maximum likelihood using an empirically determined optimal substitution model – Le Gascuel 2008 with a gamma distribution (5categories) and invariant sites (LG + G+I) computed with the MEGA version 6.1 package (Tamura et al., 2013). Data was bootstrap re-sampled 100 times; values of >=70% are shown next to the branches. This phylogenetic tree and corresponding sequence alignment areavailable to download from the Resources page.

Relationships with other taxa

Members of the Flaviviridae have been placed into RNA virus supergroup II, a group that also includes members of the Tombusviridae (plant),members of the Luteovirus genus in the Luteoviridae (plant), Leviviridae (bacterial virus) and a series of recently described insect-derivedflavi-like viruses, many with segmented genomes (Shi et al., 2015). However, the virion structure and other viral structural and nonstructuralgenes in these other virus groups are distinct and likely non-homologous.

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Genus: Flavivirus

Distinguishing features

The 5′-end of the genome possesses a type I cap (m GpppAmp) not seen in viruses of the other genera. Most flaviviruses are transmitted tovertebrate hosts by arthropod vectors, mosquitoes or ticks, in which they replicate actively. Some flaviviruses transmit between rodents orbats without known arthropod vectors.

Virion

Morphology

Virions are 50 nm in diameter and spherical in shape (Figure 1.Flavivirus). Two virus forms can be distinguished. Mature virions contain twovirus encoded membrane-associated proteins, E and M. Intracellular immature virions contain the precursor prM, which is proteolyticallycleaved into M during maturation (Stadler et al., 1997). In certain instances, partially mature/immature forms are also released from infectedcells. The virion structures of dengue virus (DENV) and West Nile virus (WNV) have been determined by X-ray crystallography (Kuhn et al.,2002, Mukhopadhyay et al., 2003). The envelope protein, E, is a dimeric, rod-shaped molecule that is oriented parallel to the membrane anddoes not form spike-like projections in its neutral pH conformation (Yu et al., 2008). Image reconstructions from cryo-electron micrographs(Figure 1.Flavivirus) have shown that the virion envelope has icosahedral symmetry, in which E protein dimers are organized in aherringbone-like arrangement.

Figure 1.Flavivirus. Three-dimensional cryo-electron microscopic reconstructions of immature (left) and mature (right) particles of anisolate of dengue virus (courtesy of M. Rossmann). Shown is a surface rendering of immature dengue virus at 12.5Å resolution (left) andmature dengue virus at 10Å resolution (right). The viruses are depicted to scale, but not coloured to scale. Triangles outline one icosahedralunit.

Physicochemical and physical properties

Virion Mr has not been precisely determined. Mature virions sediment at about 200S and have a buoyant density of about 1.19 g cm insucrose (Kokorev et al., 1976). Viruses are stable at slightly alkaline pH 8.0 but are readily inactivated by exposure to acidic pH, temperaturesabove 40 °C, organic solvents, detergents, ultraviolet light and gamma-irradiation.

Nucleic acid

The virion RNA of flaviviruses is a positive-sense infectious ssRNA of 9.2–11.0 kb. The 5′-end of the genome possesses a type I cap (m-7GpppAmp) where the A is followed by a highly conserved G nucleotide. The 3′-ends lack a terminal poly(A) tract and terminate with theconserved dinucleotide CU.

Proteins

Virions contain three structural proteins: capsid (C, 11 kDa), the major envelope protein (E, 50 kDa), , and either prM (26 kDa), in immaturevirions, or M (8 kDa), in mature virions. The E protein is the viral haemagglutinin, which mediates both receptor binding and acid pH-dependent fusion activity after uptake by receptor-mediated endocytosis. Seven nonstructural proteins are synthesized in infected cells: NS1(46 kDa), NS2A (22 kDa), NS2B (14 kDa), NS3 (70 kDa), NS4A (16 kDa), NS4B (27 kDa) and NS5 (103 kDa). Some members of the genusharbour sequences that appear to induce a proportion of translating ribosomes to shift -1 nt and continue translating in the new reading frameto produce a 'transframe' fusion protein (Firth and Atkins 2009). When functionally utilized, this is referred to as programmed-1 ribosomalframeshifting (-1 PRF). NS1 has multiple forms and roles, with a cell-associated form functioning in viral RNA replication and a secreted formthat regulates complement activation. One such form, a NS1′ protein, is the product of a −1 ribosomal frameshift and plays a role in viral

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neuroinvasiveness (Melian et al., 2010). The N-terminal one-third of NS1 forms the viral serine protease complex together with NS2B that isinvolved in processing the polyprotein. The C-terminal portion of NS3 contains an RNA helicase domain involved in RNA replication, as wellas an RNA triphosphatase activity that is probably involved in formation of the 5′-terminal cap structure of the viral RNA. NS5 is the largestand most highly conserved protein that acts as the viral RdRP and also possesses methyltransferase activity involved in the modification ofthe viral cap structure.

Lipids

Virions contain about 17% lipid by weight; lipids are derived from host cell membranes.

Carbohydrates

Virions contain about 9% carbohydrate by weight (glycolipids, glycoproteins); their composition and structure are dependent on the host cell(vertebrate or arthropod). N-glycosylation sites are present in the proteins prM (1 to 3 sites), E (0 to 2 sites) and NS1 (1 to 3 sites).

Genome organization and replication

The genomic RNA represents the only viral messenger RNA in infected cells. It consists of a single long ORF of more than 10,000 nt thatcodes for all structural and nonstructural proteins and is flanked by NCRs at the 5′- and 3′-terminal ends (Figure 2.Flavivirus).

Figure 2.Flavivirus. Flavivirus genome organization (not to scale) and polyprotein processing. The virion RNA is about 11 kb. At the top isthe viral genome with the structural and nonstructural protein coding regions and the 5′- and 3′-NCRs. Boxes below the genome indicateviral proteins generated by the proteolytic processing cascade. P, H, and R symbols indicate the localization of the NS3 protease, the NS3RNA helicase, and the NS5 RdRP domains, respectively.

Both the 5′-NCR and the 3′-NCR contain RNA sequence motifs that are involved in viral RNA translation, replication and possibly packaging.Although RNA secondary structure and function of some elements are conserved, sequence composition, length and exact localization canvary considerably between different members of the genus, in particular between tick-borne and mosquito-borne flaviviruses. In some cases,the 3′-NCR of tick-borne encephalitis virus, for example, contains an internal poly(A) tract. Viral infection induces dramatic rearrangements ofcellular membrane structures within the perinuclear endoplasmic reticulum (ER) and causes the formation of ER-derived vesicular packetsthat most likely represent the sites of viral replication. After translation of the incoming genomic RNA, RNA replication begins with synthesis ofcomplementary negative-strands, which are then used as templates to produce additional genome-length positive-stranded molecules. Theseare synthesized by a semi-conservative mechanism involving replicative intermediates (containing double-stranded regions as well asnascent single-stranded molecules) and replicative forms (duplex RNA molecules). Translation usually starts at the first AUG of the ORF, butmay also occur at a second in-frame AUG located 12 to 14 codons downstream in mosquito-borne flaviviruses. The polyprotein is processedby cellular proteases and the viral NS2B-NS3 serine protease to give rise to the mature structural and nonstructural proteins. Protein topologywith respect to the ER and cytoplasm is determined by internal signal and stop-transfer sequences. Virus particles can first be observed in therough endoplasmic reticulum, which is believed to be the site of virus assembly. These immature virions are then transported through themembrane systems of the host secretory pathway to the cell surface where exocytosis occurs. Shortly before virion release, the prM protein iscleaved by furin or a furin-like cellular protease to generate mature virions. Infected cells also release a non-infectious subviral particle thathas a lower sedimentation coefficient than whole virus (70S rather than 200S) and exhibits haemagglutination activity.

Biology

Host range

Flaviviruses can infect a variety of vertebrate species and in many cases arthropods. Some viruses have a limited vertebrate host range (e.g.,only primates), while others can infect and replicate in a wide variety of species (mammals, birds, etc.). The usual route of infection forarthropods is when they feed on a viraemic vertebrate host, but non-viraemic transmission between vectors has also been described for tick-borne flaviviruses. A new group of unclassified viruses in the genus, including cell fusing agent virus, appear only to infect mosquitoes, andseveral more, highly genetically distinct insect-only flaviviruses have now been identified (Blitvich and Firth 2015).

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Transmission

Most flaviviruses are arthropod-borne viruses with cycles of transmission from hematophagous arthropod vectors to vertebrate hosts. About50% of known flaviviruses are mosquito-borne, 28% are tick-borne and the remainder transmit between rodents or between bats withoutknown arthropod vectors. For some flaviviruses, the transmission cycle has not yet been identified. In certain instances, flaviviruses can betransmitted to humans by blood products, organ transplantation, non-pasteurized milk or aerosols. Some tick-borne flaviviruses are known tobe transmitted directly between ticks by a process known as non-viraemic transmission. In the arthropod vectors, the viruses may also betransmitted trans-ovarially or vertically (mosquitoes, ticks) and transstadially (ticks). The mechanisms of virus transmission involving theinsect-only flaviviruses may include vertical transmission, but other mechanisms need to be considered to explain the success with whichthese viruses have dispersed globally.

Geographical distribution

Flaviviruses have a world-wide distribution but individual species are restricted to specific endemic or epidemic areas (e.g., yellow fever virusin tropical and subtropical regions of Africa and South America; dengue virus in tropical areas of Asia, Oceania, Africa, Australia and theAmericas; Japanese encephalitis virus in Southeast Asia; tick-borne encephalitis virus in Europe and Northern Asia).

Pathogenicity

More than 50% of known flaviviruses have been associated with human disease, including many important human pathogens such as yellowfever virus, dengue virus, Zika virus, Japanese encephalitis virus, West Nile virus and tick-borne encephalitis virus. The induced diseases maybe associated with symptoms of the central nervous system (e.g., meningitis, encephalitis), fever, arthralgia, rash and haemorrhagic fever.Several flaviviruses are pathogenic for domestic or wild animals (turkey, pig, horse, sheep, dog, grouse, muskrat) and cause economicallyimportant diseases.

Antigenicity

All flaviviruses are serologically-related, which can be demonstrated by binding assays such as ELISA and by haemagglutination-inhibitionusing polyclonal and monoclonal antibodies. Neutralization assays are more discriminating and have been used to identify more closelyrelated Flavivirus serocomplexes (as indicated in Figure 1.Flaviviridae), although not down to the species level. The envelope protein E is themajor target for neutralizing antibodies and induces protective immunity. The E protein also induces flavivirus cross-reactive non-neutralizingantibodies. Antigenic sites involved in neutralization have been mapped to each of the three structural domains of the E protein. The prM andNS1 proteins can also induce antibodies that protect infected animals from lethal infection.

Species demarcation criteria

Species demarcation criteria in the genus include:

Nucleotide and deduced amino acid sequence data.Antigenic characteristics.Geographic association.Vector association.Host association.Disease association.Ecological characteristics.

Species demarcation considers a combination of each of the criteria listed above. While nucleotide sequence relatedness and the resultingphylogenies are important criteria for species demarcation, the other listed criteria may be particularly useful in the demarcation of geneticallyclosely related viruses. For example far-eastern (FE) strains of tick-borne encephalitis virus exhibit distinct ecological differences whencompared with Omsk haemorrhagic fever virus despite the fact that they are genetically relatively closely related. FE strains of tick-borneencephalitis virus are associated predominantly with Ixodes persulcatus ticks in forest environments in far-east Russia, whereas Omskhemorrhagic fever virus is found in the Steppe regions of western Siberia associated particularly with Dermacentor spp. and to a lesser extentwith Ixodes spp. These viruses are also antigenically distinguishable in neutralization tests that employ convalescent sera.

