*For correspondence. E-mail: youngmin.lee@usu.edu; Tel.: +1-435-797-9667Copyright 2017, The Microbiological Society of Korea

Sang-Im Yun1 and Young-Min Lee1,2*1Department of Animal, Dairy, and Veterinary Sciences; 2Utah Science Technology and Research, College of Agriculture and Applied Sciences, Utah State University, Logan, UT 84322-4815, USA

(Received Feb 10, 2017 / Accepted Feb 15, 2017)

Journal of Microbiology (2017) Vol. 55, No. 3, pp. 204–219DOI 10.1007/s12275-017-7063-6

eISSN 1976-3794pISSN 1225-8873

REVIEW

Zika virus: An emerging flavivirus

Zika virus (ZIKV) is a previously little-known flavivirus clo-sely related to Japanese encephalitis, West Nile, dengue, and yellow fever viruses, all of which are primarily transmitted by blood-sucking mosquitoes. Since its discovery in Uganda in 1947, ZIKV has continued to expand its geographic range, from equatorial Africa and Asia to the Pacific Islands, then further afield to South and Central America and the Carib-bean. Currently, ZIKV is actively circulating not only in much of Latin America and its neighbors but also in parts of the Pacific Islands and Southeast Asia. Although ZIKV infection generally causes only mild symptoms in some infected indi-viduals, it is associated with a range of neuroimmunological disorders, including Guillain-Barré syndrome, meningoence-phalitis, and myelitis. Recently, maternal ZIKV infection du-ring pregnancy has been linked to neonatal malformations, resulting in various degrees of congenital abnormalities, mi-crocephaly, and even abortion. Despite its emergence as an important public health problem, however, little is known about ZIKV biology, and neither vaccine nor drug is avail-able to control ZIKV infection. This article provides a brief introduction to ZIKV with a major emphasis on its molecular virology, in order to help facilitate the development of diag-nostics, therapeutics, and vaccines.

Keywords: Zika virus, flavivirus, mosquito-borne virus, ar-bovirus, microcephaly, Guillain-Barré syndrome, replica-tion, pathogenesis

Introduction

DiscoveryZika virus (ZIKV) was named after the Zika Forest area of Uganda (Fig. 1), where it was first isolated from the serum of a pyrexial rhesus monkey (named Rhesus 766) in April 1947 and then from homogenates of Aedes africanus mos-

quitoes in January 1948 (Dick et al., 1952). Both isolations were made by intracerebral inoculation of the samples into Swiss albino mice. Although their results should be inter-preted with caution because of the serologic cross-reactivity with related flaviviruses, a sizable number of seroepidemio-logical surveys for ZIKV began to suggest its wide geographic distribution in a band of equatorial African countries, in-cluding the Central African Republic, Egypt, Gabon, Kenya, Nigeria, Senegal, Sierra Leone, Tanzania, and Uganda (Smi-thburn, 1952; Smithburn et al., 1954b; Dick, 1953; Macnamara, 1954; Geser et al., 1970; Robin and Mouchet, 1975; Fagbami, 1977; Jan et al., 1978; Saluzzo et al., 1981, 1982; Adekolu-John and Fagbami, 1983; Monlun et al., 1993), and in parts of Asia, including India, Indonesia, Malaysia, Pakistan, the Philip-pines, Thailand, and Vietnam (Smithburn, 1954; Smithburn et al., 1954a; Hammon et al., 1958; Pond, 1963; Darwish et al., 1983; Olson et al., 1983). However, ZIKV received little atten-tion in the medical literature until the early 2000s because it was reported to be associated with only about a dozen cases of mild self-limiting febrile illnesses in such endemic areas of Africa and Asia (Hayes, 2009), namely Indonesia, Nigeria, and Uganda (Macnamara, 1954; Simpson, 1964; Moore et al., 1975; Fagbami, 1979; Olson et al., 1981).

Historical outbreaksMore recently, in the last decade, ZIKV has gained global attention (Kindhauser et al., 2016). In April 2007, the first major ZIKV outbreak outside of Africa and Asia occurred on Yap Island in the Federated States of Micronesia, located in the northwestern Pacific Ocean (Fig. 1); this outbreak was associated with fever, rash, arthralgia, and conjunctivitis (Lanciotti et al., 2008; Duffy et al., 2009). A post-outbreak serological survey for ZIKV antibodies in Yap Island sug-gested that approximately three-quarters of the ~7,000 peo-ple aged 3 and older had recently been exposed to ZIKV, but only one of every five infected people had developed clinical disease related to ZIKV infection, with no serious complica-tions being seen (Duffy et al., 2009). After the Yap outbreak, only single cases or small clusters of acute ZIKV infection in native residents and foreign travelers had been sporadi-cally diagnosed until the mid-2010s in endemic countries in Southeast Asia, i.e., Cambodia, Indonesia, Malaysia, Thailand, and the Philippines (Heang et al., 2012; Kwong et al., 2013; Fonseca et al., 2014; Tappe et al., 2014, 2015; Alera et al., 2015; Buathong et al., 2015; Perkasa et al., 2016). In October 2013, the second large ZIKV outbreak started in French Polynesia in the south-central Pacific Ocean (Fig. 1); French Polynesia consists of 118 islands (grouped into five

