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Virus Research 127 (2007) 151–157

Genetic variability and molecular evolution of Hepatitis A virus

Juan Cristina a,∗, Mauro Costa-Mattioli b

a Laboratorio de Virologıa Molecular, Centro de Investigaciones Nucleares, Facultad de Ciencias, Igua 4225, 11400 Montevideo, Uruguayb Department of Biochemistry, McGill University, McIntyre Medical Building, Montreal, Quebec H3G 1Y6, Canada

Available online 27 February 2007

bstract

Hepatitis A virus (HAV), the causative agent of type A viral hepatitis, was first identified about three decades ago. Recent findings have shown thatAV possess several characteristics that make it unique among the family Picornaviridae, particularly in terms of its mechanisms of polyproteinrocessing and virion morphogenesis. HAV circulates in vivo as distributions of closely genetically related variants referred to as quasispecies.

AV exploits all known mechanisms of genetic variation to ensure its survival, including mutation and recombination. Only one serotype and

ix different genetic groups (three humans and three simian) have been described. HAV mutation rate is significantly lower as compared to otherembers of the family Picornaviridae. The mode of evolution appears, at least in part, to contribute to the presence of only one known serotype.2007 Published by Elsevier B.V.

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eywords: Hepatitis A virus; Genetic variability; Evolution; Recombination

. Introduction

The disease described as “jaundice” in ancient Greek, Romannd Chinese literature is thought to be viral hepatitis (Nainan etl., 2006). Hepatitis A, a term first introduced by Krugman et al.1967), is now known to be caused by infection with Hepatitis

virus (HAV). In 1973, HAV was first identified in the stools ofnfected persons (Feinstone et al., 1973), which eventually ledo development of diagnostic tests (Nainan et al., 2006).

Continued progress in the cell culture propagation of HAV,nd the developments of methods for the successful inactivationf the virus, led to the production and licensure of safe andery effective vaccines for prevention of hepatitis A by the early990s (Werzberger et al., 1992; Innis et al., 1994).

Transmission of HAV occurs mainly through the faecal-oraloute where insufficient sanitation or poor hygienic conditionsavor the pollution of water and food, especially shellfish (Hadlert al., 1980).

HAV causes occasional, dramatic disease outbreaks of acuteepatitis, as well as isolated severe cases of fulminant hepatitis,ith fatal outcomes in otherwise healthy adults. However, it has

ever been associated with chronic liver disease (Martin andemon, 2006).

∗ Corresponding author. Tel.: +59 82 525 09 01; fax: +59 82 525 08 95.E-mail address: cristina@cin.edu.uy (J. Cristina).

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168-1702/$ – see front matter © 2007 Published by Elsevier B.V.oi:10.1016/j.virusres.2007.01.005

Although the incidence of hepatitis A in developed countries,ike the USA, have fallen dramatically since the introduction ofaccines, HAV remains more than a troublesome nuisance, sincelmost 6000 cases were reported in 2004, representing an esti-ated 60,000 cases nationwide (Martin and Lemon, 2006). A

uite different picture is observed in other regions of the world,uch as South America, where systematic immunological sur-eys indicate that over 90% of the low-income population bearserological evidence of infection by HAV at the age of 18, andepatitis A is responsible for more than 50% of all acute hep-titis cases reported to national reference centers (Abuzwaida etl., 1987; Gaspar et al., 1992; Montano, 2002).

Travellers account for 10–15% of all reported cases of hepati-is, with an annual estimated rate of 3.6–7.2 per 100 individuals.pidemological studies indicate that the morbility and mortal-

ty rate of hepatitis A among travelers is 500 times higher thanhose of cholera, 10 times those of typhoid fever and three timesigher than hepatitis B (Lenfant, 1994).