Louping ill virus and tick-borne encephalitis virus provide another example of viruses where, despite their close genetic relationships andsimilar host ranges, they display different ecologies (moorlands versus forests), pathogenicities (red grouse, sheep/goats versus humans) andgeographical distributions (UK versus Europe/Eurasia), thus justifying their classification as members of the distinct species, Louping ill virusand Tick-borne encephalitis virus .

On the other hand, the four dengue virus serotypes all belong to a single species ( Dengue virus), despite being phylogenetically andantigenically quite distinct. This is justified by the fact that they co-circulate in the same geographical areas and ecological habitats, and thatthey exploit identical vectors, exhibit similar life cycles and disease manifestations (Table 1.Flavivirus).

Table 1.Flavivirus. Flaviviruses grouped by vector and host.

Virus species Virus name Accession number Virus abbreviation

Tick-borne, mammalian hostGadgets Gully virus Gadgets Gully virus DQ235145 GGYV

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Kyasanur Forest disease virus Kyasanur Forest disease virus AY323490 KFDV Alkhumra hemorrhagic fever virus AF331718 AHFVLangat virus Langat virus AF253419 LGTVLouping ill virus Louping ill virus Y07863 LIV British subtype D12937 LIV-Brit Irish subtype X86784 LIV-Ir Spanish subtype DQ235152 LIV-Spain Turkish sheep encephalitis virus subtype DQ235151 TSEV Greek goat encephalitis virus subtype DQ235153 GGEVOmsk hemorrhagic fever virus Omsk hemorrhagic fever virus AY193805 OHFVPowassan virus Powassan virus L06436 POWV deer tick virus AF311056 DTVRoyal Farm virus Royal Farm virus DQ235149 RFVTick-borne encephalitis virus European subtype U27495 TBEV-Eur Far Eastern subtype X07755 TBEV-FE Siberian subtype L40361 TBEV-SibTick-borne, seabird hostMeaban virus Meaban virus DQ235144 MEAVSaumarez Reef virus Saumarez Reef virus DQ235150 SREVTyuleniy virus Tyuleniy virus KF815939 TYUVProbably tick-borneKadam virus Kadam virus DQ235146 KADVMosquito-borne, Aroa virus groupAroa virus Aroa virus AY632536 AROAV Bussuquara virus AF013366 BSQV Iguape virus AF013375 IGUV Naranjal virus AF013390 NJLVMosquito-borne, Dengue virus groupDengue virus Dengue virus 1 U88536 DENV-1 Dengue virus 2 U87411 DENV-2 Dengue virus 3 M93130 DENV-3 Dengue virus 4 AF326573 DENV-4Mosquito-borne, Japanese encephalitis virus groupCacipacore virus Cacipacoré virus KF917536 CPCVJapanese encephalitis virus Japanese encephalitis virus M18370 JEVKoutango virus Koutango virus AF013384 KOUVMurray Valley encephalitis virus Alfuy virus AF013360 ALFV Murray Valley encephalitis virus AF161266 MVEVSt Louis encephalitis virus St. Louis encephalitis virus DQ525916 SLEVUsutu virus Usutu virus AY453411 USUVWest Nile virus Kunjin virus D00246 KUNV West Nile virus M12294 WNVYaounde virus Yaoundé virus AF013413 YAOVMosquito-borne, Kokobera virus groupKokobera virus Kokobera virus AY632541 KOKV Stratford virus AF013407 STRVMosquito-borne, Ntaya virus groupBagaza virus Bagaza virus AY632545 BAGVIlheus virus Ilhéus virus AY632539 ILHV Rocio virus AF013397 ROCVIsrael turkey meningoencephalitis virus Israel turkey meningoencephalitis virus AF013377 ITVNtaya virus Ntaya virus JX236040 NTAVTembusu virus Tembusu virus JF895923 TMUVZika virus Zika virus AY632535 ZIKVMosquito-borne, yellow fever virus groupSepik virus Sepik virus DQ837642 SEPVWesselsbron virus Wesselsbron virus EU707555 WESSVYellow fever virus yellow fever virus X03700 YFVProbably mosquito-borne, Kedougou virus group Kedougou virus Kédougou virus AY632540 KEDVProbably mosquito-borne, Edge Hill virus group Banzi virus Banzi virus DQ859056 BANV

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Bouboui virus Bouboui virus DQ859057 BOUVEdge Hill virus Edge Hill virus DQ859060 EHVJugra virus Jugra virus DQ859066 JUGVSaboya virus Potiskum virus DQ859067 POTV Saboya virus DQ859062 SABVUganda S virus Uganda S virus DQ859065 UGSVUnknown vector, Entebbe bat virus groupEntebbe bat virus Entebbe bat virus DQ837641 ENTV Sokuluk virus AF013405 SOKVYokose virus Yokose virus AB114858 YOKVUnknown vector, Modoc virus groupApoi virus Apoi virus AF160193 APOIVCowbone Ridge virus Cowbone Ridge virus AF013370 CRVJutiapa virus Jutiapa virus KJ469371 JUTVModoc virus Modoc virus AJ242984 MODVSal Vieja virus Sal Vieja virus AF013401 SVVSan Perlita virus San Perlita virus AF013402 SPVUnknown vector, Rio Bravo virus groupBukalasa bat virus Bukalasa bat virus AF013365 BBVCarey Island virus Carey Island virus AF013368 CIVDakar bat virus Dakar bat virus AF013371 DBV Montana myotis leukoencephalitis virus Montana myotis leukoencephalitis virus AJ299445 MMLVPhnom Penh bat virus Batu Cave virus AF013369 BCV Phnom Penh bat virus AF013394 PPBVRio Bravo virus Rio Bravo virus AF144692 RBV

Member species

★ Exemplar isolate of the speciesSpecies Virus name Isolate Accession

numberRefSeqnumber Available sequence Virus

Abbrev.★ Apoi virus Apoi virus ApMAR AF160193 NC_003676 Complete genome APOIV★ Aroa virus Aroa virus BeAn 4073 AY632536 NC_009026 Complete genome AROAV

Aroa virus Aroa virus VenA-1809 AF013362 Partial genome AROAVAroa virus Bussuquara virus BeAn 4073 AF013366 Partial genome BSQVAroa virus Iguape virus SP An71686 AF013375 Partial genome IGUVAroa virus Naranjal virus 25008 AF013390 Partial genome NJLV

★ Bagaza virus Bagaza virus DakAr B209 AY632545 NC_012534 Complete genome BAGV★ Banzi virus Banzi virus SAH 366 DQ859056 NC_043110 Complete genome BANV★ Bouboui virus Bouboui virus DAK AR B490 DQ859057 NC_033693 Complete genome BOUV★ Bukalasa bat virus Bukalasa bat virus UGBP-111 AF013365 NC_043111 Partial genome BBV★ Cacipacore virus Cacipacoré virus BeAn 3276000 KF917536 NC_026623 Complete genome CPCV★ Carey Island virus Carey Island virus P70-1215 AF013368 NC_043112 Partial genome CIV★ Cowbone Ridge virus Cowbone Ridge virus W-10986 AF013370 NC_043113 Partial genome CRV★ Dakar bat virus Dakar bat virus 209 AF013371 NC_043114 Partial genome DBV★ Dengue virus dengue virus type 2 16681 U87411 NC_001474 Complete genome DENV-2

Dengue virus dengue virus type 1 45AZ5 U88536 Complete genome DENV-1Dengue virus dengue virus type 3 H87 M93130 Complete genome DENV-3Dengue virus dengue virus type 4 814669 AF326573 Complete genome DENV-4

★ Edge Hill virus Edge Hill virus YMP 48 DQ859060 NC_030289 Complete genome EHV★ Entebbe bat virus Entebbe bat virus UgIL-30 DQ837641 NC_008718 Complete genome ENTV

Entebbe bat virus Sokuluk virus LEIV-400K AF013405 Partial genome SOKV★ Gadgets Gully virus Gadgets Gully virus Aus DQ235145 NC_033723 Complete genome GGYV★ Ilheus virus Ilhéus virus Original AY632539 NC_009028 Complete genome ILHV

Ilheus virus Rocio virus H-34675 AF013397 Partial genome ROCV★ Israel turkey meningoencephalomyelitis

virus Israel turkey meningoencephalomyelitis virus AF013377 NC_043115 Partial genome ITV

★ Japanese encephalitis virus Japanese encephalitis virus JaOArS982 M18370 NC_001437 Complete genome JEV★ Jugra virus Jugra virus P-9-314 DQ859066 NC_033699 Complete genome JUGV★ Jutiapa virus Jutiapa virus JG-128 KJ469371 NC_026620 Complete genome JUTV★ Kadam virus Kadam virus Uganda DQ235146 NC_033724 Complete genome KADV★ Kedougou virus Kédougou virus DakAar D1470 AY632540 NC_012533 Complete genome KEDV★ Kokobera virus Kokobera virus AusMRM 32 AY632541 NC_009029 Complete genome KOKV

Kokobera virus Stratford virus AUSC-338 AF013407 Partial genome STRV★ Koutango virus Koutango virus Dak Ar D1470 AF013384 NC_043116 Partial genome KOUV★ Kyasanur Forest disease virus Kyasanur Forest disease virus AY323490 NC_039218 Complete genome KFDV

Kyasanur Forest disease virus Alkhumra hemorrhagic fever virus 1176 AF331718 Complete genome AHFV★ Langat virus Langat virus TP21 AF253419 NC_003690 Complete genome LGTV★ Louping ill virus louping ill virus 369/T2 Y07863 NC_001809 Complete genome LIV

Louping ill virus louping ill virus-British subtype LI/31 D12937 Partial genome LIV-BritLouping ill virus louping ill virus-Irish subtype LI/MA54 X86784 Partial genome LIV-IrLouping ill virus louping ill virus-Spanish subtype 87/2617 DQ235152 Complete coding

genome LIV-Spain

Louping ill virus Turkish sheep encephalitis virus subtype TTE80 DQ235151 Complete codinggenome TSEV

Louping ill virus Greek goat encephalitits virus subtype Vergina DQ235153 Complete codinggenome GGEV

★ Meaban virus Meaban virus France DQ235144 NC_033721 Complete genome MEAV★ Modoc virus Modoc virus M544 AJ242984 NC_003635 Complete genome MODV★ Montana myotis leukoencephalitis virus Montana myotis leukoencephalitis virus USA AJ299445 NC_004119 Complete genome MMLV

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★ Murray Valley encephalitis virus Murray Valley encephalitis virus 18629 AF161266 NC_000943 Complete genome MVEV

Murray Valley encephalitis virus Alfuy virus MRM-3929 AF013360 Partial genome ALFV★ Ntaya virus Ntaya virus IPDIA JX236040 NC_018705 Complete genome NTAV★ Omsk hemorrhagic fever virus Omsk hemorrhagic fever virus Bogoluvovska AY193805 NC_005062 Complete genome OHFV★ Phnom Penh bat virus Phnom Penh bat virus CAMA-38D AF013394 Partial genome PPBV

Phnom Penh bat virus Batu Cave virus P70-1459 AF013369 Partial genome BCV★ Powassan virus Powassan virus LB L06436 NC_003687 Complete genome POWV