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Fig. 1. Temporal and geographical distribution of ZIKV (1947 to February 1, 2016). Highlighted are the discovery of ZIKV (Uganda, 1947) and three his-torical ZIKV outbreaks: Yap Island (2007), French Polynesia (2013-2014), and Latin America (2015-2016). Reproduced from Kindhauser et al. (2016).

archipelagoes), 67 of which are inhabited, with a total pop-ulation of ~277,000 (Cao-Lormeau et al., 2014). This out-break resulted in an estimated 28,000 individuals seeking medical treatment for suspected ZIKV infection (Besnard et al., 2014; ECDC, 2014; Musso et al., 2014). Unlike the pre-vious outbreaks of ZIKV, a striking 20-fold increase in the incidence of Guillain-Barré syndrome was observed for the first time during this outbreak (ECDC, 2014; Oehler et al., 2014; Cao-Lormeau et al., 2016; Watrin et al., 2016), raising concerns about an association between ZIKV infection and Guillain-Barré syndrome. In addition to the French Polynesia outbreak, multiple relatively small-scale outbreaks were also recorded during 2013-2014 in a cluster of South Pacific is-lands (Cao-Lormeau and Musso, 2014; Musso et al., 2014, 2015), including the Cook Islands (Roth et al., 2014), New Caledonia (Dupont-Rouzeyrol et al., 2015), and Easter Island (Tognarelli et al., 2016). In early 2015, four studies reported the detection of ZIKV RNA among 19 acute-phase serum samples from local resi-dents and visitors to northeastern Brazil who presented with common symptoms of fever, rash, myalgias/arthralgia, and conjunctivitis (Campos et al., 2015; Cardoso et al., 2015; Zammarchi et al., 2015; Zanluca et al., 2015), indicating the first autochthonous transmission of ZIKV in the Western Hemisphere (Fig. 1). By December 2015, between 440,000 and 1,300,000 suspected cases of ZIKV infection were re-corded in at least 14 of the 26 states in Brazil (WHO, 2015; Hennessey et al., 2016). In addition to an increased number of instances of Guillain-Barré syndrome, a significant in-crease in the incidence of microcephaly among newborn infants was recognized for the first time during the ZIKV outbreak in Brazil (Schuler-Faccini et al., 2016), suggesting a possible association between ZIKV infection and micro-cephaly. In Brazil, fewer than 200 cases of microcephaly were

documented annually prior to 2015, but from mid-2015 to January 2016, more than 4000 suspected cases of microce-phaly were reported, although some of these cases may rep-resent over- or mis-diagnosis (Victora et al., 2016). Further evidence for a ZIKV-microcephaly association came from two retrospective studies in French Polynesia conducted af-ter the 2013-2014 ZIKV outbreak (Cauchemez et al., 2016; Jouannic et al., 2016). Since its introduction into Brazil, ZIKV has spread ex-plosively across South and Central America and the Carib-bean Islands, and there has been mounting evidence of its epidemiological link to various neurological disorders (e.g., Guillain-Barré syndrome) and neonatal malformations (e.g., microcephaly). As a result, the World Health Organization announced a “Public Health Emergency of International Concern” for the nine months beginning in February 2016 (WHO, 2016a, 2016b). As of January 12, 2017, the mosquito- borne transmission of ZIKV had been reported in 48 Pan- American countries and territories (PAHO/WHO, 2017), with an accumulated number of >170,000 confirmed and >510,000 suspected cases (PAHO/WHO, 2016). Also, the congenital syndrome associated with ZIKV infection had been documented in 22 Pan-American countries and terri-tories (PAHO/WHO, 2017), with a total of >2,300 confirmed cases (PAHO/WHO, 2016). As predicted (Bogoch et al., 2016; Fauci and Morens, 2016; Lessler et al., 2016; Musso and Gubler, 2016), ZIKV expanded its geographic range to North America, emerging first in southern Florida in July 2016 and causing 211 confirmed cases of local mosquito- borne transmission in that area by January 2017 (CDC, 2017a, 2017c). Thereafter, Texas became the second state in the United States (US) to confirm a locally transmitted case of ZIKV in November 2016 and had identified five ad-ditional locally acquired cases by January 2017 (CDC, 2017a,

