The molecular epidemiology of hepatitis E virus infection

Virus Research 161 (2011) 31–39

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Review

The molecular epidemiology of hepatitis E virus infection夽 Michael A. Purdy ∗ , Yury E. Khudyakov Centers for Disease Control and Prevention National Center for HIV/Hepatitis/STD/TB Prevention Division of Viral Hepatitis MS-A33 1600 Clifton Rd NE Atlanta, GA 30333, United States

a r t i c l e

i n f o

Article history: Available online 11 May 2011 Keywords: Hepatitis E virus Molecular epidemiology Genotypes subtypes Transmission Population dynamics

a b s t r a c t Molecular characterization of various hepatitis E virus (HEV) strains circulating among humans and animals (particularly swine, deer and boars) in different countries has revealed substantial genetic heterogeneity. The distinctive four-genotype distribution worldwide of mammalian HEV and varying degrees of genetic relatedness among local strains suggest a long and complex evolution of HEV in different geographic regions. The population expansion likely experienced by mammalian HEV in the second half of the 20th century is consistent with an extensive genetic divergence of HEV strains and high prevalence of HEV infections in many parts of the world, including developed countries. The rate and mechanisms of humanto-human transmission and zoonotic transmission to humans vary geographically, thus contributing to the complexity of HEV molecular evolution. Published by Elsevier B.V.

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic diversity: genotypes and subgenotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evolutionary history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic relatedness among HEV strains in different transmission contexts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic relatedness among geographically distinct human and swine strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic disassociation between local human and swine HEV strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Potential human source of “zoonotic” HEV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The “true” number of human HEV infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transmission rates and HEV genetic relatedness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Enterically transmitted non-A, non-B hepatitis was first recognized in a large outbreak of jaundice in India in the 1950s (Wong et al., 1980). Similar large-scale epidemics were subsequently reported elsewhere in the Indian Subcontinent, and in Central Asia, Africa, and Mexico. These were determined to be due to infections with hepatitis E virus (HEV) (Okamoto, 2007). Hepati-

夽 Disclaimer: This information is distributed solely for the purpose of predissemination peer review under applicable information quality guidelines. It has not been formally disseminated by the Centers for Disease Control and Prevention/Agency for Toxic Substances and Disease Registry. It does not represent and should not be construed to represent any agency determination or policy. ∗ Corresponding author. Tel.: +1 404 639 2332; fax: +1 404 639 1228. E-mail address: [email protected] (M.A. Purdy). 0168-1702/$ – see front matter Published by Elsevier B.V. doi:10.1016/j.virusres.2011.04.030

tis E is largely an acute, self-limiting disease. Clinical attack rates are highest among adults in their second and third decades of life. There is a positive correlation between age and anti-HEV positivity, with the highest seropositivity rates observed in adults over sixty years old (Trautwein et al., 1995). The hepatitis E mortality rate among the general population during an outbreak is <3%. For unknown reasons, pregnant women during their third trimesters are more susceptible to infection and fulminant hepatitis than the general population, with fatality rates approaching 20% (Tsega et al., 1993). HEV belongs to the genus Hepevirus in the Hepeviridae family. It is a non-enveloped, positive-sense, single-stranded RNA virus (Reyes et al., 1990), and has a 7.2-kb-long genome, which is capped and polyadenylated. The genome contains three open reading frames (ORFs). The longest and 5 -most ORF1 codes for non-

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structural proteins, the next longest ORF2 encodes the viral capsid, and the remaining ORF3, which overlaps the capsid gene, codes for a small protein of unknown function, which may have regulatory properties (Chandra et al., 2008).