Powassan virus deer tick virus ctb30 AF311056 Complete codinggenome DTV

★ Rio Bravo virus Rio Bravo virus RiMAR AF144692 NC_003675 Complete genome RBV★ Royal Farm virus Royal Farm virus Afghanistan DQ235149 NC_039219 Complete genome RFV★ Saboya virus Saboya virus Dak AR D4600 DQ859062 NC_033697 Complete genome SABV

Saboya virus Potiskum virus IBAN 10069 DQ859067 Complete codinggenome POTV

★ Saint Louis encephalitis virus St. Louis encephalitis virus Kern217 DQ525916 NC_007580 Complete genome SLEV★ Sal Vieja virus Sal Vieja virus 38TWM-106 AF013401 NC_043117 Partial genome SVV★ San Perlita virus San Perlita virus 71V-1251 AF013402 NC_043118 Partial genome SPV★ Saumarez Reef virus Saumarez Reef virus Australia DQ235150 NC_033726 Complete genome SREV★ Sepik virus Sepik virus MK7148 DQ837642 NC_008719 Complete genome SEPV★ Tembusu virus Tembusu virus JS804 JF895923 NC_015843 Complete genome TMUV★ Tick-borne encephalitis virus tick-borne encephalitis virus-European subtype Neudoerfl U27495 NC_001672 Complete genome TBEV-Eur

Tick-borne encephalitis virus tick-borne encephalitis virus - Far Easternsubtype Sofjin X07755 Partial genome TBEV-FE

Tick-borne encephalitis virus tick-borne encephalitis virus-Siberian subtype Vasilchenko L40361 Complete genome TBEV-Sib★ Tyuleniy virus Tyuleniy virus LEIV-6C KF815939 NC_023424 Complete genome TYUV★ Uganda S virus Uganda S virus Uganda DQ859065 NC_033698 Complete genome UGSV★ Usutu virus Usutu virus Vienna 2001 AY453411 NC_006551 Complete genome USUV★Wesselsbron virus Wesselsbron virus SAH177 EU707555 NC_012735 Complete genome WESSV★West Nile virus West Nile virus 956 M12294 NC_001563 Complete genome WNV

West Nile virus Kunjin virus MRM61C D00246 Complete genome KUNV★ Yaounde virus Yaoundé virus DakArY 276 AF013413 Partial genome YAOV★ Yellow fever virus yellow fever virus 17D X03700 NC_002031 Complete genome YFV★ Yokose virus Yokose virus Oita 36 AB114858 NC_005039 Complete genome YOKV★ Zika virus Zika virus MR 766 AY632535 NC_012532 Complete genome ZIKV

Zika virus Zika virus Pf13/251013-18 KY766069 Complete genome ZIKV

Zika virus Zika virus H/PF/2013 KJ776791 Complete genome ZIKVZika virus Zika virus PRVABC59 KX377337 Complete genome ZIKV

Virus names, the choice of exemplar isolates, and virus abbreviations, are not official ICTV designations.

Related, unclassified viruses

Virus name Accession number Virus abbreviationMammalian tick-borneKarshi virus DQ235147 KSIVMosquito-borneFitzroy River virus KM361634 FRVSpondweni virus DQ859064 SPOVT’Ho virus EU879061Insect-specific flavivirusesAedes flavivirus AB488408 AEFVAedes galloisi flavivirus AB639347 AGFVAnopheles flavivirus KX148546 AnFVCalbertado virus EU569288 CLBOVcell fusing agent virus M91671 CFAVCuacua virus KX245154 CuCuVCulex flavivirus GQ165808 CXFVCulex theileri flavivirus HE574574 CXthFVCuliseta flavivirus KT599442 CsFVEcuador Paraiso Escondido virus KJ152564 EPEVHanko virus JQ268258 HaFVKamiti River virus AY149905 KRVMercadeo virus KP688058 MECDVmosquito flavivirus KC464457 MoFVNakiwogo virus GQ165809 NAKVNienokoue virus JQ957875 NiFVPalm Creek virus KC505248 PCFVParramatta River virus KT192549 PaRVQuảng Bình virus FJ644291 QBVXishuangbanna aedes

flavivirusKU201526 XFV

Yamadai flavivirus AB981186 YDFVViruses with no known arthropod vector

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Barkedji virus KC496020 BJVCháoyáng virus FJ883471 CHAOVDonggang virus JQ086551 DONVIlomantsi virus KC692067 ILOVKampung Karu virus KY320648 KPKVLammi virus FJ606789 LAMVLa Tina virus KY320649 LTNVLong Pine Key virus KY290256 LPKVMarisma mosquito virus MF139576 MMVNanay virus MF139575 NANVNgoye virus DQ400858 NGOVNhumirim virus KJ210048 NHUVnounané virus EU159426 NOUVTamana bat virus AF285080 TABVSegmented flavi-like viruses

Jingmen tick virus

KJ001579;KJ001580;KJ001581;KJ001582

JMTV

Mogiana tick virus

JX390986;KY523073;JX390985;KY523074

MGTV

Alongshan virus

MH158415;MH158416;MH158417;MH158418

ASV

Guaico Culex virus

KM461666;KM461667;KM461668;KM461669;KM461670

GCXV

Shuangao insect virus 7

KR902717;KR902718;KR902719;KR902720

SAIV7

Wuhan flea virus

KR902713;KR902714;KR902715;KR902716

WHFV

Wuhan aphid virus 1

KR902721;KR902722;KR902723;KR902724

WHAV1

Wuhan aphid virus 2

KR902725;KR902726;KR902727;KR902728

WHAV2

Wuhan cricket virus

KR902709;KR902710;KR902711;KR902712

WHCV

Virus names and virus abbreviations are not official ICTV designations.

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Genus: Hepacivirus

Summary

The best characterised member of the Hepacivirus genus is hepatitis C virus (HCV), classified as a member of the species Hepacivirus C(Smith et al., 2016). HCV infects humans and is an important aetiological agent of chronic hepatitis. A further hepacivirus, GB virus-B (GBV-B),highly divergent in sequence from HCV, was first described in tamarins in 1995 in which it establishes acute infections and liver pathologycomparable to that of HCV (Simons et al., 1995b). Several other hepaciviruses have recently been described that infect Old World colobusmonkeys, various rodents and bats species, European and African cattle, horses and donkeys (Sibley et al., 2014, Walter et al., 2017, Cormanet al., 2015, Baechlein et al., 2015, Kapoor et al., 2011, Burbelo et al., 2012, Kapoor et al., 2013b, Drexler et al., 2013, Firth et al., 2014, Quanet al., 2013). These viruses have been assigned to further hepacivirus species and show distinct host ranges - Hepacivirus A (non-primatehepacivirus, NPHV) infecting horses and possibly dogs, Hepacivirus B (GBV-B) infecting tamarins and potentially other New World primates,Hepacivirus D infecting colobus monkeys, Hepacivirus E, Hepacivirus F, Hepacivirus G, Hepacivirus H, Hepacivirus I and Hepacivirus Jinfecting various species of rodents, Hepacivirus K, Hepacivirus L and Hepacivirus M infecting bats and Hepacivirus N infecting cows. Acurrently unclassified, more divergent hepacivirus-like sequence has been assembled from liver tissue of a gracile shark (Wenlin shark virus).

Collectively, these other hepaciviruses are much less well characterised virologically and clinically than HCV (Hepacivirus C) and thedescriptions below primarily refer to HCV; any available information on other members is referred to where appropriate.

Distinguishing features

Hepatitis C virus (HCV) is transmitted between humans, principally via exposure to contaminated blood; the transmission routes of otherhepaciviruses are poorly understood but are unlikely to be parenteral. There is no known invertebrate vector for HCV or any other hepacivirus.Hepaciviruses differ from members of the genera Flavivirus and Pestivirus by their limited ability to be propagated in cell culture; only a fewadapted strains of HCV, including JFH1, efficiently infect the only susceptible cultured cell line, human hepatoma cell line (Huh7). Cell cultureof other hepaciviruses has not been achieved to date. In the HCV precursor protein, the NS2-3 junction is auto-catalytically cleaved by Zn-dependent NS2-3 protease activity; a similar mechanism is likely for other hepaciviruses.

Virion

Morphology

Virions of HCV are about 50 nm in diameter, as determined by filtration and electron microscopy. They are spherical in shape with a lipidenvelope, as determined by electron microscopy and inactivation by chloroform. The viral core is spherical and about 30 nm in diameter.Detailed structural properties of HCV or other hepaciviruses have not been determined.

Physicochemical and physical properties

Virion Mr has not been determined. The buoyant density in sucrose of HCV is predominantly about 1.06 g cm for virus recovered fromserum during acute infections while more dense forms (ca. 1.15–1.18 g cm ) predominate when recovered from the serum of chronicallyinfected individuals (Thomssen et al., 1993). Lower density banding results from physical association of the virion with serum very-low-densitylipoproteins (VLDLs)(Thomssen et al., 1992). Higher density virions are those bound to serum antibodies. Of these different particle typesfound in humans, the lowest density particles are the most infectious (paricles < 1.1 g cm-3)(Bradley et al., 1991). A buoyant density range inisosmotic iodixanol gradients of 1.01–1.10 g cm has been measured for HCV recovered from hepatoma cells infected with HCV. The Sis equal to or greater than 150S. The virus is stable in buffer at pH 8.0–8.7. Virions are sensitive to heat, organic solvents and detergents(Feinstone et al., 1983).

Nucleic acid

Hepacivirus virions contain a single positive-sense, infectious ssRNA (Figure 1. Hepacivirus). The genome of HCV is about 9.6 kb, while forother hepaciviruses the range is 8.9–10.5 kb. The 5′-NCR of HCV possesses a type IV IRES (Honda et al., 1999) of approximately 340 nt.The divergent GBV-B, a member of Hepacivirus B, has a genome organization similar to HCV, but with a more extensive 5′-NCR (445 nt) alsocontaining a type IV IRES (Muerhoff et al., 1995). Type IV IRESs are similarly present in members of most other hepacivirus species althoughthe rodent hepaciviruses that are members of Hepacivirus F and Hepacivirus J possess an IRES showing sequence homology to those ofpegiviruses (Smith et al., 2016, Drexler et al., 2013). The 3′-NCR of HCV contains a sequence-variable region of about 50 nt, apolypyrimidine-rich region (average of 100 nt), and a highly conserved 98 nt 3′-terminal region with three stem-loop RNA secondary structures(Tanaka et al., 1995) The 3′-NCRs of members of other hepacivirus species show little or no conservation in sequence or predicted RNAstructure to that of HCV. There are at least two seed sites in the HCV 5′-NCR for the liver abundant microRNA miR-122; this virus-hostinteraction is required for efficient HCV replication (Jopling et al., 2005). One or two miR-122 seed sites are present in hepaciviruses of otherspecies (Smith et al., 2016).