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Fig. 2. Phylogenetic relationships among 73 viruses of the genus Flavivirus (A) and among 29 isolates of ZIKV (B). The phylogenetic tree of the genus Flavivirus is reproduced from Gould and Solomon (2008). The phylogenetic tree of ZIKV was produced by the neighbor-joining method (bootstrapped 1,000 times) using the nucleotide sequence of complete or near-complete genomes. Shown are the strain name, country and year of isolation. Highlighted are the two genetic lineages, African (green) and Asian (orange), together with the eighteen 2015-2016 Latin American epidemic strains (red) that belong to the Asian lineage. The scale bar indicates the number of nucleotide substitutions per site. Note that MR-766 has been fully sequenced by four research groups, and their nucleotide sequences are not identical, most likely because of differences in the cultivation history of the virus.

2017c). As of January 10, 2017, the US Zika Pregnancy Regi-stry database had collected information on 37 newborn in-fants with birth defects and 5 pregnancy losses (CDC, 2017b). Since December 14, 2016, the active autochthonous trans-mission of ZIKV has been considered ongoing not only in much of Latin America and its neighbors but also in parts of the Pacific Islands and Southeast Asia (CDC, 2016; ECDC, 2017). It is highly likely that ZIKV will become endemic in the entire Western Hemisphere, as well as in Africa and Southeast Asia in the Eastern Hemisphere.

Virology

TaxonomyAccording to the tenth report of the International Commit-tee on Taxonomy of Viruses, the family Flaviviridae is cur-rently divided into four genera: (1) Flavivirus, which com-prises a list of 53 species, each transmitted to vertebrates by

mosquitoes, ticks, or no known arthropod vector; (2) Hepaci-virus, which comprises a single species, hepatitis C virus, the members of which are grouped into seven genotypes; (3) Pegivirus, which comprises two species, pegivirus A (e.g., he-patitis G virus, simian pegivirus-chimpanzee, and six geno-types of human pegiviruses) and pegivirus B (e.g., bat pegi-virus-68); and (4) Pestivirus, which comprises four species, border disease virus, classical swine fever virus, and bovine viral diarrhea viruses 1 and 2 (Simmonds et al., 2017). Within the Flaviviridae family, ZIKV is a member of the mosquito- borne flaviviruses (Gubler et al., 2007; Gould and Solomon, 2008), as are several major human and animal pathogens, including Japanese encephalitis virus (JEV), West Nile virus (WNV), Murray Valley encephalitis virus, St. Louis encepha-litis virus, Usutu virus, dengue virus (DENV), and yellow fever virus (YFV) (Fig. 2A).

Genetic diversityThere is very limited information regarding the genetic di-

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Fig. 3. Cryo-EM structure of ZIKV. (A) A surface-shaded and depth-cued illustration of ZIKV. A black triangle indicates one asymmetric unit. The relative positions of the 5-, 3-, and 2-fold interfaces of symme-try are indicated. (B) A cross-section image of ZIKV. The radial density gradient is col-or-coded: <130 Å (blue), 131-150 Å (cyan), 151-190 Å (green), 191-230 Å (yellow), and>231 Å (red). The nucleocapsid (blue) does not follow icosahedral symmetry. (C) An arrangement of the viral M and E proteins on the surface of a mature virion. A total of 180 E monomers form 30 protein rafts of three E homodimers in a herringbone pat-tern. (D) Structures of the ZIKV M and E proteins. The top panel shows the E dimer viewed down the 2-fold axis. Three domainsof each E monomer are color-coded: do-main I (DI, red), domain II (DII, yellow), and domain III (DIII, blue). The fusion loop located at the tip of DII is indicated in green. The Asn-154 glycan and the variableloop surrounding it are highlighted. The bottom panel shows the M-E dimer, inclu-ding the three E ectodomains (color-coded as top panel), the E-stem and E-transmem-brane (TM) domains (light pink), and the M-loop and M-transmembrane (TM) do-mains (light blue). Reproduced from Sirohiet al. (2016).