2. Genetic diversity: genotypes and subgenotypes Sequence analysis of HEV strains involved in the earliest recognized hepatitis E outbreaks on the Indian Subcontinent and in Central Asia showed that these strains were closely related (Khuroo, in this issue). The HEV strain subsequently isolated from an outbreak in Telixtac, Mexico, in 1986 had a nucleotide sequence similarity of 76% and 77% with prototypic strains from Burma and Pakistan, respectively (Huang et al., 1992). These findings suggested that there were at least two genotypes of human HEV, now assigned genotypes 1 and 2. Both HEV genotypes could also establish infection in a variety of experimentally infected primates (Okamoto, 2007). Later, HEV was isolated from domestic swine (Meng et al., 1997). This strain exhibited a nucleotide sequence similarity of about 79–80% with genotypes 1 and 2, and was designated genotype 3. Since then swine HEV has been identified in many countries worldwide. Thereafter, Wang et al. (1999) discovered an additional HEV genotype in the sera of Chinese patients with acute hepatitis, designated as genotype 4. This genotype was observed to infect swine also (Meng, 2011; Wang et al., 2002). Subsequently, both genotypes 3 and 4 have been found to infect a broader range of hosts including deer and boars, with genotype 3 also infecting rabbits (Balayan, 1997; Johne et al., 2010; Sonoda et al., 2004; Zhao et al., 2009). These four HEV genotypes have been referred to as mammalian HEV. Genotypes 1 and 2 appear to be anthroponotic whereas genotypes 3 and 4 although enzootic are zoonotic (Teo, 2010). All four genotypes belong to a single serotype (Emerson et al., 2004). The recent discovery of novel lineages of HEV in rabbits (Geng et al., in press; Zhao et al., 2009), rats (Johne et al., 2010), and a wild boar (Takahashi et al., 2011) has expanded further mammalian HEV diversity. It has been suggested that the HEV sequences found in rabbits represent a novel genotype (Geng et al., in press; Zhao et al., 2009). However, additional phylogenetic analysis indicated that rabbit HEV belongs in genotype 3 (Bilic et al., 2009; Johne et al., 2010). This review focuses on the molecular epidemiology of HEV genotypes 1–4, which infect humans. The scientific consensus up to the end of the 20th century was that hepatitis E was endemic in developing countries, but seen only in industrialized countries when imported by travelers. However, molecular analysis of HEV strains in case-patients with acute nonA, non-B and non-C hepatitis but without history of recent travel to HEV-endemic countries showed that HEV is endemic worldwide. Genotype 1 particularly circulates among humans in tropical and subtropical countries in Asia and Africa, genotype 2 in Mexico and West and South Africa, and genotype 4 in East and Southeast Asia, whereas genotype 3 circulates worldwide (Okamoto, 2007). More extensive genetic analysis of HEV strains showed that more than one HEV genotype can circulate in human population in some geographic regions. However, genotype distribution among HEV strains may change over time. For example, two HEV genotypes, 1 and 4, infect humans in China, with genotype 4 now overtaking genotype 1 in predominance. Genotype 1 was associated with one of the largest recorded hepatitis E outbreaks that occurred in Xinjiang between 1986 and 1988 (Aye et al., 1992). Although this genotype continues to be found in Xinjiang and the rest of China, its detection rate appears to be much reduced over time (Tai et al., 2009). In 2000, genotype 4 was reported in China for the first time (Chen et al., 2005) and since then its detection rate in pigs and humans has increased (Lam et al., 2009; Ma et al., 2010;

Yu et al., 2009). It has also been noted that the number of sporadic cases of genotype 4 hepatitis E is increasing (Tai et al., 2009). Lu et al. (2006) comprehensively analyzed the genetic diversity of HEV worldwide, and suggested that genotypes 1, 2, 3 and 4 could be classified into five, two, ten and seven subgenotypes, respectively. Genotypes and subgenotypes are important molecular epidemiological tools, the efficient application of which ultimately depends on the accuracy of classification approaches. However, multiple genotype/subgenotype definitions have been applied to HEV (Arankalle et al., 1999; Lu et al., 2006; Worm et al., 2002), thus complicating the use of this classification. Underlying issues relate to the length of sequences examined and the genomic regions analyzed, since different research groups have analyzed multiple regions of different sizes (Chen and Meng, 2004; Lu et al., 2006; Schlauder and Mushahwar, 2001; Zhai et al., 2006). For example, Lu et al. (2006) had to examine not only full-length genomes but sequences from two separate ORF1 regions and three separate ORF2 regions to complete their analysis of subgenotypes. Further, although specified regions were used in this study, not all the sequences were of same length; e.g., sequences of 148 and 287nt derived from the same ORF1 region. HEV sequences in GenBank come from multiple regions across the genome, and in some cases there is little commonality in fragment size even when a common region is sequenced. An examination of these sequences shows that most are shorter than 550nt, with some sequences being as short as 69nt (e.g. AB193019). Just as it was recognized that the nomenclature of hepatitis C virus needed to be standardized to ensure that epidemiological, evolutionary and clinical observations among strains are classified correctly according to genotype- and subgenotype-specific genetic differences (Simmonds et al., 2005), a similar standardization is needed for HEV.