. Molecular virology of HAV

HAV has been classified within the family Picornaviridae,n a separate genus, the genus Hepatovirus. For this reason, our

nderstanding of HAV has benefited from several studies car-ied out in other prototype members of this family, as Poliovurs.owever, in recent years, there have been a growing numberf features that are unique to hepatoviruses, including important

152 J. Cristina, M. Costa-Mattioli / Virus Research 127 (2007) 151–157

Fig. 1. HAV genome organization. On top a scheme of the HAV genome is shown. The positive-strand (messenger-sense) RNA genome contains a single openr iral pi tural

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eading frame encoding a polyprotein that is proteolytially processed by the vndicated by a red arrow. Structural proteins (P1) are indicated in blue, nonstruc

etails of the processing of the viral polyprotein, the morphogen-sis of the virion, and the interactions of the virus with host cellsMartin and Lemon, 2006, 2002). Recently, medium-resolutionmages of the HAV particle have been obtained by cryo-electron

icroscopy. These studies suggest that the HAV particle hasignificant differences in its structure compared with other mem-ers of the family Picornaviridae. In particular, no well-definedcanyon” surrounding the particle’s five-fold-axes, a prominenteature and the site of cellular receptor binding in other picor-aviruses, is observed in HAV particles (Martin and Lemon,006).

HAV is a positive-strand RNA virus (Hollinger and Emerson,001). The genome itself is approximately 7500 nucleotide inength and contains a single large open-reading frame encod-ng a polyprotein in which the major capsid proteins representhe amino-terminal third, with the remainder of the polypro-ein comprising a series of nonstructural proteins required forAV RNA replication: 2B, 2C, 3A and 3B (also known as VPg,

hat is covalently linked to the 5′ end of the genomic RNA andhat probably serves as the protein primer for RNA synthesis),Cpro (a cysteine protease responsible for most post-translationalleavage events within the polyprotein), and 3Dpol (the viralNA-dependent, RNA polymerase) (see Fig. 1).

Proteolytic processing of the polyprotein occurs simultane-usly with translation and is largely carried out by the 3Cpro

rotease. Synthesis of the RNA follows the assembly of a large,acromolecular replicase complex containing the nonstructural

iral protein spanning the 2B-3Dpol segment of the polyprotein,nd occurs on membranes that are usurped for this purpose fromhe cellular endoplasmic reticulum (Gosert et al., 2000).

The primary polyprotein cleavage event occurs at the 2A/2Bunction, and differs from other well-studied picornaviruses inhat it is mediated by the 3Cpro protease (Martin et al., 1995).he resulting P1-2A structural precursor is further cleaved by

he viral protease to generate two capsid protein precursors:

P0 (VP4–VP2) and VP1-2A (also known as pX, Anderson

nd Ross, 1990), as well as the mature VP3 capsid protein (seeig. 1). VP1-2A is a critical structural intermediate in virion mor-hogenesis (Cohen et al., 2002). Cleavage at the HAV VP1/2A

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rotease 3Cpro. A yet-to-be-identified cellular protease cleavage in VP1-2A isproteins (P2 and P3) are indicated in yellow and green, respectively.

unction occurs late in the process of virion morphogenesisnd results from the action of an unknown cellular proteaseMartin et al., 1999; Graff et al., 1999). However, the matureA protein has never been identified directly in infected cells.oreover, infectious virus can be recovered from recombinantAV genomes containing exogenous protein-coding sequences

nserted in-frame at the 2A/2B junction and flanked by consen-us 3Cpro cleavage sites, indicating that the nonstructural 2Aolypeptide does not function in cis as a 2AB precursor (Beardt al., 2001). Viral RNA replication is not impaired when the C-erminal 60% of the 2A sequence is deleted (Cohen et al., 2002).hus, the nonstructural 2A protein segment is not required forNA synthesis. This aspect of HAV biology is very different

rom other well-known members of the family Picornaviridae,ike Poliovirus.