versity of ZIKV, particularly before early 2015, when the Latin American outbreak started in Brazil. As of January 25, 2017, the GenBank database contained the nucleotide sequences of 591 ZIKV isolates/strains; of these, 444 had been released since January 1, 2015, and only 93 were the complete or near-complete genome sequences (Kuno and Chang, 2007; Lanciotti et al., 2008; Baronti et al., 2014; Berthet et al., 2014; Barzon et al., 2016; Cunha et al., 2016; Ellison et al., 2016; Giovanetti et al., 2016; Ladner et al., 2016; Lednicky et al., 2016; Liu et al., 2016; Yun et al., 2016a; Zhu et al., 2016). As presented in Fig. 2B, recent phy-logenetic analyses using a limited but considerable number of the complete or near-complete genome sequences has revealed that ZIKVs are clustered into the two major ge-netic lineages (African and Asian) that are geographically and temporally distinct (Haddow et al., 2012; Faria et al., 2016; Lanciotti et al., 2016; Mlakar et al., 2016; Wang et al., 2016). The Asian lineage has expanded its geographic range since the early 2000s, causing the three historical ZIKV out-breaks: (1) the 2007 Yap Island outbreak, which most likely originated in Southeast Asia (Lanciotti et al., 2008; Duffy et al., 2009; Haddow et al., 2012); (2) the 2013-2014 French Polynesia outbreak, which presumably resulted from the introduction of a strain similar to the Cambodia (2010) and Yap Island (2007) strains (Cao-Lormeau et al., 2014); and (3) the 2015-2016 Latin America outbreak, which was prob-ably caused by the spread of an Asian lineage-derived strain from the Pacific Islands (Faria et al., 2016).

Virus structureElectron microscopy (EM) has shown ZIKV to be an en-

veloped, spherical virus with a diameter of ~50 nm (Hamel et al., 2015; Barreto-Vieira et al., 2016) (Fig. 3A). Inside the mature flavivirus particle, multiple copies of the α-helical capsid (C) protein form an unstructured nucleocapsid com-plexed with one copy of the genomic RNA (Dokland et al., 2004; Ma et al., 2004). The inner nucleocapsid is enclosed in a lipid bilayer derived from the modified membrane of the endoplasmic reticulum (ER), with the two surface proteins membrane (M) and envelope (E) anchored in the bilayer through their two C-terminal transmembrane domains (Kuhn et al., 2002; Mukhopadhyay et al., 2003; Zhang et al., 2003a, 2013b) (Fig. 3B). Recently, three-dimensional cryo-EM struc-tures of the mature ZIKVs have been solved at 3.7-3.8 Å re-solution (Kostyuchenko et al., 2016; Sirohi et al., 2016), re-vealing that the overall architecture of ZIKV is similar to that of other structurally well-defined flaviviruses, such as WNV (Mukhopadhyay et al., 2003; Zhang et al., 2013a) and DENV (Zhang et al., 2013b). Specifically, the smooth outer layer of the mature ZIKVs is constituted by 180 copies of the E protein, arranged as 90 antiparallel homodimers in a “herringbone” pattern with icosahedral symmetry (Fig. 3C). Each E monomer has three topological parts (Fig. 3D): (1) an elongated ectodomain, which possesses both receptor bin-ding and membrane fusion activity; (2) a “stem” region, which contains two α-helices lying almost flat on the viral membrane underneath the ectodomain; and (3) a “trans-membrane” region, which has an antiparallel coiled-coil hairpin (Stiasny et al., 1996; Allison et al., 1999; Zhang et al., 2003a, 2004). Also, the E ectodomain forms three structural domains (Fig. 3D): (i) domain I, which is located at the cen-ter of the ectodomain; (ii) domain II, which contains the hy-drophobic fusion loop at its distal end (Allison et al., 2001);

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Fig. 4. Genome structure, polyprotein processing, and protein properties of ZIKV. (A) Genome structure. The positive-sense genomic RNA of ZIKV PRVABC- 59 is composed of a 5’NCR, a single long ORF, and a 3’NCR. (B) Polyprotein processing. The viral ORF encodes a 3,423-amino-acid polyprotein, which is co- or post-translationally processed by host- and virus-encoded proteases (as indicated) into three structural (C, prM, and E) and at least seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. Vertical black bars represent one or two transmembrane domains located at the junctions of C/prM (designated “anchor”), prM/E, E/NS1, and NS4A/NS4B (designated “2K”). Also indicated are the putative cleavage sites conserved among flavivi-ruses and the lengths of the cleavage products. (C) Hydropathy plot of the 3,423-amino-acid polyprotein of ZIKV PRVABC-59. The average hydropathy scores were calculated using the Kyte-Doolittle method, and protein regions scoring above 0 are considered to be hydrophobic.

and (iii) domain III, which is implicated in receptor bin-ding and antibody neutralization (Chen et al., 1997; Crill and Roehrig, 2001; Beasley and Barrett, 2002; Lee and Lobigs, 2002; Wu et al., 2003; Kaufmann et al., 2006; Lee et al., 2006; Barba-Spaeth et al., 2016; Dai et al., 2016; Stettler et al., 2016). Of note, ~10 amino acids around the N-linked glycosylation site at Asn-154 in domain I of ZIKV E protrude from the surface of the virion, suggesting that the glycan at Asn-154 may serve as an attachment site for the virus to host cells (Sirohi et al., 2016). Previous work on WNV indicated that Asn-154 glycosylation enhanced viral neuroinvasiveness in mice (Beasley et al., 2005).