3. Evolutionary history The discovery of a genetically distinct avian HEV (Haqshenas et al., 2001) indicates a very long evolutionary history for the HEV group of viruses. Recently, the evolutionary history of mammalian HEV was reported (Purdy and Khudyakov, 2010). The times to the most recent common ancestors (tMRCAs) for all four HEV genotypes were calculated using sequences from the non-overlapped region of ORF2 in a Bayesian analysis. The calculated root for the HEV genotypes 1–4 tree with or without outgroups falls between clades composed of genotypes 1 and 2, and of genotypes 3 and 4. This finding showed that the most common recent ancestor for modern mammalian HEV existed between 536 and 1344 years ago. This progenitor appears to have given rise to anthropotropic and enzootic variants of HEV, which evolved into genotypes 1 and 2 and genotypes 3 and 4, respectively. The anthropotropic ancestor of genotypes 1 and 2 existed about 367–656 years ago, and the enzootic ancestor of genotypes 3 and 4 from 417 to 679 years ago. While the genotype 3 ancestor existed about 265–342 years ago and the genotype 4 ancestor from 131 to 266 years ago, the genotype 1 ancestor existed as recently as 87–199 years ago. The estimates for the tMRCAs are about 25% longer when ORF1 was used. The tMRCA for modern genotype 2 could not be calculated since only one genotype 2 whole-genome sequence was available. Bayesian inference was also used to show that the effective number of infections in Japan associated with genotypes 3 and 4 are on the rise (Tanaka et al., 2006). A more extensive population dynamics study suggests that genotypes 1, 3 and 4 experienced a population expansion during the 20th century (Purdy and Khudyakov, 2010). Genotype 1 has increased in effective population size only ∼30–35 years ago. Genotypes 3 and 4 appear to have experienced an increase in population size starting late in the 19th century until ca. 1940–1945, with genotype 3 having undergone

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additional rapid expansion until ca.1960. The effective population size for both genotypes 3 and 4 rapidly declined to pre-expansion levels starting in ca. 1990. For genotype 4, although the effective population size of Japanese strains has declined recently, the Chinese strains have maintained a constant effective population size since the 1950s, suggesting that this genotype experienced different evolutionary histories in these two countries.

4. Genetic relatedness among HEV strains in different transmission contexts HEV causes predominantly acute, self-resolving infections. Since genomic mutations occur only during viral replication, transmission among hosts plays a crucial role in determining the rate of HEV evolution and is a major focus of the molecular epidemiology. The molecular epidemiology of HEV infections and, in particular, HEV transmission is not well understood. HEV genotypes 1 and 2 are associated with water-borne transmission (Teo, 2010), with waterborne outbreaks of jaundice due to hepatitis E reported from India (Khuroo, 1980; Vivek et al., 2010), Vietnam (Corwin et al., 1996), Uganda (Teshale et al., 2010) and China (Aye et al., 1992) known to affect hundreds and thousands of people. Water-borne transmissions present a special challenge for genetic analysis of HEV strains. During these outbreaks drinking water can be expected to be contaminated with more than one genetically distinct HEV strain and provide exposure to large populations. Consistent with this consideration, heterogeneous HEV strains have been identified during water-borne outbreaks in Sudan (Guthmann et al., 2006) and India (Vivek et al., 2010). The HEV strains identified in Darfur, Sudan, and in Chad during a large water-borne outbreak belonged to genotypes 1 and 2, suggesting more than one source of infection (Nicand et al., 2005; Guthmann et al., 2006). The hepatitis E outbreak related to sewage draining into a river in Nellore in southern India was also associated with several genetically close HEV genotype 1 strains (Vivek et al., 2010). However, water-borne outbreaks may be associated with a single HEV strain as was recently observed during the outbreak in Alandi village in Maharashtra, India, in 2006 (Verma and Arankalle, 2010). Nonetheless, the HEV variants identified during this outbreak investigation used a short ORF1 region, and minority strains might not be identified. HEV genotypes 3 and 4, by contrast to genotype 1, have been hypothesized to be transmitted zoonotically (Fu et al., 2010a,b; Meng et al., 1997; Meng, 2011). Indeed, the recent decade of research has brought about examples of food-borne HEV transmission related to ingestion of raw or undercooked meat and offal from swine (Lewis et al., 2010; Teo, 2010). It would be expected that the source, infected with a single viral strain, transmits this strain to a recipient, and, therefore, viral genomic sequences identified in both hosts should be identical or nearly identical. However, such molecularly confirmed observations of direct transmission from source to recipient are sparse. Although few food-borne transmissions are documented, they provide genetic proof of HEV strains shared between the animal source and human host. For example, genetic analysis of HEV strains in livers of infected pigs sold in a local grocery store in Hokkaido, Japan, showed that some HEV variants found in porcine organs were identical to HEV genotype 3 and 4 strains recovered from the local human cases (Yazaki et al., 2003). More direct evidence for food-borne transmission was obtained by Tei et al. (2003), who reported on a hepatitis outbreak among family members and some of their friends. Epidemiologic investigation of these cases suggested they became infected with HEV genotype 3 after eating uncooked deer meat. Comparison of HEV RNA sequences obtained from these patients and a frozen portion of the suspect deer meat showed that all these sequences were identical. Another study examined the HEV RNA sequences from wild