Proteins

The HCV virion comprises at least three proteins: the nucleocapsid core protein C (p19-21), and two envelope glycoproteins, E1 (gp31) andE2 (gp70). An additional protein, p7 (believed to have properties of an ion channel protein important in viral assembly) is incompletely cleavedfrom a precursor of E2 to yield E2-p7 and p7 (Shanmugam and Yi 2013) but it is not known whether these are virion structural components. InGB virus-B, a corresponding protein, p13, is cleaved to p7 and p6 proteins (Takikawa et al., 2006). The two envelope glycoproteins can

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associate as non-covalent heterodimers; recent data, however, indicate that they are covalently linked in virions (Vieyres et al., 2010).Nonstructural proteins include NS2, a 21 kDa protein that, before cleavage, is part of a Zn-dependent cysteine protease that bridges NS2 andNS3 and mediates autocatalytic cleavage of the NS2/NS3 junction, and is involved with virus assembly and release, NS3, a 70 kDa proteinwith additional serine protease, helicase and NTPase activities; the NS3 protease cleaves the remaining junctions between nonstructuralproteins, NS4A , a 6 kDa cofactor essential for trans NS3 serine protease activity, NS4B, a 27 kDa protein that induces a membranousreplication complex at the endoplasmic reticulum, NS5A, a serine phosphoprotein of unknown specific function, but critical for viral replicationand assembly, that exists in 56 and 58 kDa forms, depending on the degree of phosphorylation, and NS5B, a 68 kDa protein with RdRPactivity.

The genomes of other hepacivirus species show similar organisations to those of HCV and GB virus-B, with predicted cleavage sites in thecoding region potentially producing core, E1, E2, p7/p13, NS2, NS3, NS4A, NS4B, NS5A and NS5B proteins homologous to and comparablein size to those of HCV and GB virus-B. One exception is a large insertion of intrinsically disordered amino acid sequence in the NS5A gene ofthe colobus monkey hepacivirus (Hepacivirus D) (Lauck et al., 2013).

Lipids

Hepacivirus virions have a lipid bi-layer envelope. Historically, based on the removal of the viral envelope and loss of infectivity of HCVfollowing exposure to solvents or detergents (Feinstone et al., 1983), the presence of lipids was inferred. Recently, it has become apparentthat the host lipid metabolism plays a critical role in the virus life cycle of HCV and likely other hepaciviruses.

Carbohydrates

The E1 and E2 glycoproteins of all hepaciviruses contain numerous N-linked glycosylation sites (1–4 in E1, 2–11 in E2), and carbohydrate isassociated with the products of these two genes. E1 and E2 are transmembrane, type I glycoproteins, with C-terminal retention signals thatanchor them within the lumen of the endoplasmic reticulum. These signals are apparently masked when budding occurs allowing the virion tomove through the secretory pathway. Recent data obtained in culture systems indicate that N-linked glycans of HCV E1 remain in the high-mannose chains lacking complex carbohydrate, whereas those of E2 are modified (Op De Beeck et al., 2004 ). Glycosylation influences E1–E2 heterodimer formation, folding and assembly and the release of virions (Meunier et al., 1999).

Genome organization and replication

The hepacivirus genome contains a single large ORF encoding a polyprotein of about 3,000 aa (Figure 1. Hepacivirus). The gene order is 5′-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-3′. Immediately downstream of the three structural proteins (C, E1, E2) is a small protein, p7in HCV, p13 in GB virus-B and comparably small predicted proteins in other hepaciviruses, followed by the nonstructural proteins in the 3′-portion of the ORF. Replication occurs in association with intracytoplasmic membranes. Replicative forms of HCV and of the recentlydescribed non-primate hepacivirus (NPHV, Hepacivirus A; (Pfaender et al., 2015)) have been detected in liver tissue. The genomic RNA istranslated into a polyprotein that is rapidly processed both co- and post-translationally by host and viral proteases. Translation initiation occursvia an IRES within the 5′-NCR. Translocation of the structural glycoproteins to the endoplasmic reticulum probably occurs via internal signalsequences. Cleavage of the structural proteins is mediated by host cell signal peptidases, and signal peptide peptidase. With the exception ofthe p7/NS2 signalase cleavage, viral proteases cleave all non-structural protein junctions. Virus assembly is believed to occur by budding intovesicles from the endoplasmic reticulum.

Figure 1.Hepacivirus. Hepacivirus genome organization (not to scale) and polyprotein processing. For members of the species Hepatitis Cvirus, the RNA is about 9.6 kb. The 5′-NCR is about 340 nt, the 3′-NCR about 250 nt, and the ORF about 9 kb. HCV has a p7 proteinbetween E2 and NS2. The host and viral proteases involved in cleavage of the polyprotein are indicated. The cleavage by host signalpeptide peptidase (at the C-terminus of the core protein) is indicated by a green arrow; the cleavages by host signal peptidase (remainingsites) are indicated by filled arrows. The locations of the NS2-3 protease, NS3 protease, NS3 RNA helicase and NS5B RdRP are indicatedby P′, P″, H and R, respectively.

Biology

Host range

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Humans are the natural host and apparent reservoir of HCV although the virus can be transmitted experimentally to chimpanzees. No othernatural host has been identified. The natural host for GB virus-B is not known although it can experimentally infect the New World primatespecies marmosets and tamarins and the more distantly related owl monkeys (Bukh et al., 2001). Members of other species of hepacivirusesinfect a wide range of other mammalian species, both wild and domestic, including colobus monkeys (Sibley et al., 2014), cows (Baechlein etal., 2015), horses (Burbelo et al., 2012), donkeys (Walter et al., 2017) and a range of rodent and bat species (Kapoor et al., 2011, Kapoor etal., 2013b, Drexler et al., 2013, Firth et al., 2014, Quan et al., 2013). The host specificity of these variants for their mammalian hosts isundetermined, although at least one rodent species, the bank vole (Moyodes glareolus) can be infected with two highly divergent hepacivirusvariants (RMU10-3382/GER/2010 and NLR07-oct70/NEL/2007)(Drexler et al., 2013) belonging respectively to the species Hepacivirus J andHepacivirus F. This observation is indicative of a potential degree of cross-species transmission.

Transmission

HCV is transmitted almost exclusively by parenteral exposure to blood, blood products and objects contaminated with blood. Effectivescreening of blood donors and implementation of inactivation procedures have virtually eliminated the transmission of HCV by blood andblood products, but other routes of exposure, principally by blood-contaminated syringes, are now the most important recognized risk factors.Sexual and mother-to-child transmission has been documented but is relatively uncommon. Other routes of transmission are suspected formembers of other hepacivirus species; in a recent study of thoroughbred horses, evidence for vertical transmission of NPHV was obtainedfrom one of four mares to their foals, while further infections occurred in the post-natal period (Gather et al., 2016).

Geographical distribution

HCV has a worldwide distribution with about 3% of the world population infected with HCV, equivalent to 170 million chronic infections, with 3–4 million new infections each year. Antibody prevalences are 0.1–2% in developed countries but as high as 20% in some developingcountries, possibly reflecting the historic use of contaminated needles and syringes. Horses infected with members of Hepacivirus A havebeen reported from four continents with viraemia frequencies ranging from 3–10%, indicative of a wide geographical distribution (Burbelo etal., 2012, Pfaender et al., 2015, Lyons et al., 2012, Figueiredo et al., 2015, Lu et al., 2016, Matsuu et al., 2015).

Pathogenicity

The effects of HCV infection in humans range from subclinical to acute and chronic hepatitis, liver cirrhosis and hepatocellular carcinoma.Persistent infection occurs in 60–80% of cases, and in about 20% of the cases the infection progresses over many years to chronic activehepatitis and cirrhosis. Patients with liver cirrhosis have an approximately 5% risk per year of developing hepatocellular carcinoma.

Persistent HCV infection has been linked by epidemiological studies to primary liver cancer, cryptogenic cirrhosis and some forms ofautoimmune hepatitis. Extrahepatic manifestations of HCV infection include mixed cryoglobulinemia with associated membrano-proliferativeglomerulonephritis and, possibly, porphyria cutanea tarda, Sjögren’s-like syndromes and other autoimmune conditions.

Similarly to HCV infections in humans, GB virus-B causes hepatitis and replicates in the liver of tamarins and owl monkeys, but infection isself-limited and has not been demonstrated in humans or chimpanzees. Only one strain of GB virus-B has been identified to date, in contrastto thousands of often quite divergent variants of HCV. The pathogenicity of other hepaciviruses infecting non-human primates, rodents, bats,cattle, and horses is poorly characterized although the presence of miR-122 seed sites in each species characterised to date predictswidespread hepatotropism among members of this genus (Jopling et al., 2005). Recent evidence suggests that infection of horses with NPHVmay be associated with higher rates of spontaneous clearance than observed for HCV in humans (Pfaender et al., 2015, Lyons et al., 2014)and only mild inflammatory liver disease (Pfaender et al., 2015) with minimal elevation of liver enzymes ( Lyons et al., 2014). Infectionoutcomes have been suggested to be dependent on the breed and age of the horse (Ramsay et al., 2015).

Cell tropism

HCV has been reported to replicate in several cell lines derived from hepatocytes and lymphocytes, but virus growth has only been sufficientfor practical application of these systems in a human hepatoma cell line, Huh7 cells and derivatives thereof. In vivo, HCV replicates inhepatocytes and possibly lymphocytes. The cellular or tissue tropism of other hepaciviruses is poorly characterized although there is evidencethat GB virus-B, NPHV, and bovine hepacivirus are hepatotropic; the presence of binding sites for miR-122 (Jopling et al., 2005) in otherdescribed hepaciviruses is consistent with widespread hepatotropism, given the restriction of expression of this miRNA to liver tissue.

Antigenicity

Virus-specific antibodies to recombinant-expressed structural proteins of HCV (C, E1 and E2) and nonstructural proteins (principally NS3,NS4 and NS5) have been detected in individuals infected with HCV. Both linear and conformational epitopes are believed to be involved in thehumoral immune response of the host to infection. Significant antigenic diversity throughout the genome is reflected in heterogeneity in thehumoral immune response. In HCV, high variability is found in the N-terminal 27 aa of E2 (hypervariable region 1; HVR1). The HVR1 containsan HCV neutralization epitope and escape variants of HVR1 are positively selected by the host humoral immune response (Mondelli et al.,2003). Other neutralization epitopes have been identified in E2 outside of HVR1 in E2 and also in E1 ( Fafi-Kremer et al., 2012, Edwards et al.,2012, Keck et al., 2004). With the development of intra- and intergenotypic genotype 1-7 JFH1-based recombinant viruses with strain-specificstructural proteins, it is now possible to carry out in vitro virus neutralization assays to address the antigenic diversity of HCV ( Gottwein et al.,2011). Serological responses to GB virus-B infection are less well-characterized; antibodies to NS3 become detectable after a delay of severalweeks in acutely infected tamarins but titres decline rapidly following virus clearance (Beames et al., 2001, Schaluder et al., 1995).

Cell-mediated immune responses to all HCV proteins have been detected ( Klenerman and Thimme 2012); it is believed that such responses

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are associated with amelioration or resolution of infection. T cell responses in marmosets that may similarly correlate with protection from viruschallenge have been detected in the NS3 and NS4A proteins of GB virus-B (Woollard et al., 2008).

Species demarcation criteria

Species in the Hepacivirus genus have been assigned based on their members’ genetic divergence, where viruses showing greater than 0.25amino acid p-distances in a conserved region of NS3 (positions 1,123–1,566 as numbered in the HCV genotype 1a H77 reference sequenceAF011751), and greater than 0.3 in the NS5B region (amino acid positions 2536–2959) are considered to be members of different species(Smith et al., 2016). Members of different hepacivirus species show characteristically restricted host ranges, typically infecting different hostspecies. There are, however, a few exceptions; the bank vole may be infected by members of both species Hepacivirus F and Hepacivirus J.