Genome organization and gene expressionZIKV contains a non-segmented, linear, single-stranded posi-tive-sense RNA genome, typically 10,807 nucleotides in length (Yun et al., 2016a), although some differences in length have been reported among different isolates and even among the prototype MR-766 strains with different passage histories (Lanciotti et al., 2008; Haddow et al., 2012; Baronti et al., 2014; Berthet et al., 2014). The genomic RNA has a type I cap structure (m7GpppAmG) at its 5’ end (Ray et al., 2006; Dong et al., 2014), followed by a 5’ non-coding region (NCR) of 106-107 nucleotides, a single open reading frame (ORF) of 10,272 nucleotides, and a 3’ NCR of 428-429 nucleotides, with no poly-A tail at the 3’ end (Fig. 4A). In flaviviruses, the presence of a properly methylated 5’ cap on the viral genomic RNA plays an important role in viral translation/replication and evasion of host restriction in viral replication (Dong et al., 2008a; Daffis et al., 2010; Zust et al., 2011; Klema et al.,

2015). The 5’NCR is required for modulating viral transla-tion and RNA replication through the long-distance RNA- RNA interaction with the 3’NCR (Iglesias and Gamarnik, 2011; Brinton and Basu, 2015); the 3’NCR is also involved in counteracting the host cell’s response to virus infection by serving as a substrate for host cell exoribonucleases to gen-erate short non-coding subgenomic flaviviral RNAs (sfRNAs), thereby contributing to viral replication and pathogenesis (Pijlman, 2014; Clarke et al., 2015; Charley and Wilusz, 2016). As outlined in Fig. 4B, the ORF encoded in the genomic RNA is translated into a polyprotein of 3,423 amino acids, which is predicted to be cleaved co- or post-translationally into at least 10 major individual proteins (Lindenbach et al., 2013): three structural (C, prM, and E) and seven nonstruc-tural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) pro-teins positioned from N- to C-terminus in the polyprotein (Kim et al., 2015). The proteolytic processing of the flavivi-rus polyprotein is mediated by a combination of four dif-ferent proteases: (1) the host signal peptidase at the junc-tions of C-“anchor”/prM, prM/E, E/NS1, and NS4A-“2K”/ NS4B (Castle et al., 1985; Wengler et al., 1985; Mason et al., 1987; Chambers et al., 1990b; Lobigs, 1993; Stocks and Lobigs, 1998); (2) the viral NS3 serine protease, which re-quires NS2B as a cofactor, at the junctions of C/“anchor”, NS2A/NS2B, NS2B/NS3, NS3/NS4A, NS4A/“2K”, and NS4B/ NS5 (Castle et al., 1986; Mason et al., 1987; Speight et al., 1988; Chambers et al., 1990b; Preugschat and Strauss, 1991; Lin et al., 1993; Amberg et al., 1994; Yamshchikov and Com-pans, 1994); (3) the host furin or furin-like protease at the junction of pr/M (Stadler et al., 1997); and (4) an unknown

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Fig. 5. Predicted primary and secondary/tertiary RNA structures found in the 5’- and 3’-terminal sequences of ZIKV PRVABC-59. The 5’-terminal sequences contain the predicted SLA, SLB, cHP, and DCS-PK structures (A), and the 3’-terminal sequences contain the predicted SL1, SL2, DB, sHP, and 3’SL struc-tures (B). The three pairs of complementary sequences involved in viral genome circularization are 5’/3’UAR (red), 5’/3’DAR (blue), and 5’/3’CS (green). Nucleotide position refers to the complete nucleotide sequence of ZIKV PRVABC-59 (GenBank accession number KX377337).

host protease at the junction of NS1/NS2A (Falgout et al., 1989; Hori and Lai, 1990; Falgout and Markoff, 1995). Basi-cally, the predicted cleavage sites in the ZIKV polyprotein conform to the rules established for closely related mosquito- borne flaviviruses (Rice et al., 1985; Chambers et al., 1990a). Also, the hydropathy profile of the ZIKV polyprotein (Fig. 4C) agrees well with the current membrane topology model of the flavivirus polyprotein (Assenberg et al., 2009). In addition to the expression of a single polyprotein pre-cursor and its proteolytic processing, in JEV and WNV, a -1 ribosomal frameshifting event has been shown to occur, presumably at codons 8-9 of NS2A in the genomic RNA (Firth and Atkins, 2009; Melian et al., 2010; Kim et al., 2015); this frameshift results in the production of a 52-amino acid C-terminally extended form of NS1 (designated NS1’). This frameshifting was originally predicted to be induced by two stimulatory elements, a conserved slippery heptanucleotide (YCCUUUU) and a potential RNA pseudoknot located a few nucleotides downstream of the slippery sequence (Firth and Atkins, 2009). In the case of a highly pathogenic wild- type JEV strain, SA14, the predicted pseudoknot consists of two directly adjacent, presumably coaxially stacked stem loops (SL1 and SL2) with a 5-nucleotide loop region (3585CUG-