Most picornaviruses have four polypeptides within their cap-id, including a small VP4 protein located at the amino terminusf the polyprotein (see Fig. 1). The HAV polyprotein appearso possess a very short VP4 polypeptide segment at its aminoerminus, this putative VP4 moiety has never been demonstratedirectly in purified virus preparation (Martin and Lemon, 2006).oreover, whereas N-terminal myristoylation of other picor-

avirus VP4 proteins is important for virion morphogenesis, theAV VP4 sequence does not contain a similarly placed myris-

oylation signal (Tesar et al., 1993). Why the assembly of theAV capsid seems to be different from other picornaviruses isnknown. One possible hypothesis is that this may be relatedo the unique intrahepatic lifestyle of HAV (Martin and Lemon,006).

Similar to other members of the family Picornaviridae, ando other hepatitis viruses, such as Hepatitis C virus (HCV), the′ non-coding region of the genomic RNA of HAV harbors annternal ribosome entry site (IRES), which directly bridges theibosome to the viral RNA. Thus, HAV translation occurs in aap-independent fashion (see Fig. 1) (Weitz et al., 1986; Glass

t al., 1993; Brown et al., 1994). In an IRES-driven translationsubset of translation initiation factors, but not the cap-bindingrotein, eukaryotic initiation factor 4e (eIF4E) are involvedn this process (reviewed by Belsham and Sonenberg, 2000;

J. Cristina, M. Costa-Mattioli / Virus Research 127 (2007) 151–157 153

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sJbdfig2ogtRttocfrom humans (I–III) and three from a simian origin (IV–VI)Genotypes I and III are the most prevalent genotypes isolatedfrom humans. The three simian genotypes were each defined

Fig. 3. Phylogenetic tree analysis of HAV using full-length VP1 sequences. Thephylogenetic tree shown was obtained by the neighbor-joining method (Saitouand Nei, 1987) under the Kimura-two parameter distance model (Felsenstein,1993). Strains in the tree are shown by their names and their types are indicatedin parenthesis. Numbers at the branches show boostraps percentages obtainedafter 1000 replications of bootstrap sampling. Bar at the bottom of the figure

ig. 2. HAV genome regions used for genetic analysis. The thick red lines belowifferent HAV isolates.

elletier and Sonenberg, 1989). Paradoxically, recent studiesuggest that HAV-IRES-driven translation is dependent of eIF4EBorman et al., 2001; Ali et al., 2001).

Clinical isolates of Hepatitis A virus (HAV) replicate ineffi-iently in cell culture unless mutations are acquired throughouthe genome. An Ala-to-Val substitution in the nonstructural pro-ein 2B (2B-216) is known to have a major impact on replicationn cell culture (see also Fig. 1). In contrast, in vivo, virus withither Ala or Val at 2B-216 replicate equally efficiently whenested in chimpanzee and in tamarins (Emerson et al., 1993).

. HAV genetic diversity

The pioneer work on HAV genetic variability was based onhe study of discrete, selected partial HAV genomic regions, suchs the C terminus of VP3 (Jansen et al., 1990), the N terminusf VP1 (Robertson et al., 1991) or the putative VP1-2A junctionegion (Jansen et al., 1990; Robertson et al., 1992) (see Fig. 2).

Since HAV and poliovirus share many genomic features, theifferent HAV strains were grouped by comparing the VP1-2Aunction, using the method of Rico-Hesse and coworkers, a cri-erion used at that time for genetic classification of Poliovirustrains (Rico-Hesse et al., 1987). In 1992, using this approach,enetic analysis of 152 HAV strains recovered around the worldesulted in the designation of seven genotypes of HAV (I–VII).his initial study by Robertson et al. significantly influenced

he direction of future research in the field (Robertson et al.,992). However, the majority of strains included in these stud-es were isolated in the USA and Asia, leaving other regionsf the world, which have a hyperendemic pattern of HAV, suchs South America, North and Central Africa and India, underepresented. Moreover, by using the traditional method of geno-yping, three HAV antigenic variants reported recently cannot beetected (Costa-Mattioli et al., 2002, 2003; Sanchez et al., 2002).