This demarcation point places hepaciviruses infecting humans and horses into different species (Hepacivirus A and Hepacivirus C) whileassigning the 7 relatively divergent genotypes of HCV described to date (Smith et al., 2014) to a single species. These HCV genotypes differfrom each other by about 30–35% at the nt level (Simmonds et al., 2005). Within each genotype, there are a number of subtypes, differingfrom each other by about 15–25% at the nt level. Although genotypes are distinct genetically, discrimination of subtypes is less clear,particularly in areas of high diversity such as sub-Saharan Africa and Southeast Asia. Because systematic serological typing by virusneutralization has not been performed to date, and because major genotypes do not have any other taxonomic characteristics except, insome cases, geographic distribution and differences in treatment response, it was considered appropriate to classify the seven genotypes ofHCV as members of same species (Hepacivirus C).

Although HCV was the first hepacivirus to be discovered and the type species of its genus, it has been assigned to the species Hepacivirus Crather than Hepacivirus A to avoid potential confusion between its name and the letter assigned for its species assignment. Other species arenamed according to the date of publication of a complete coding sequence with the exception of the assignment of GB virus-B to HepacivirusB, again to match the virus name and species letter.

Member species

★ Exemplar isolate of the speciesSpecies Virus name Isolate Accession number RefSeq number Available sequence Virus Abbrev.

★ Hepacivirus A non-primate hepacivirus NZP1 KP325401 NC_038425 Complete genome NPHV★ Hepacivirus B GB virus-B B U22304 NC_038426 Complete genome GBV-B★ Hepacivirus C hepatitis C virus genotype 1a H77 AF009606 NC_004102 Complete genome HCV1a

Hepacivirus C hepatitis C virus genotype 1a PT M62321 Complete genome HCV1aHepacivirus C hepatitis C virus genotype 1b J D90208 Complete coding genome HCV1bHepacivirus C hepatitis C virus genotype 2a HC-J6 D00944 Complete coding genome HCV2aHepacivirus C hepatitis C virus genotype 2b HC-J8 D10988 Complete coding genome HCV2bHepacivirus C hepatitis C virus genotype 3a NZL1 D17763 Complete coding genome HCV3aHepacivirus C hepatitis C virus genotype 3k JK049 D63821 Complete coding genome HCV3kHepacivirus C hepatitis C virus genotype 4a ED43 GU814265 Complete coding genome HCV4aHepacivirus C hepatitis C virus genotype 5a EUH1480 Y13184 Complete coding genome HCV5aHepacivirus C hepatitis C virus genotype 6a euhk2 Y12083 Complete coding genome HCV6aHepacivirus C hepatitis C virus genotype 6g JK046 D63822 Complete coding genome HCV6gHepacivirus C hepatitis C virus genotype 7a QC69 EF108306 Complete coding genome HCV7a

★ Hepacivirus D guereza hepacivirus 1/BWC08 KC551800 Complete coding genome GHV★ Hepacivirus E rodent hepacivirus 339 KC815310 NC_021153 Complete genome RHV-E★ Hepacivirus F rodent hepacivirus 25842 KC411784 NC_038427 Complete coding genome RHV-F★ Hepacivirus G Norway rat hepacivirus 1 C12 KJ950938 NC_025672 Complete coding genome NRHV1

Hepacivirus G Norway rat hepacivirus 1 rn-1 KX905133 Complete genome NRHV1★ Hepacivirus H Norway rat hepacivirus 2 E43 KJ950939 NC_025673 Complete genome NRHV2★ Hepacivirus I rodent hepacivirus 3/RSA/2008 KC411806 NC_038428 Complete coding genome RHV-I★ Hepacivirus J rodent hepacivirus 3382/GER/2010 KC411777 NC_038429 Complete coding genome RHV-J★ Hepacivirus K bat hepacivirus PDB-829 KC796074 NC_038430 Complete coding genome BHV-K★ Hepacivirus L bat hepacivirus PDB-112 KC796077 NC_031916 Complete coding genome BHV-L★ Hepacivirus M bat hepacivirus PDB-491.1 KC796078 NC_038431 Complete coding genome BHV-M★ Hepacivirus N bovine hepacivirus GER/2014 KP641127 NC_038432 Complete coding genome BoHV

Virus names, the choice of exemplar isolates, and virus abbreviations, are not official ICTV designations.

Related, unclassified viruses

Virus name Accession number Virus abbreviationWenling shark virus KR902729 WLSV-MHS-2

Virus names and virus abbreviations are not official ICTV designations.

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Genus: Pegivirus

Distinguishing features

Pegiviruses have been detected in a variety of mammalian hosts, with transmission of human pegiviruses occurring by sexual, parenteral andmaternal routes. Invertebrate vectors have not been discovered.

Pegiviruses show distant sequence relatedness to other members of the family Flaviviridae, forming a distinct cluster based on phylogeneticanalysis of the RdRP (Figure 1.Flaviviridae). In addition to their separate phylogenetic position, they show several differences in genomeorganization from members of the Hepacivirus, Flavivirus, and Pestivirus genera. Most pegiviruses possess an IRES element that isstructurally unrelated to those of hepaciviruses and pestiviruses and they do not encode a protein homologous to the nucleocapsid proteinfound in members of the other genera (Quan et al., 2013, Muerhoff et al., 1995, Stapleton et al., 2011). Infections with human pegivirus(HPgV) are frequently persistent but, with the exception of an association with non-Hodgkin’s lymphomac (Krajden et al., 2010, Chang et al.,2014), are not associated with the development of any identifiable disease. Pegivirus infections of other mammalian species are persistentand non-pathogenic, apart from the report of Theiler’s disease in horses infected with Theiler’s disease associated virus (Chandriani et al.,2013).

Virion

Morphology

Virions of pegiviruses have not been visualized; the lack of an encoded core protein suggests that they may be structurally distinct from othermembers of the Flaviviridae. The virion size of HPgV was estimated to be 50–100 nm based on sequential filtration through filters ofdecreasing pore sizes.

Physicochemical and physical properties

The buoyant density of HPgV from human serum on both sucrose and CsCl density centrifugation ranged from 1.05–1.13 g cm (Xiang etal., 1998, Melvin et al., 1998). Treatment of HPgV with detergent did not recover a denser, non-enveloped form of the virion, consistent withthe lack of a viral nucleocapsid (Melvin et al., 1998). In the absence of an established cell culture model for pegiviruses, no information iscurrently available on their stability or inactivation characteristics.

Nucleic acid

Pegivirus virions contain a single positive-sense, potentially infectious ssRNA ranging from 8.9–11.3 kb (Figure 1. Pegivirus). The 5′-NCRcontains an IRES element of between 300-550 nt. No miR-122 binding sites have been identified in 5′-NCR sequences of HPgV or amongmembers of other pegivirus species (Smith et al., 2016). Most pegiviruses possess an IRES broadly similar in structure but not in sequence tothe type I IRES elements of picornaviruses (Quan et al., 2013); however, the more divergent human hepegivirus (HHPgV), a member ofPegivirus H, as well as members of Pegivirus F and Pegivirus J have type IV IRES elements structurally resembling those of hepacivirusesand pestiviruses, but again with almost no sequence identity between them (Kapoor et al., 2015).

Proteins

Functional studies of most pegivirus proteins have not been performed and information on their likely function in replication and virusassembly has largely been inferred from comparison with homologous genes in hepaciviruses. Most pegiviruses lack or possess no obvioushomologue of the core protein of hepaciviruses and other members of the Flaviviridae, so how pegivirus virions are assembled remainsuncertain. However, typically pegiviruses are predicted to encode shorter, although somewhat variable length, basic proteins containingmultiple arginine and leucine amino acids immediately upstream of the signalase site before E1; these may play some role in RNA packagingduring virion assembly. E1 and E2 proteins are believed to be envelope glycoproteins, while NS3 and NS5B contain motifs common tohelicase and polymerase proteins in viruses of other genera of the Flaviviridae (reviewed in (Stapleton et al., 2011)). The NS3-4A region hasbeen shown to be proteolytically active for processing the nonstructural region of the human pegivirus polyprotein (Belyaev et al., 1998).

Lipids

The virion structure of pegiviruses is unknown, but the presence of predicted hydrophobic transmembrane regions in the E1 and E2glycoproteins is consistent with the presence of viral envelope, likely derived by budding of pegiviruses from infected cells, analogously toother members of the family Flaviviridae.

Carbohydrates

The E1 and E2 glycoproteins have variable numbers of potential N-linked glycosylation sites, with members of the more divergent speciesPegivirus F, Pegivirus H, and Pegivirus J possessing a larger number of sites, a feature more typical of hepaciviruses ( Kapoor et al., 2015).

Genome organization and replication

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In common with other members of the Flaviviridae, the genome contains a single ORF. Structural proteins are processed by cellularproteases, while the NS3-4A viral protease cleaves the nonstructural region of the polyprotein in the same gene order as hepaciviruses(Figure 1.Pegivirus).

Figure 1.Pegivirus. Genome organization of pegiviruses. Pegivirus genomes range from approximately 8.9–11.3 kb; those with longergenomes code for additional predicted structural proteins, X and Y (lower diagram). The genome encodes a polyprotein that is co- and post-translationally cleaved into individual viral proteins. Structural proteins common to all pegiviruses are the envelope glycoproteins (E1 andE2), and non-structural proteins are NS2–NS5B. No protein homologous to the core protein of other members of the Flaviviridae has beenidentified in pegiviruses although some possess a predicted, basic protein upstream of E1 of unknown function (Y). Several pegiviruses alsohave a predicted additional glycoprotein downstream of E2 (X). Cleavage of structural proteins by cellular signal peptidases, of NS2/NS3 bythe NS2–NS3 autoprotease and of the remaining NS proteins by the NS3–NS4A protease complex is comparable to hepaciviruses. Allpegiviruses possess a long 5ʹ-noncoding region with predicted IRES function; most pegiviruses have a type I picornavirus-like IRES whileothers have a type IV IRES type structurally related to those of hepaciviruses and pestiviruses.

Biology

Host range

Pegiviruses can be detected in a wide range of mammalian species (humans, non-human primates, pigs, horses and a range of rodent andbat species). Members of Pegivirus A infect New World monkeys (Muerhoff et al., 1995, Bukh and Apgar 1997) and bats, while members ofPegivirus C infect humans, chimpanzees (Adams et al., 1998, Birkenmeyer et al., 1998) and Old World monkeys (Sibley et al., 2014, Bailey etal., 2016). Members of Pegivirus E (equine pegivirus (Kapoor et al., 2013a) and Pegivirus D (Theiler’s disease associated virus (Chandriani etal., 2013)) infect horses, members of Pegivirus K infect pigs (Baechlein et al., 2016) and members of Pegivirus B, Pegivirus F, Pegivirus G,Pegivirus I and Pegivirus J infect a wide range of bat and rodent species (Kapoor et al., 2013b, Quan et al., 2013, Epstein et al., 2010)(Epsteinet al., 2010; Kapoor et al., 2013b; Quan et al., 2013).

Very limited information is available on the potential of pegiviruses to transmit between different host species. However, chimpanzees can beexperimentally infected by inoculation with HPgV (Pegivirus C) but not by the New World primate virus, GBV-A (Pegivirus A) (Bukh et al.,1998) and rhesus macaques can be infected with a baboon isolate of Pegivirus C (Bailey et al., 2015).