GC3589) of SL1 base-paired with its complementary sequence (3621GCCAG3625), six nucleotides downstream of SL2 (Yun et al., 2016b). In both JEV and WNV, the expression of NS1’ has been shown to be abolished by altering the slip-pery sequence or destabilizing the predicted pseudoknot, consequently reducing viral virulence in mice (Melian et al., 2010; Ye et al., 2012). In the case of ZIKV, the two fra-meshift-stimulating elements are absent from the viral ge-nomic RNA, but it remains to be determined experimentally whether NS1’ is expressed in ZIKV-infected cells.

Conserved primary sequences and RNA secondary structuresA complex of cis-acting RNA sequences and structures plays critical roles in viral translation and RNA replication as well as viral pathogenicity (Bidet and Garcia-Blanco, 2014; Villordo et al., 2016). As an initial step toward understand-ing these RNA-RNA interactions in ZIKV replication, RNA secondary structure prediction using the Mfold program (Zuker, 2003), guided by the cis-acting RNA elements that are highly conserved in mosquito-borne flaviviruses (Paran-jape and Harris, 2010; Gebhard et al., 2011), has identified the potential cis-acting primary and higher-order RNA struc-

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Fig. 6. Replication cycle of flaviviruses. Highlighted are the eight major steps of the viral replication cycle: attachment, endocytosis, membrane fusion, transla-tion, RNA replication, assembly, maturation, and release.

tures embedded in the 5’- and 3’-terminal regions of the ge-nomic RNA of ZIKV PRVABC-59, the current Latin Ameri-can epidemic strain isolated in Puerto Rico from a patient in 2015 (Lanciotti et al., 2016). As depicted in Fig. 5A, five major RNA elements predicted at the 5’-terminal region are: (1) the 5’-end stem-loop A (SLA), which binds viral NS5 protein and promotes RNA synthesis (Thurner et al., 2004; Filomatori et al., 2006; Dong et al., 2008b; Lodeiro et al., 2009); (2) a string of three short conserved sequences termed the 5’ sequence upstream of the AUG region (5’UAR), the 5’ sequence downstream of the AUG region (5’DAR), and the 5’ cyclization sequence (5’CS), which is involved in viral genome cyclization through base-pairing with complemen-tary sequences located in the 3’NCR (Khromykh et al., 2001; Alvarez et al., 2005; Song et al., 2008; Zhang et al., 2008; Friebe and Harris, 2010; Friebe et al., 2011); (3) the stem- loop B (SLB), which is located just downstream of SLA, with the 5’UAR embedded within it (Filomatori et al., 2006); (4) a hairpin in the C protein-coding region (cHP), which is critical for recognition of the translation start codon and for RNA synthesis (Clyde and Harris, 2006; Clyde et al., 2008); and (5) a 3-stem pseudoknot termed the sequence downstream of the 5’CS-pseudoknot (DCS-PK), which en-hances viral RNA replication by regulating genome cycliza-tion (Liu et al., 2013). As illustrated in Fig. 5B, five well-defined RNA elements are predicted in the 3’-terminal region of ZIKV genomic RNA: (i) the 3’-end long stem-loop (3’SL), which is required for viral RNA replication (Zeng et al., 1998; Tilgner and Shi, 2004; Elghonemy et al., 2005; Yu and Markoff, 2005; Yun et al., 2009; Davis et al., 2013) and is known to interact with a number of cellular proteins (Davis et al., 2007; Yocupicio-