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boars, a deer and four patients, who contracted hepatitis E after eating raw deer meat. The sequences displayed a sequence similarity of 99.7% and, although direct transmission from deer to humans could not be established, the sequence similarity suggested inter-species HEV transmission between boar and deer, and possible food-borne transmission to humans (Takahashi et al., 2004). In another report, a woman, whose husband was a boar hunter, ate boar meat from one of his kills 11 and 8 weeks before she became ill with hepatitis E. HEV RNA sequences from her serum and the consumed boar meat juice displayed a sequence similarity of 99.95%, confirming transmission of HEV from the boar to the woman (Li et al., 2005). Additional evidence of food-borne transmission was obtained from Hokkaido where HEV genotype 4 strains recovered from a series of sporadic hepatitis E cases from 2004 to 2009, some of which were fulminant, exhibited a high degree of sequence similarity to each other (Kang et al., 2010; Sugawara et al., 2009; Yazaki et al., 2006). Blood-borne transmission is rarely observed for viruses causing short enteric infections. However, in recent years several cases of molecularly confirmed transfusion-related hepatitis E were reported. The first case of transfusion transmission for an indigenous HEV genotype 4 strain was reported from Japan (Matsubayashi et al., 2004). Thereafter, five cases of HEV transmission associated with blood transfusion were reported in nonhyperendemic countries; including Japan, the United Kingdom, and France (Boxall et al., 2006; Colson et al., 2007; Matsubayashi et al., 2008; Mitsui et al., 2004; Tamura et al., 2007). Cases of proven direct human-to-human transmission present an important opportunity for analyzing genetic relatedness among HEV strains passed from one human host to another. Matsubayashi et al. (2004) reported complete genetic identity between sequences of a 326nt genomic region of the HEV genotype 4 strains recovered from a blood donor and recipient. A few years later, the same group of researchers identified another case of transfusion-related hepatitis E (Matsubayashi et al., 2008) and showed that the complete genome sequences of HEV genotype 4 strains found in the donor and recipient differed at only one position. This finding strongly indicates that these two patients shared the same HEV strain. It is interesting, though, to note that the donor was infected with HEV through consumption of barbecued pig meat. Another patient, who shared this meal with the donor, also acquired acute hepatitis E infection. The 6588nt HEV sequences recovered from this patient and the donor were found to differ at nine sites, indicating that these two strains were genetically very close. Thus, this report describes the first case of transfusion transmission of the same HEV strain from a donor who was infected through the zoonotic food-borne route. All reported cases of transfusion-related hepatitis E were caused by HEV strains of genotypes 3 and 4, indicating a potential for human-to-human transmission of these “zoonotic” genotypes. Nonetheless, the contribution of blood-borne transmission to the overall human HEV infections seems small.

5. Genetic relatedness among geographically distinct human and swine strains The effective application of viral genetic analysis to the molecular epidemiological investigation of HEV transmissions requires a clear understanding of the genetic heterogeneity of viral strains circulating in different geographic regions. However, it was only recently that such information has become available. In addition to the HEV genotypes, finer-resolution genetic analysis showed a close genetic relatedness among locally circulating animal and human HEV strains. HEV genotype 1 is prevalent in hyperendemic countries. The HEV genotype 1 strains have been predominantly characterized during outbreaks. The degree of genetic relatedness among local