Recently, Costa-Mattioli et al. (2002) performed phyloge-etic studies using full-length VP1 sequences (900 nucleotides;ee Fig. 2), as an alternative method to the percentage of identity

ithin a short (168 nucleotides) selected region of the genome.These studies revealed the presence of five distinct genetic

roups, all of them supported by high bootstrap values (Costa-attioli et al., 2002). It is important to note that the only

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genome scheme indicate the different genomic regions used to characterize the

equences not included in these studies were the one of strainM-55 (genotype VI) and those representing genotype IIIB,ecause none of them were available in a public sequenceatabase at that time. Based on these studies, a novel classi-cation of HAV genotypes was proposed to include six differentenotypes (see also Fig. 3; reviewed by Costa-Mattioli et al.,003). Strikingly, a higher degree of genetic relation than previ-usly expected was observed between the previously describedenotypes II and VII (Costa-Mattioli et al., 2002), suggestinghat they may be one or two sub-genotypes of the same type.ecent work performed by Lu and co-workers on establishing

he genetic relations of genotype II strain CF53/Berne confirmedhe hypothesis that genotypes II and VII are two sub-genotypesf genotype II (Lu et al., 2004) (see Fig. 3). Therefore, weoncluded that HAV has six different genotypes: three isolated

hows distances. Strain 9F94, previously described as genotype II using theutative VP1/2A region, and strain SLF88, previously described genotype VII,re shown in bold. As it can be seen in the tree, the least variation observed ismong these two strains, who now represent two different sub-genotypes of theame genotype (genotype II).

154 J. Cristina, M. Costa-Mattioli / Virus Research 127 (2007) 151–157

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ig. 4. Geographic distribution of HAV genotypes. Genotypes isolated from huA21 (genotype IIIA), isolated from Panamanian Owl monkeys, is shown in gr

y unique nucleotide sequences from the P1 regions of HAVtrains recovered from species of Old World monkeys. In addi-ion, all simian HAVs have a distinct signature sequence at theP3/VP1 junction which distinguishes these strains from humanAVs (Brown et al., 1989; Nainan et al., 1991; Tsarev et al.,991). Genotype IV was recovered from a cynomolgus macaqueMacaca fasicularis) imported from the Philippines (Nainan etl., 1991). The prototype strain of genotype V, AGM27, was iso-ated from an African green monkey (Cercopithecus aethiops)mported from Kenya (Tsarev et al., 1991). Genotype VI was alsosolated from a cynomolgus macaque (M. fasicularis) importedrom Indonesia (Robertson et al., 1992).

. Distribution of HAV genotypes

Different HAV genotypes have a different geographic distri-ution (Nainan et al., 2006). The current worldwide distributionf HAV genotypes are shown in Fig. 4. Genotype I is most preva-ent worldwide, and sub-genotype IA is more common thanB. Sub-genotypes IA and IB are most often found in Northnd South America, Europe, China and Japan (Robertson et al.,992; Costa-Mattioli et al., 2001a,b). For sub-genotype IIIA,he prototype strain, PA21 (Brown et al., 1989), was originallysolated from captured Panamanian Owl monkeys and thoughto be a simian virus. However, once nucleotide sequence analy-is was performed many years later, the unique nucleotide andmino acid sequence patterns which differentiate human fromimian HAV were not found in this strain (Nainan et al., 1991;

obertson et al., 1992) (see Fig. 4).

Cocirculation of multiple genotypes or sub-genotypes haseen reported in some regions of the world, as IA and IB in Southfrica (Taylor, 1997), Brazil (Villar et al., 2004) and France

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are shown in black, genotypes isolated from simians are shown in red. Strain

Costa-Mattioli et al., 2001a,b) and sub-genotypes IA and IIIAn India (Hussain et al., 2005). Cocirculation of sub-genotypesA and IB have been observed also in the USA, although mostB isolates were found to be from travelers returning from otherountries (Nainan et al., 2005).