Transmission

HPgV is transmitted between humans by sexual transmission, exposure to contaminated blood, and from mother to child. Horizontaltransmission has neither been confirmed nor refuted (Bhattarai and Stapleton 2012). HPgV viraemia frequencies are higher in injecting drugusers and in haemophiliacs with a history of exposure to non-virally inactivated clotting factor concentrates, indicating an efficient parenteralroute of transmission. HPgV viraemia frequencies are also higher in people with sexually transmitted diseases and without a history ofparenteral exposure (Scallan et al., 1998); human pegivirus infection is also a frequent co-infection with human immunodefiency virus 1 (HIV-1). Among HIV-infected subjects, co-infection with HPgV does not correlate with HIV transmission risk; however, hepatitis C virus (HCV) andHCV-HPgV co-infection are significantly associated with a parenteral mode of HIV acquisition (Bourlet et al., 1999), indicating the likelihood ofsexual routes of transmission.

HHPgV (Pegivirus H) additionally shows evidence for parenteral routes of transmission with infections largely confined to intravenous drugusers and recipients of blood or blood products (Kapoor et al., 2015, Coller et al., 2016, Bonsall et al., 2016, Berg et al., 2015); however, othermodes of transmission have not been extensively studied.

Geographical distribution

Infection of humans with HPgV occurs worldwide and it is likely that it is ubiquitous in human populations. Prevalence studies in developedcountries indicate between 1–4 % of healthy blood donors are viraemic for HPgV and another 5–13 % have anti-E2 antibodies, indicating prior

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infection. Rates of infection with HPgV in developing countries are higher, with viraemia frequencies in the general population frequentlyexceeding 10%. Infection frequencies of pegiviruses infecting non-human hosts are incompletely described. Infections with HHPgV appear tobe confined to those with parenteral exposure, more reminiscent of HCV. Relatively low frequencies (1%–2%) of viraemia of other pegiviruseshave been described in horses for members of Pegivirus D and Pegivirus E (Lu et al., 2016, Lyons et al., 2014, Kapoor et al., 2013a, de Souzaet al., 2015) and pigs for members of Pegivirus K) (Baechlein et al., 2016).

Pathogenicity

Infections with HPgV in humans are considered non-pathogenic, to the extent that viraemic blood donations are not excluded fromtransfusion. The pathogenicity of pegiviruses infecting other hosts is unknown although experimental infection of New World primates withsimian pegiviruses does not induce liver disease. However, it has been reported that infection of horses with members of Pegivirus D isassociated with Theiler’s disease in horses (Chandriani et al., 2013).

Cell tropism

Pegiviruses infecting humans or new world primates cannot be readily detected in the liver of infected hosts, whereas they are present athigher viral loads in circulating lymphocytes, including T and B lymphocytes (Kobayashi et al., 1999, Tucker et al., 2000). However, based onautopsy studies in humans (Tucker et al., 2000, Radkowski et al., 1999), and the animal model of nonhuman pegivirus infection in rhesusmacaques, pegivirus replication occurs primarily in the bone marrow (Bailey et al., 2015). The tissue or cellular tropism of pegiviruses infectingother hosts is unknown. Like the hepaciviruses, pegiviruses differ from members of the genera Flavivirus and Pestivirus by their limited abilityto be propagated in cell culture (Chivero and Stapleton 2015).

Antigenicity

Pegivirus antigenicity is poorly characterized in the absence of in vitro neutralization assays or experimental animal models. Antibody to theE2 glycoprotein of HPgV can be detected in humans and is associated with clearance of viraemia (Feucht et al., 1997, Tacke et al., 1997).These E2 antibodies reduce the rate of re-infection following liver transplantation (Tillmann et al., 1998). Recent data show the immunemodulating effects of E2 protein on T cell activation and NK cell signalling, which may contribute to the absence of serological reactivity toother HPgV proteins (Chivero et al., 2015).

Species demarcation criteria

Species in the Pegivirus genus are now classified based on their genetic divergence (Smith et al., 2016) rather than their host range(Stapleton et al., 2011). Assignment thresholds are based on amino acid sequence divergence in conserved regions of NS3 and NS5B;pegiviruses showing greater than 0.31 amino acid p-distances in a conserved region of NS3 (positions 888–1635 as numbered in the HPgVreference sequence U22303), and greater than 0.31–0.36 in the NS5B region (amino acid positions 2398–2916) are considered to beseparate species (Smith et al., 2016). In general, members of different pegivirus species infect different hosts with the notable exception ofPegivirus A, into which are assigned pegiviruses infecting New world primates (GB virus-A) and African bats.

Pegivirus species names have been assigned in alphabetical sequence largely based on their order of discovery, the exception beingPegivirus C which was chosen to match the virus name GB virus C (GBV-C). Isolates of this species are also known as hepatitis G virus(HGV) (Linnen et al., 1996, Simons et al., 1995a), although more recently the name human pegivirus (HPgV) has been proposed and adoptedas there is now no evidence that infections are associated with hepatitis (Stapleton et al., 2011), nor did this virus infect the surgeon, GB, fromwhom infective material was passaged in primates and the virus first described. A second, more divergent group of pegiviruses, termedhuman hepegivirus (HHPgV) or HPgV-2 (Kapoor et al., 2015, Berg et al., 2015) has been assigned to Pegivirus H (Smith et al., 2016).

Member species

★ Exemplar isolate of the speciesSpecies Virus name Isolate Accession number RefSeq number Available sequence Virus Abbrev.

★ Pegivirus A GB virus-A; simian pegivirus A/T1053 U22303 Complete coding genome GBV-A; SPgVPegivirus A GB virus-A; simian pegivirus Alab U94421 Complete genome GBV-A; SPgV

★ Pegivirus B GB virus-D D/68 GU566734 NC_030291 Complete genome GBV-DPegivirus B GB virus-D D/93 GU566735 Partial genome GBV-D

★ Pegivirus C human pegivirus genotype 2 PNF2161 U44402 NC_001710 Complete coding genome HPgVPegivirus C human pegivirus genotype 1 EA U63715 Complete coding genome HPgVPegivirus C human pegivirus genotype 1 CG01BD AB003289 Complete coding genome HPgVPegivirus C human pegivirus genotype 2 R10291 U45966 Complete genome HPgVPegivirus C human pegivirus genotype 2 765 AY196904 Complete genome HPgVPegivirus C human pegivirus genotype 3 K2141 D87713 Complete genome HPgVPegivirus C human pegivirus genotype 4 MY14 AB021287 Complete coding genome HPgVPegivirus C human pegivirus genotype 5 D50 AY949771 Complete coding genome HPgVPegivirus C human pegivirus genotype 6 G05BD AB003292 Complete coding genome HPgVPegivirus C simian pegivirus-chimpanzee Ctro AF070476 Complete genome SPgV-ch

★ Pegivirus D Theiler’s disease associated virus horseA1 KC145265 NC_038433 Complete coding genome TDAV★ Pegivirus E equine pegivirus C0035 KC410872 NC_020902 Complete coding genome EPgV★ Pegivirus F bat pegivirus F PDB-1698 KC796080 NC_038434 Complete coding genome BPgV-F★ Pegivirus G bat pegivirus G PDB-620 KC796076 NC_038435 Complete coding genome BPgV-G★ Pegivirus H human hepegivirus; human pegivirus 2 AK-790 KT439329 NC_038436 Complete coding genome HHPgV★ Pegivirus I bat pegivirus I PDB-1715 KC796088 NC_038437 Complete coding genome BPgV-I★ Pegivirus J rodent pegivirus CC61 KC815311 NC_021154 Complete coding genome RPbV★ Pegivirus K porcine pegivirus 903/GER/2013 KU351669 NC_034442 Complete coding genome PPgV

Virus names, the choice of exemplar isolates, and virus abbreviations, are not official ICTV designations.

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Genus: Pestivirus

Distinguishing features

Compared to the other viruses in the Flaviviridae, pestiviruses encode two unique gene products, namely N and E (Tautz et al., 2015).The first protein of the ORF, nonstructural protein N , which possesses an autoproteolytic activity and is responsible for its release from thenascent polyprotein (Gottipati et al., 2014, Rumenapf et al., 1998, Stark et al., 1993), is not essential for virus replication in cell culture(Tratschin et al., 1998). One of the three viral envelope glycoproteins, E , possesses an intrinsic RNase activity (Krey et al., 2012, Schneideret al., 1993). Both of these unique proteins of the pestiviruses are involved in repression of the host type I IFN response ( Schweizer andPeterhans 2001, Meyers et al., 2007, Ruggli et al., 2005, Ruggli et al., 2009, Python et al., 2013, Zurcher et al., 2014, Hilton et al., 2006, LaRocca et al., 2005). Two biotypes of pestiviruses, cytopathogenic (cp) and non-cytopathogenic (noncp) viruses, are distinguished by theirability to cause cytopathic effects in cell culture (Tautz et al., 2015, Becher and Tautz 2011).

Virion

Morphology

Virions are 40–60 nm in diameter and spherical in shape (Figure 1.Pestivirus) (Laude 1979). The virion envelope has 10–12 nm ring-likesubunits on its surface. The structure and symmetry of the core have not been characterized.

Figure 1.Pestivirus. Negative-contrast electron micrograph of particles of an isolate of bovine viral diarrhea virus 1. The bar represents 100nm. (From M. König, with permission.)

Physicochemical and physical properties

Virion Mr has not been determined precisely. Buoyant density in sucrose is 1.10–1.15 g cm ; S is 140–150S (Laude 1979, Maurer et al.,2004). Virion infectivity is stable over a relatively broad pH range, but unstable at temperatures above 40 °C. Organic solvents and detergentsrapidly inactivate these viruses (Depner et al., 1992).

Nucleic acid

The virion RNA is a positive-sense, infectious molecule of ssRNA of 11.3–13.0 kb encoding a single ORF ( Becher et al., 2014, Becher et al.,1998, Collett et al., 1988, Meyers et al., 1989). The 5′-NCR contains an IRES and is about 370–385 nt ( Poole et al., 1995). The 3′-NCR, ofabout 185–273 nt, is complex and contains a region with variable sequences and a highly conserved terminal region (Pankraz et al., 2005, Yuet al., 1999). For some cp pestivirus strains, a small and variable segment of host cell or viral nucleic acid is integrated into particular regions(often within NS2 or directly upstream of NS3) of the viral genome, sometimes accompanied by viral gene duplications or deletions (Tautz etal., 2015, Becher and Tautz 2011). Other cp pestiviruses contain only viral gene duplications involving all or part of the N and NS3 to NS4Bprotein-coding regions, resulting in genomic RNA of up to about 16.5 kb. In all cases, the single large ORF is maintained. Finally, cp virusesmay also arise by deletion of large portions of their genomes. Such defective genomes can be rescued by co-infecting intact helper viruses(Tautz et al., 2015, Becher and Tautz 2011, Abbas et al., 2013).