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HEV genotype 1 strains circulating between epidemics has only recently become elucidated. In Lucknow, India, HEV genotype 1 strains were sequenced from sewage drains during a period when there was no known outbreak of hepatitis E (Ippagunta et al., 2007). Among 192 tested samples, 79 were found HEV PCR positive. HEV sequences were obtained from 6 sewage samples, all being identical. In Eastern China, the diversity among local HEV genotype 1 strains was also observed to be limited: among 47 isolates recovered from patients admitted to hospitals in Nanjing and Taizhou, 13 belonged to genotype 1, all of which shared only 3 sequences that were >98% homologous to each other (Zhang et al., 2010). HEV genotype 3 is prevalent worldwide, including Europe. Analysis of HEV strains in different European countries showed phylogenetic clustering of genotype 3 strains into geographic specific clades. An analysis in Sweden confirmed high genetic similarity among HEV strains from patients and swine from the same geographic area (Norder et al., 2009). With more extensive sampling of swine than human HEV sequences, many swine HEV variants were found to be phylogenetically very close to each other or even identical in the studied geographic region, whereas human HEV variants collected from the same locale were more distant from each other and from the swine sequences (Norder et al., 2009). Another unique study of HEV strains recovered from pigs in twelve farms, wild boars in nine counties and humans with hepatitis E was conducted in Sweden (Widen et al., 2010). It showed that pigs in each Swedish farm were infected with one, or at most two distinct genotype 3 strains. Most of the HEV strains from the farmed swine and wild boars belonged to subtype 3f, previously found to be the most common subtype circulating among domestic swine in France, the Netherlands and Spain (Kaba et al., 2009; LegrandAbravanel et al., 2009; Peralta et al., 2009; Rutjes et al., 2009; van der Poel et al., 2001). HEV strains from human cases also belonged mainly to subtype 3f. All HEV strains were clustered in the phylogenetic tree preferentially according to geographic origin. It is interesting to note that the sequences identified in this study were country- and even county-specific and allowed for the identification of the geographic origin of HEV strains (Widen et al., 2010). The close genetic relatedness among HEV strains from humans and pigs was similarly observed in Europe and Japan (Dalton et al., 2007b; Fogeda et al., 2009; Forgach et al., 2010; Lopes Dos Santos et al., 2010; Meng et al., 1997; Reuter et al., 2009; Schlauder et al., 1998; Yazaki et al., 2003). In Japan, such relatedness was detected among HEV genotype 4 strains as well (Yazaki et al., 2003). These observations of geographically specific genetic relatedness among HEV strains circulating among humans and swine provide strong evidence for autochthonous hepatitis E in Europe and Japan, and prove a genetic linkage between human and animal infections. The close genetic relatedness among human and swine HEV strains in Europe and Japan, and evidence of direct zoonotic transmission of HEV through consumption of raw or undercooked animal meat and offal (Li et al., 2005; Takahashi et al., 2004; Tei et al., 2003) suggest that domestic and wild animals constitute a significant reservoir of HEV, with humans being infected through food consumption and the environment. Domestic swine are specifically considered the prime reservoir for human HEV infections in many countries of the world. In the Netherlands, about 7.5 × 106 pigs are raised annually (Bouwknegt et al., 2009). Taking into consideration that HEV RNA was detected in feces from more than half of the Dutch pig farms examined (Rutjes et al., 2007) and HEV is highly transmissible among pigs through contact-exposure (Bouwknegt et al., 2008), it can be appreciated how HEV infection in swine might be a significant source of human infections in that country. Nevertheless, these observations do not suggest how humans acquire HEV infections. For example, an extensive survey of HEV strains circulating in the Netherlands did not identify any common source for HEV infection in human population (Borgen et al., 2008).

A direct zoonotic transmission of HEV from pigs to humans was not observed in this study; neither was there evidence for foodborne transmission. Rather, the data indicated that the identified human genotype 3 infections were most probably acquired via an indirect route of transmission. The identification of identical HEV sequences in serum of an infected person and surface water near the home of the patient suggested that HEV genotype 3 infection may be acquired from environmental contamination (Borgen et al., 2008). The source of that contamination was not known.