Early studies of HAV isolates from cell culture have shownittle genetic variation between the different strains, most likelyssociated to cell culture cross-contamination problems. How-ver, more recent PCR studies based on strains isolated fromlinical specimens (stools, livel suspension and serum) havehown more genetic heterogeneity. In regions of the world suchs the USA, Japan, and China, HAV-related isolates tend to clus-er, suggesting an endemic spread. A high degree of geneticonservation was shown during the infection prior of an indi-idual (Robertson et al., 1992) or even among different isolatesith a common source of infection (Grinde et al., 1997; Chudy

t al., 1999; De Serres et al., 1999; Arauz-Ruiz et al., 2001; Diazt al., 2001; Tallo et al., 2003). In contrast, a higher degree ofeterogeneity than reported previously has been found in strainssolated in South America (Costa-Mattioli et al., 2001a, 2002; Deaula et al., 2002; Mbayed et al., 2002). Moreover, these strainso not cluster in according to geographic origin, as reported forsolates in Europe (Costa-Mattioli et al., 2002).

. Recombination in HAV

Over the last two decades it has become increasingly clear

aterial with one another. Genetic exchange by homologous andon-homologous recombination is a phenomenon that is com-on among RNA viruses and may lead to hybrid or defective

nterfering RNA molecules (Lai, 1992; Nagy and Simon, 1997).

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Kew and Nottay (1984) were the first to report the isolationf a naturally occurring recombinant Poliovirus that containedequences derived from all three serotypes of Poliovirus vaccinetrains as a result of two crossovers. Recombination among vac-ine and wild-type Polioviruses has been reported as a naturaleans of evolution of Poliovirus (Guillot et al., 2000). This has

lso been observed in entroviruses (Santti et al., 1999). In thease of HAV, genetic exchange among strains had been observedn cell culture (Lemon et al., 1991; Beard et al., 2001; Gauss-

uller and Kusov, 2002) but for many years it was supposed noto occur in nature. This view was challenged by the report of aase of dual infection of a young childcare provider (AUX-23)ith HAV strains belonging to different sub-genotypes (de Paula

t al., 2003). Interesting, AUX-23 was hired at a childcare cen-re in which HAV IA and IB were circulating. These particularonditions may have facilitated the double infection.

The first HAV recombinant strain isolated from an infectedatient was reported in 2003 (Costa-Mattioli et al., 2003). Theecombinant isolate, 9F94, comes from a little girl who wasospitalized in France after a 3 month holiday in Morocco.ccordingly, the putative parental strains SLF88 (now classi-ed as genotype II) and MBB (genotype IB) were also originally

solated in North Africa, a region of high endemicity for HAVnfection and one in which multiple genotypes co-circulateMelnick, 1995).

The recombination event in strain 9F94 took place in theP1 capsid protein (Costa-Mattioli et al., 2003). This finding

ndicates that capsid recombination may play a significant rolen shaping the genetic diversity of HAV and, as such, can havemportant implications for its evolution, biology and control.evertheless, the frequency and possible implications of HAV

apsid recombination events for the generation of pathogenicAV strains are not clear at present.

. HAV mode of evolution

As other RNA viruses, HAV exists in vivo as distributionsf closely related variants referred to as quasispecies (Sanchezt al., 2003a; Costa-Mattioli et al., 2006). Quasispecies dynam-cs is characterized by continuous generation of variant viralenomes, competition among them, and selection of the fittestutant distributions in any given environment. Understanding

he principles that shape the evolution of viral quasispecies isecoming increasingly important to model disease progressionnd to design preventive and therapeutic strategies to controliral disease (Domingo, 2005; Domingo et al., 2005).