Proteins

Virions contain four structural proteins: a basic nucleocapsid core protein, C (14 kDa) and three envelope glycoproteins, E (gp44/48), E1(gp33) and E2 (gp55). All three glycoproteins exist as intermolecular disulfide-linked complexes: E homodimers, E1-E2 heterodimers andE2 homodimers (Rumenapf et al., 1991, Thiel et al., 1991, Weiland et al., 1992, Weiland et al., 1990). The E protein possesses an intrinsicRNase activity. Pestiviruses encode eight nonstructural (NS) proteins among which N (23 kDa), p7 (7 kDa) and NS2 (40 kDa) are not

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necessary for RNA replication (Behrens et al., 1998, Tautz et al., 1999). N is a proteinase that auto-catalytically releases itself from thenascent polyprotein. Nonstructural protein p7 is presumed to have a role in virus maturation (Harada et al., 2000, Elbers et al., 1996). NS2-3(120 kDa) is a multifunctional protein of which the N-terminal 40% (NS2) is hydrophobic and contains a zinc finger motif that binds divalentmetal ions (De Moerlooze et al., 1990, Lackner et al., 2006). NS2 is a cysteine protease that is responsible for processing of NS2-3 to give riseto NS2 and NS3 (Lackner et al., 2006). NS3 (80 kDa) acts as both a serine protease involved in polyprotein processing and an RNAhelicase/NTPase involved in RNA replication (Tautz et al., 1997, Tautz et al., 2000, Warrener and Collett 1995, Wiskerchen and Collett 1991).NS2-3 is found after infection with all pestiviruses. In cells infected with cp pestiviruses, large amounts of NS3 can be detected. For noncpbovine viral diarrhea virus (BVDV), noncp Border disease virus (BDV) and classical swine fever virus (CSFV) strains, efficient NS2-3 cleavageis limited to the first eight hours of infection and at later time points the cleavage products NS2 and NS3 are difficult to detect (Lackner et al.,2004). The NS4A (7 kDa) protein acts as a cofactor to the NS3 protease activity ( Tautz et al., 2000). The role of NS4B (33 kDa) is unknown.NS5A (58 kDa) represents a phosphorylated protein and also plays still to be further characterized roles in RNA replication and virionmorphogenesis (Chen et al., 2012, Isken et al., 2014, Tellinghuisen et al., 2006, Xiao et al., 2009). NS5B (75 kDa) possesses RdRP activity(Zhong et al., 1998, Steffens et al., 1999, Choi et al., 2006).

Lipids

The viruses are enveloped, but no reports have described the lipid composition.

Carbohydrates

All virus envelope glycoproteins contain N-linked glycans ( Thiel et al., 1991).

Genome organization and replication

The genomic RNA contains a single large ORF encoding a polyprotein of about 3,900 aa that is preceded by a 5′-NCR of 370–385 nt andfollowed by a 3′-NCR of 185–273 nt. The gene order is 5′-N -C-E -E1-E2-p7-NS2-3(NS2-NS3)-NS4A-NS4B-NS5A-NS5B-3′ (Figure2.Pestivirus) (Tautz et al., 2015, Abbas et al., 2013).

Figure 2.Pestivirus. Pestivirus genome organization (not to scale) and polyprotein processing. The RNA is 11.3–13.0 kb, comprising a 5′-NCR of 370–385 nt, the single ORF of about 11.7 kb and the 3′-NCR of 185–273 nt. Virus nonstructural proteins are indicated as NS. Thesymbols P′, P″, P′″, H and R indicate the localization of the N protease, the NS2 protease, the NS3 protease, the NS3 RNA helicase andthe NS5B RdRP, respectively. The proteases and proteolytic steps involved in the generation of individual proteins are indicated. In noncpBVDV viruses, NS2-3 cleavage is detectable early after infection whereas in cp BVDV viruses both NS2-3 and NS3 are producedcontinuously.

Pestivirus replication is initiated by receptor-mediated endocytosis involving more than one cell surface molecule and the viral glycoproteinsE and E2. CD46 has been shown to function as a cellular receptor for BVDV but is not by itself sufficient to mediate infection ( Maurer et al.,2004, Krey et al., 2006a, Krey et al., 2006b, Krey et al., 2005). After endocytosis and uncoating, the genome RNA serves as mRNA; there areno subgenomic mRNA molecules. Translation initiation occurs by a cap-independent internal initiation mechanism involving a type IV IRESwithin the 5′-NCR of the RNA (Lozano and Martinez-Salas 2015). Polyprotein processing occurs co- and post-translationally by both cellularand viral proteases (Tautz et al., 2015). Nonstructural protein N , the first protein of the ORF, auto-proteolytically removes itself from thenascent polyprotein by cleavage at the N /C site. Downstream cleavages that produce structural proteins C, E , E1 and E2 as well as p7are mediated by cellular signal peptide peptidase and signal peptidase(s) (Elbers et al., 1996, Bintintan and Meyers 2010, Heimann et al.,2006, Rumenapf et al., 1993). Glycoprotein translocation to the endoplasmic reticulum occurs by an internal signal sequence, within the C-terminal region of the C protein. Cleavage between E2 and p7 is not complete, leading to two intracellular forms of E2 with different C-termini(Elbers et al., 1996). Depending on the pestivirus biotype, NS2-3 either remains mostly intact or is found at reduced levels together with highamounts of its N- and C-terminal products NS2 and NS3 (Lackner et al., 2004). The increased generation of NS3 in cp pestiviruses is in mostcases due to gene insertion, deletion, duplications or rearrangements (Tautz et al., 2015, Becher and Tautz 2011). The NS3/NS2-3 serineprotease activity is responsible for all processing events downstream of NS3. NS4A facilitates cleavages by the NS3 protease of sites 4B/5Aand 5A/5B (Tautz et al., 2015).

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RNA replication probably occurs in association with intracytoplasmic membranes, presumably in a replication complex composed of viral RNAand viral nonstructural proteins. Nonstructural proteins NS3, 4A, 4B, 5A and 5B are necessary for RNA replication; only NS5A can be providedin trans (Grassmann et al., 2001). Replicative forms of viral RNA have been detected ( Gong et al., 1996). The ratio of positive- to negative-sense RNA in cells 12 hours post-infection is about 10. RNA synthesis is resistant to actinomycin D. Virus maturation and release is poorlyunderstood. Budding of virions occurs at ER membranes. Pestivirus particles have been shown in intracellular vesicles and the Golgi complexand during exocytosis (Schmeiser et al., 2014). Considerable amounts of infectious virus remain cell-associated. Host cell RNA and proteinsynthesis continues throughout infection.

Biology

Pestiviruses infect pigs and ruminants, including cattle, sheep, goats and wild ruminants (Becher et al., 1997, Vilcek and Nettleton 2006).Moreover, pestivirus sequences have been detected in samples from bats and rats by next generation sequencing, but infectious pestiviruseshave not yet been isolated from these host species (Firth et al., 2014, Postel et al., 2015, Harasawa et al., 2000, Schirrmeier et al., 2004,Vilcek et al., 2005, Kirkland et al., 2007, Wu et al., 2012, Hause et al., 2015, Postel et al., 2016). There are no invertebrate hosts. Transmissionoccurs by direct and indirect contact (e.g., nasal or urine secretion, faeces, contaminated food, etc.) and transplacentally. Infections may besubclinical or produce a range of clinical conditions including acute diarrhea, acute hemorrhagic syndrome, acute fatal disease, and a wastingdisease. Transplacental infection can result in foetal death, congenital abnormalities, or lifelong persistent infection (Moennig and Plagemann1992). Fatal mucosal disease can occur in cattle persistently infected with noncp viruses when a cp virus is generated by mutation orintroduced by superinfection (Meyers and Thiel 1996). Pestivirus infections of livestock are economically important worldwide ( Moennig andBecher 2015, Houe 2003).

Experimental infection models have not been established for bovine or ovine pestiviruses outside their natural mammalian hosts; CSFV canbe adapted to propagate in rabbits (Tautz et al., 2015). Cells derived from natural host species (bovine, porcine, ovine) support virusreplication. Most virus isolates are noncp and can establish persistent infections in cell culture. Infectious noncp BVDV is often present inbovine serum products used for cell culture (Buttner et al., 1997). Cp pestiviruses induce extensive cytopathology and apoptosis, and formvirus plaques under appropriate conditions (Hilbe et al., 2013, Birk et al., 2008, Grummer et al., 2002, Jordan et al., 2002). Nohemagglutinating activity has been found associated with pestiviruses.

Antigenicity

Pestiviruses are antigenically related and cross-reactive epitopes have been documented for all species investigated (Tautz et al., 2015,Moennig et al., 1987). Separate antigenic determinants defined by monoclonal antibodies have also been identified. Antigenic variation isparticularly pronounced among isolates of BVDV and BDV (Becher et al., 2003, Postel et al., 2015). The N-terminal portion of E2 contains anantigenically hypervariable region (van Rijn et al., 1994, van Rijn et al., 1992). Monoclonal antibody binding patterns are generally consistentwith the genetic relatedness of viruses.

Infected animals mount potent antibody responses to two structural glycoproteins (E , E2) and to the NS2-3/NS3 protein, while antibodyresponses to other virus-encoded polypeptides are weak or not detectable. E and E2 are able to induce protection independently andmonoclonal antibodies reactive with these proteins can neutralize virus infectivity (Weiland et al., 1992, Weiland et al., 1990, Hulst et al., 1993van Zijl et al., 1991 , Reimann et al., 2004).

Species demarcation criteria

Species demarcation criteria in the genus include:

Nucleotide and deduced amino acid sequence relatedness.Antigenic relatedness.Host of origin.

Pestiviruses have been assigned to eleven different species based on phylogenetic analysis of conserved amino acid sequences in theregions 189–418, 1,547–2,321, 2,397–2,688 and 3,312– 3,837 (numbered according to the first amino acid of the polyprotein if BVDV-1 Sd-1,M96751) (Figure 3.Pestivirus) (Smith et al., 2017). Pestivirus species are named according to the format Pestivirus A, Pestivirus B, etc.replacing the previous names as follows: Pestivirus A replaces Bovine viral diarrhea virus 1 , Pestivirus B replaces Bovine viral diarrhea virus2, Pestivirus C replaces Classical swine fever virus and Pestivirus D replaces Border disease virus. Additional species in the genus includePestivirus E (pronghorn antelope virus), Pestivirus F (porcine pestivirus, Bungowannah virus), Pestivirus G (giraffe pestivirus), Pestivirus H(Hobi-like pestivirus, atypical ruminant pestivirus), Pestivirus I (Aydin-like pestivirus, sheep pestivirus), Pestivirus J (rat pestivirus) andPestivirus K (atypical porcine pestivirus) (Firth et al., 2014, Postel et al., 2015, Hause et al., 2015, Postel et al., 2016). Convalescent animalsera generated against members of a given species (e.g., Pestivirus A) generally show a several-fold higher neutralization titre againstviruses of the same species than against viruses from the other species (Becher et al., 2003, Postel et al., 2015). Finally, differences in host oforigin and disease can assist in species demarcation.

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Figure 3.Pestivirus. Phylogenetic tree of pestivirus amino acid positions 3,312–3,899. Maximum likelihood distances were calculated usinga JTT+G model in MEGA 6 (Tamura et al., 2013) using up to 15 of the most divergent sequences for each species, with elimination ofsequences <1 % divergent. Branches supported by >70 % of bootstrap replicates are indicated. Virus names and species assignments areindicated to the right. This phylogenetic tree and corresponding sequence alignment are available to download from the Resources page.

For example, Pestivirus A and Pestivirus C are considered different species because their members have: (i) amino acid sequences that arephylogenetically distinct in three different subgenomic regions (ii) at least 10-fold difference in neutralization titre in cross-neutralization testswith polyclonal immune sera, and (iii) host range, in that under natural conditions members of Pestivirus C infect only pigs while members ofPestivirus A infect ruminants as well as pigs.

Members of the species Pestivirus A can be further subdivided into at least seventeen genotypes, while three genotypes (CSFV-1, CSFV-2,and CSFV-3) are recognized among members of Pestivirus C (Yesilbag et al., 2014, Postel et al., 2012). Genotypes of these Pestivirusspecies can be further divided into subgroups.