Monroy et al., 2007; Vashist et al., 2009; Gomila et al., 2011); (ii) a series of three short conserved sequences (3’UAR, 3’DAR, and 3’CS) that are involved in the long-range intra-genomic RNA-RNA interaction with their complementary sequences (5’UAR, 5’DAR, and 5’CS) for viral genome cir-cularization, a prerequisite for RNA replication (Khromykh et al., 2001; Alvarez et al., 2005; Song et al., 2008; Zhang et al., 2008; Friebe and Harris, 2010; Friebe et al., 2011); (iii) a small hairpin (sHP), which is located immediately upstream of 3’SL and partially overlapping the sequences of the 3’UAR and 3’DAR, thus suggesting a role in switching between the linear and circular forms of viral genomic RNA during rep-lication (Song et al., 2008; Villordo et al., 2010); (iv) a dumb-bell-shaped structure (DB) with its distal loop involved in pseudoknot formation, which has an important role in vi-ral RNA replication (Olsthoorn and Bol, 2001; Yun et al., 2009); and (v) a pair of duplicated stem-loops (SL1 and SL2), each of which is involved in multiple pseudoknot inter-actions and promotes sfRNA production and host adap-tion (Funk et al., 2010; Chapman et al., 2014; Villordo et al., 2015; Akiyama et al., 2016). As with other mosquito- and tick-borne flaviviruses (Pijl-man et al., 2008; Funk et al., 2010; Silva et al., 2010), ZIKV is known to produce sfRNAs as the result of incomplete degradation of the genomic RNA by the cellular 5’ 3’ ex-oribonuclease 1 (XRN1), which stalls at a well-defined but highly structured RNA, such as SL1, SL2, or DB (Akiyama et al., 2016). JEV sfRNAs are thought to play an important role in modulating the transition between translation and replication of the viral genomic RNA (Lin et al., 2004; Fan et al., 2011). Also, WNV sfRNAs have been shown to be critical for viral cytopathogenicity in cells and pathogeni-

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city in mice (Pijlman et al., 2008; Liu et al., 2014). In several mosquito-borne flaviviruses (JEV, WNV, and DENV), these RNAs can dysregulate the host cell’s antiviral and stress re-sponses (Roby et al., 2014), including RNAi pathways (Sch-nettler et al., 2012; Moon et al., 2015; Goertz et al., 2016), mRNA decay pathways (Moon et al., 2012), and the type I interferon response (Schuessler et al., 2012; Chang et al., 2013; Bidet et al., 2014; Manokaran et al., 2015). Further studies are needed to determine the precise roles of sfRNAs in the replication and pathogenesis of ZIKV.

Viral replicationVery little is known about how ZIKV enters susceptible cells and replicates inside them. Here, we provide an over-view of the replication cycle of flaviviruses to point to pro-mising avenues for ZIKV research. As schematically shown in Fig. 6, most flaviviruses initiate their infection of target cells by attaching to the cell surface, with virions binding to nonspecific host factors (Chen et al., 1997; Navarro-Sanchez et al., 2003; Tassaneetrithep et al., 2003; Davis et al., 2006; Pokidysheva et al., 2006). It has been reported that ZIKV enters the cell using adhesion factors, such as the lectin DC- SIGN and diverse members of the phosphatidylserine re-ceptor family (Hamel et al., 2015). This attachment step most likely precedes a specific interaction of the viral E glyco-protein with an as-yet unknown cellular receptor(s) (Perera- Lecoin et al., 2013; Pierson and Kielian, 2013) in order to gain access into the cell’s interior via rapid clathrin-medi-ated endocytosis (Chu and Ng, 2004; Chu et al., 2006; van der Schaar et al., 2007, 2008; Acosta et al., 2008; Mosso et al., 2008). Upon internalization, the viruses are transported to the endosomes, where the low pH causes specific conforma-tional changes in their E glycoproteins that trigger the fusion between viral and endosomal membranes. Comparison of the pre- and post-fusion atomic structures of the E ectodo-main (Rey et al., 1995; Modis et al., 2003, 2004; Bressanelli et al., 2004; Zhang et al., 2004; Kanai et al., 2006; Nybakken et al., 2006; Nayak et al., 2009), together with their biochemical properties (Allison et al., 1995; Corver et al., 2000; Stiasny et al., 2002, 2004, 2007; Liao et al., 2010), generated the current model for flavivirus membrane fusion: Mechanically, the fusion is preceded by a low pH-induced dissociation of the antiparallel E homodimers, exposure of the fusion loops, and their insertion into the target membrane, followed by a global structural rearrangement into a parallel E homotrimer capable of inducing membrane fusion (Kaufmann and Ross-mann, 2011; Stiasny et al., 2011; Harrison, 2015). Upon membrane fusion, the viral genomic RNA is released from the virion and enters the cytosol, where it functions as an mRNA and directs the synthesis of a polyprotein in association with the ER. The polyprotein is cleaved by cel-lular and viral proteases to generate three structural pro-teins and at least seven nonstructural proteins in equimolar amounts (Lindenbach et al., 2013; Kim et al., 2015). Fol-lowing translation, the viral positive-sense genomic RNA serves as a template for the synthesis of its complementary negative-sense RNAs; successively, the newly made neg-ative-sense RNAs act as templates for the synthesis of new positive-sense genomic RNAs (Iglesias and Gamarnik, 2011; Selisko et al., 2014). RNA replication occurs in the viral re-