Over time, RNA virus evolution is conditioned by perturba-ions of population equilibrium, that may not be equal amongndividual hosts, and therefore, multiple viral sublineages mayapidly be established that differ in the number of rounds of repli-ation (and history of environmental perturbations), and mayo-circulate in the same geographical area.

To study HAV evolution over time in a specific geographic

egion, recent studies were carried out on HAV genotype I strainssolated in France from 1983 to 2001, using a non-hierarchical

ethod developed to study closely related components of mutantpectra of viral quasispecies (Baccam et al., 2001). This method

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Research 127 (2007) 151–157 155

as used to compare epidemiologically related, consensusAV sequences. These studies have identified different sub-opulations of HAV variants that coexist in time and in differentnvironments (Costa-Mattioli et al., 2006). Clades isolated fromifferent years, reemerged and were even associated with epi-emic strains. These findings suggest that beyond mutations andenetic recombination, HAV exploits this variation strategy inominance to promote and ensure its survival (Costa-Mattioli etl., 2006).

The coexistence of different subpopulations are consistentith the presence in each HAV isolate of a mutant spectrum

Sanchez et al., 2003a), which provides a repertoire of variantshat, while constituting a minority in an infected individual, mayecome dominant following transmission to a new host indi-idual. These findings fit the general picture of quasispeciesynamics (Domingo, 2005; Domingo et al., 2005); with thealient antigenic stability of HAV that is probably related totructural constraints of the viral capsid (Sanchez et al., 2003b).

The first estimations of HAV mutation rate was done byanchez et al. (2003a), who determined a rate of 1 × 10−3 to× 10−4 substitutions per site. This figures are much lower

han that found in other members of the family Picornaviridae,s for instance FMDV (2.7 × 10−2, Gurumurthy et al., 2002),oliovirus type 1 (3.36 × 102, Kew et al., 1998) or Enterovirus0 (2.2 × 10−2, Takeda et al., 1994). Nevertheless, further stud-es are needed in order to establish substitutions/site/year inonophyletic natural populations of HAV.Genetic studies using full-length VP1 and capsid sequences

ave shown the presence of different patterns in the intragenicistributions of synonymous substitutions in the VP1 protein,uggesting that synonymous divergence could be random in theP1 gene (Costa-Mattioli et al., 2002, 2003). Nevertheless, theistribution on non-synonymous substitutions along the VP1rotein shows a completely different situation, with extremelyow rates of substitutions compared to those of synonymousubstitutions. Thus, the pattern of divergence observed for HAVP1 is probably due to selective forces that do not allow amino

cid replacements, despite the relative high rates of synonymousubstitutions observed all over the gene. This is in contrast withhe situation found in multiple serotype members of the fam-ly Picornaviridae, like foot-and-mouth disease virus (FMDV),hich is subjected to positive selection (Haydon et al., 2001).or these reasons, the mode of evolution of HAV may explain, at

east in part, the presence of only one known serological groupf HAV.

. Conclusions

Hepatitis A virus (HAV), the causative agent of type A viralepatitis, is an ancient human virus that was first identifiedlmost 35 years ago. The structure of HAV, its tissue tropism,ts genetic distance from other members of the family Picor-aviridae, its mechanisms of polyprotein processing and virion

orphogenesis, indicate that HAV is unique within this fam-

ly, and this may likely contribute to its pathobiology. HAVxploits all known mechanisms of genetic variation to ensurets survival, including mutation and recombination. The study

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56 J. Cristina, M. Costa-Mattioli /

f other genome regions besides VP1, as well as completeenome sequences, may be very useful in determining the fre-uency of intra- and intertypic recombination in the field and themergence of possible new genetic or antigenic variants. Quasis-ecies dynamics studies on different regions of the world may belso very helpful in order to understand the co-circulation of dif-erent HAV sub-populations in different geographic regions. Inddition, further studies on HAV mutation rates would facilitatehe understanding of HAV evolution.

cknowledgement

JC acknowledge support from PEDECIBA, Uruguay.

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