Proposals for additional pestivirus species should be based on the complete genome sequence of at least one virus isolate, while data onantigenic relatedness and host range should also be considered. An incomplete genome sequence of an isolate from a bat (JQ814854)therefore remains unclassified.

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Member species

★ Exemplar isolate of the speciesSpecies Virus name Isolate Accession number RefSeq number Available sequence Virus Abbrev.

★ Pestivirus A bovine viral diarrhea virus 1 SD-1 M96751 Complete genome BVDV1Pestivirus A bovine viral diarrhea virus 1 NADL M31182 Complete genome BVDV1

★ Pestivirus B bovine viral diarrhea virus 2 XJ-04 FJ527854 Complete genome BVDV2Pestivirus B bovine viral diarrhea virus 2 890 U18059 Complete genome BVDV2

★ Pestivirus C classical swine fever virus Alfort/187 X87939 NC_038912 Complete genome CSFV★ Pestivirus D Border disease virus X818 AF037405 NC_003679 Complete genome BDV★ Pestivirus E pronghorn antelope pestivirus AY781152 NC_024018 Complete genome PAPeV★ Pestivirus F porcine pestivirus Bungowannah EF100713 NC_023176 Complete genome PPeV★ Pestivirus G giraffe pestivirus H138 AF144617 NC_003678 Complete genome GPeV★ Pestivirus H HoBi-like pestivirus Th/04_KhonKaen FJ040215 NC_012812 Complete genome HoBiPeV★ Pestivirus I Aydin-like pestivirus 04-TR JX428945 NC_018713 Complete genome AydinPeV★ Pestivirus J rat pestivirus NrPV/NYC-D23 KJ950914 NC_025677 Complete genome RPeV★ Pestivirus K atypical porcine pestivirus 515 KR011347 NC_038964 Complete genome APPeV

Virus names, the choice of exemplar isolates, and virus abbreviations, are not official ICTV designations.

Related, unclassified viruses

Virus name Accession number Virus abbreviationbat pestivirus JQ814854 BaPV

Virus names and virus abbreviations are not official ICTV designations.

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Authors: Flaviviridae

Rebecca Rico-Hesse*Flaviviridae Study Group ChairDepartment of Molecular Virology & MicrobiologyBaylor College of MedicineOne Baylor PlazaHouston, TX 77030USATel: 1-713-798-3010E-mail: rebecca.rico-hesse@bcm.edu

Tatjana Avsic-ZupancUniversity of Ljubljana | Faculty of MedicineINSTITUT OF MICROBIOLOGY AND IMMUNOLOGYZaloška 4,1000 LjubljanaSloveniaE-mail: tatjana.avsic@mf.uni-lj.si

Bradley BlitvichCollege of Veterinary Medicine1800 Christensen DriveAmes, Iowa 50011-1134Tel: 515-294-1242E-mail: blitvich@iastate.edu

Jens BukhCopenhagen Hepatitis C Program (CO-HEP)Department of Infectious Diseases and Clinical Research CentreCopenhagen University Hospital, Hvidovre, andDepartment of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of CopenhagenKettegaard Alle 30, DK-2650 HvidovreDenmarkTel: +45-23-41-89-69E-mail: jbukh@sund.ku.dk

Van-Mai Cao-LormeauInstitut Louis MalardéPO Box 3098713 PapeeteTahitiFrench PolynesiaE-mail: mlormeau@ilm.pf

Allison ImrieFaculty of Health and Medical SciencesUniversity of Western AustraliaCrawley, WAAustraliaE-mail: allison.imrie@uwa.edu.au

Amit KapoorCenter for Vaccines and ImmunityThe Ohio State University700 Children's Drive Room WA4015ColumbusOH 43205USAE-mail: Kapoor.102@osu.edu

Laura D KramerNYS Department of Health, Wadsworth CenterEmpire State PlazaP.O. Box 509AlbanyNew York 12201-0509USATel: 518 485-6632E-mail: laura.kramer@health.ny.gov

Brett D Lindenbach

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Microbial PathogenesisDepartment of Microbial Pathogenesis295 Congress AveNew Haven,CT 06536-0812USATel: 203.785.4705E-mail: brett.lindenbach@yale.edu

Peter Simmonds[Previous Flavivirdae Study Group Chair]Nuffield Department of MedicineUniversity of OxfordPeter Medawar BuildingSouth Parks RoadOxford, OX1 3SYUnited KingdomTel: +44 (0) 1865 281 233E-mail: Peter.Simmonds@ndm.ox.ac.uk

Donald B. SmithCentre for Immunity, Infection and EvolutionUniversity of EdinburghWest Mains Road,Edinburgh, EH9 3FLUnited KingdomTel: +44-131-650-7331E-mail: D.B.Smith@ed.ac.uk

Pedro Fernando da Costa VasconcelosSeção de Arbovirologia e Febres HemorrágicasInstituto Evandro ChagasAnanindeua,ParaBrazilE-mail: pedrovasconcelos@iec.pa.gov.br

* to whom correspondence should be addressed

Authors of a previous version of this Report:

Paul BecherInstitute of VirologyDepartment of Infectious DiseasesUniversity of Veterinary Medicine HannoverBuenteweg 17D-30559 HannoverGermanyTel: +49-511-953-8840E-mail: paul.becher@tiho-hannover.de

Ernest GouldUnité des Virus EmergentsFaculté de Médecine Timone5ème étage Aile Bleu27, Bd Jean Moulin13385 Marseille Cedex 05Tel: +44-7806-939165E-mail: eag@ceh.ac.uk

Gregor MeyersFriedrich Loeffler InstituteSüdufer 10D-17493 Greifswald-RiemsGermanyTel: +49-3835-171156E-mail: Gregor.Meyers@fli.bund.de

Thomas P. MonathBioProtection Systems/NewLink Genetics Corp.94 Jackson Rd. Suite 108Devens MA 01434

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USAPhoine: +1 515-598-2568tmonath@linkp.com

A. Scott Muerhoff Abbott Laboratories100 Abbott Park RoadDept. 09QS, AP20-4Abbott Park, IL 60064-6015USATel: +1-224-668-1077E-mail: scott.muerhoff@abbott.com

Alexander G. PletnevLaboratory of Infectious DiseasesNational Institute of Allergy and Infectious DiseasesNational Institutes of Health33 North DriveBethesda, MD 20892United States of AmericaTel: +1-301-402-7754E-mail: apletnev@niaid.nih.gov

Jack T. StapletonDepartments of Internal Medicine and MicrobiologyUniversity of Iowa, SW54, GH200 Hawkins Drive, UIHCIowa City, IA 52242USATel: +1-319-356-3168E-mail: jack-stapleton@uiowa.edu

The chapter in the Ninth ICTV Report, which served as the template for this chapter, was contributed by Simmonds, P., Becher, P., Collett, M.S.,Gould, E.A., Heinz, F.X., Meyers, G., Monath, T., Pletnev, A., Rice, C.M., Stiasny, K., Thiel, H.-J., Weiner, A. and Bukh, J.

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Resources: Flaviviridae

Websites:

ICTV Flaviviridae Study Group (dengue virus and hepatitis C virus subtypes):

https://talk.ictvonline.org/ictv_wikis/flaviviridae/w/sg_flavi

Sequence alignments and tree files:

Figure 1.Flaviviridae:

Tree file (newick format)

Alignment file (FASTA format)

Figure 3.Pestivirus:

Tree file (newick format)

Alignment file - amino acid (FASTA format)

Alignment file - nucleotide (FASTA format)

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Further reading: Flaviviridae

Gottwein, J. M., Scheel, T. K., Callendret, B., Li, Y. P., Eccleston, H. B., Engle, R. E., Govindarajan, S., Satterfield, W., Purcell, R. H.,Walker, C. M. & Bukh, J. (2010). Novel infectious cDNA clones of hepatitis C virus genotype 3a (strain S52) and 4a (strain ED43): geneticanalyses and in vivo pathogenesis studies. J Virol 84, 5277-5293. [PubMed]

Grard, G., Moureau, G., Charrel, R. N., Holmes, E. C., Gould, E. A. & de Lamballerie, X. (2010). Genomics and evolution of Aedes-borneflaviviruses. J Gen Virol 91, 87-94. [PubMed]

Gubler, D., Kuno, G. & Markoff, L. (2007). Flaviviruses. In Fields Virology, pp. 1153-1252. Edited by D. M. Knipe & P. M. Howley. Philadelphia:Lippincott Williams and Wilkins.

Hartlage, A. S., Cullen, J. M. & Kapoor, A. (2016). The Strange, Expanding World of Animal Hepaciviruses. Annu Rev Virol 3, 53-75. [PubMed]

Junglen, S., Kopp, A., Kurth, A., Pauli, G., Ellerbrok, H. & Leendertz, F. H. (2009). A new flavivirus and a new vector: characterization of a novelflavivirus isolated from uranotaenia mosquitoes from a tropical rain forest. J Virol 83, 4462-4468. [PubMed]

Lemon, S. M., Walker, C. M., Alter, M. J. & Yi, M. (2007). Hepatitis C Virus. In Fields Virology, pp. 1253-1304. Edited by D. M. Knipe & P. M.Howley. Philadelphia: Lippincott Williams and Wilkins.

Lindenbach, B. D., Thiel, H. J. & Rice, C. M. (2007). Flaviviridae: The Viruses and Their Replication. In Fields Virology, pp. 1101-1152. Edited byD. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams and Wilkins.

Mukhopadhyay, S., Kuhn, R. J. & Rossmann, M. G. (2005). A structural perspective of the flavivirus life cycle. Nat Rev Microbiol 3, 13-22.[PubMed]

Postel, A., Schmeiser, S., Oguzoglu, T. C., Indenbirken, D., Alawi, M., Fischer, N., Grundhoff, A. & Becher, P. (2015). Close relationship ofruminant pestiviruses and classical Swine Fever virus. Emerg Infect Dis 21, 668-672. [PubMed]

Simmonds, P., Bukh, J., Combet, C., Deleage, G., Enomoto, N., Feinstone, S., Halfon, P., Inchauspe, G., Kuiken, C., Maertens, G.,Mizokami, M., Murphy, D. G., Okamoto, H., Pawlotsky, J. M., Penin, F., Sablon, E., Shin, I. T., Stuyver, L. J., Thiel, H. J., Viazov, S., Weiner,A. J. & Widell, A. (2005). Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology 42, 962-973.[PubMed]

Tautz, N., Tews, B. A. & Meyers, G. (2015). The Molecular Biology of Pestiviruses. Adv Virus Res 93, 47-160. [PubMed]

Theze, J., Lowes, S., Parker, J. & Pybus, O. G. (2015). Evolutionary and Phylogenetic Analysis of the Hepaciviruses and Pegiviruses. GenomeBiol Evol 7, 2996-3008. [PubMed]

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References: Flaviviridae

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Citation: Flaviviridae

A summary of this ICTV Report chapter has been published as an ICTV Virus Taxonomy Profile article in the Journal of General Virology, andshould be cited when referencing this online chapter as follows:

Simmonds, P., Becher, B., Bukh, J., Gould, E.A., Meyers, G., Monath, T., Muerhoff, S., Pletnev, A., Rico-Hesse, R., Smith, D.B., Stapleton, J.T.,and ICTV Report Consortium. 2017, ICTV Virus Taxonomy Profile: Flaviviridae, Journal of General Virology, 98:2–3.

Funding support

Support for the preparation of this ICTV Report chapter and associated Journal of General Virology taxonomy profile, was funded by a grant fromthe Wellcome Trust (WT108418AIA).

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