plication complexes inside the virus-induced ER-derived membranous compartments (Welsch et al., 2009; Gillespie et al., 2010; Hsu et al., 2010; Chatel-Chaix and Bartenschlager, 2014). All of the seven nonstructural proteins are directly or indirectly involved in viral RNA replication (Yun and Lee, 2006; Bollati et al., 2010; Brinton, 2013), and one or more of these proteins also plays an important role in viral as-sembly (Kummerer and Rice, 2002; Liu et al., 2003; Pijlman et al., 2006; Leung et al., 2008; Patkar and Kuhn, 2008; Wu et al., 2015; Xie et al., 2015) and evasion of the host cell in-nate immunity (Munoz-Jordan et al., 2003, 2005; Guo et al., 2005; Jones et al., 2005; Liu et al., 2005; Morrison et al., 2012; Dalrymple et al., 2015). Multiple enzymatic activities have been demonstrated by the two nonstructural proteins, NS3 and NS5 (Bollati et al., 2010). Like other flaviviruses, ZIKV NS3 consists of an N-terminal serine protease domain and a C-terminal RNA helicase domain (Jain et al., 2016; Lei et al., 2016; Phoo et al., 2016; Zhang et al., 2016b), and ZIKV NS5 is composed of an N-terminal methyltransferase do-main (Coloma et al., 2016; Coutard et al., 2016; Zhang et al., 2016a) and a C-terminal RNA-dependent RNA polymerase domain (Cox et al., 2015; Adiga, 2016). Moreover, the crys-tal structure of ZIKV NS1 has also been solved; this protein plays multiple roles in the flavivirus life cycle, contributing to viral replication, pathogenesis, and immune evasion (Song et al., 2016; Xu et al., 2016). The four hydrophobic nonstruc-tural proteins NS2A, NS2B, NS4A, and NS4B are associated with intracellular membranes, where they are involved in rearranging cytoplasmic membranes and assembling the vi-ral replication complex (Westaway et al., 1997; Mackenzie et al., 1998; Roosendaal et al., 2006; Miller et al., 2007; Mc-Lean et al., 2011; Kaufusi et al., 2014). For viral assembly, two viral glycoproteins (prM and E) form a heterodimer on the ER membrane and drive the bud-ding of the viral genomic RNA and C proteins into the ER lumen (Apte-Sengupta et al., 2014), producing the immature noninfectious virions characterized by 90 prM-E hetero-dimers protruding from the viral membrane as 60 trimeric spikes (Lorenz et al., 2002; Zhang et al., 2003b, 2007; Li et al., 2008). These particles move through the cellular secre-tory pathway, during which the trans-Golgi-resident furin or furin-like protease cleaves prM, generating M protein and the “pr” peptide (Guirakhoo et al., 1991; Stadler et al., 1997; Elshuber et al., 2003; Yu et al., 2009). This proteolytic proc-essing leads to major structural rearrangements of the M and E proteins, organizing the latter as 90 head-to-tail homo-dimers lying horizontally along the external surface of the viral membrane (Zhang et al., 2004; Li et al., 2008; Yu et al., 2008). The completely or partially mature virions are then transported to the cell surface for release by exocytosis (Junjhon et al., 2010; Pierson and Diamond, 2012).

Conclusion

ZIKV is an emerging mosquito-borne flavivirus that has been circulated silently in a number of countries across equa-torial Africa and Asia for the last half century. However, it spread eastward from those endemic areas to the Pacific Islands in 2007 and further to the Americas in 2015. Since

212 Yun and Lee

then, ZIKV has been actively circulating in these newly in-vaded areas. ZIKV infection in humans is usually asympto-matic, with only a small portion of those infected developing mild febrile illnesses. In recent years, however, ZIKV has been recognized as a previously overlooked cause of severe neurological disorders, such as Guillain-Barré syndrome and microcephaly. From a virology standpoint, ZIKV is a rela-tively little-known flavivirus, as compared to such other taxo-nomically related and clinically important mosquito-borne flaviviruses as JEV, WNV, DENV, and YFV, but it shares many biological and molecular properties with these viruses, including virion morphology, genome organization, gene ex-pression, and replication strategy. However, the pathogenesis of ZIKV infection is distinct, as are the diseases it causes. Future studies are warranted to determine the similarities and differences in replication, pathogenesis, and transmission between ZIKV and other flaviviruses, with the goal of ach-ieving better diagnosis and developing potential therapeutics and vaccines for ZIKV.

Acknowledgements

This work was supported by grants from the Utah Science Technology and Research (A34637 and A36815) and by a grant (200784-00001) from the American Board of Obstet-rics and Gynecology, the American College of Obstetricians and Gynecologists, the American Society for Reproductive Medicine, and the Society for Reproductive Endocrinology and Infertility. We thank Dr. Deborah McClellan for her careful and critical reading of the manuscript.

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