6. Genetic disassociation between local human and swine HEV strains The molecular epidemiology of hepatitis E in China is very complex. As in Europe and Japan, swine is implicated to be the principal reservoir of HEV in eastern China (Zheng et al., 2006). In Xinjiang, in western China, analysis of genotype 4 HEV strains circulating among swine and humans showed that one human genotype 4 strain shares 100% nucleotide identity with swine strains from the same district (Fu et al., 2010b), suggesting that in this region of China, as in the east, HEV transmission may occur from swine to humans, most probably through exposure to swine and their waste. In central China, however, genetic analysis of HEV strains from the general human population and swine from commercial farms showed that human and swine HEV variants belong to different genotype 4 subtypes (Zhang et al., 2009a), suggesting that cross-species transmission from swine to humans is not common or even absent. Recently, a similar distinction between human and swine cases was reported from Shanghai, situated in eastern China (Zhang et al., 2009b). A genotype 4 strain found in this study in a seven-monthold infant was shown to have 97.6% homology with HEV strain recovered from a Japanese patient who was infected during travel to Shanghai. These strains were <91% homologous to four genotype 4 swine HEV strains found in the same area, suggesting that both human infections did not result from zoonotic transmission of the local swine HEV. Several other studies described substantial diversity among local HEV genotype 4 strains in China (Fu et al., 2010b; Zhang et al., 2009a; Zhang et al., 2010). It is not common that human HEV strains match each other genetically in the same given locale in China, whereas, as was mentioned above, some HEV variants may presumably be shared by human and animal hosts, indicating the potential zoonotic transmission (Fu et al., 2010b). However, the contribution of such transmission to human infections remains to be clarified. An even wider separation between HEV strains circulating in human and animal populations exists in India, although the hepatitis E epidemiology in this country is different from China. In India, human HEV strains belong to genotype 1 and swine HEV strains belong to genotype 4 (Arankalle et al., 2002; Shukla et al., 2007). Since genotype 1 is incapable of infecting pigs (Meng et al., 1998a), cross-species HEV transmission between human and animal populations seems to be limited in this country. Only recently was a single case of human infection with HEV genotype 4 reported, from a traveler to India (Rolfe et al., 2010). The mechanism of this particular dissociation of HEV strains between human and animal hosts is not known. It may be related to historical patterns of introduction and establishment of different HEV lineages in this part of the world, existence of widespread anti-HEV immunity in the human population, difference in infectivity of genotype 4 variants among humans and animals, or difference in the number of infections caused by both genotypes in their respective hosts. In addition, HEV genotype 4 has been suggested to cause more subclinical infections than HEV genotype 1 (Purcell and Emerson, 2008), thus reducing the opportunity for detection of genotype 4 infections. However,

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recent investigation of clinical features of acute hepatitis E showed that both genotypes 3 and 4 may cause severe hepatitis (Ohnishi et al., 2006; Takahashi et al., 2009). In Europe, with the exception of a few cases of documented direct transmission, human HEV strains are infrequently found to be identical to swine HEV strains even within short genomic regions used for phylogenetic analysis. A recent study of endemic HEV strains in Hungary (Reuter et al., 2009) showed sequence identity between HEV variants identified in one human case and roe-deer, indicating a potential for the zoonotic transmission of HEV genotype 3. However, this study also revealed three clusters of identical human HEV sequences, which were distinct from swine sequences identified in this country. By contrast, and as noted earlier, some swine HEV strains from the same farms in Sweden were found to be very closely genetically related or identical to each other, whereas they were found genetically different from human strains in same geographic region (Norder et al., 2009). A similar observation of the genetic distinction among local human and animal HEV variants was reported from the Netherlands where a molecular survey of the HEV genotype 3 strains from human, swine and environmental samples revealed four distinct subgenotypic clusters (Rutjes et al., 2009). However, distribution of human HEV strains among these clusters was significantly uneven. One major subtype 3c cluster contained 43% of human strains whereas another major subtype 3f cluster included only 5% of human strains. These findings suggest separation between Dutch human and swine HEV. It was hypothesized that HEV 3c strains were more pathogenic to humans, more stable in the environment, or were shed in higher numbers (Rutjes et al., 2009). If this separation of human and swine HEV variants between two genotype 3 subtypes is confirmed, it should be very important for understanding molecular evolution of genotype 3 in the Netherlands and other non-hyperendemic countries. The aforementioned molecular data on genetic distinction between local human and swine HEV strains suggest that human and swine HEV populations may evolve independently. The extent of independence seems to vary in different regions of the world. The blurring of distinction between human and swine HEV variants in Europe, Japan and some parts of China may reflect a strong dependence of human infections on continuous HEV introduction from the animal reservoir. Under-sampling of HEV isolates may also contribute to the apparent distinction between the local animal and human HEV isolates. Moreover, the magnitude of genetic diversity among HEV isolates may be underestimated because of the use of short genomic regions in molecular epidemiological investigations of hepatitis E, which substantially reduces the amount of genetic information and prevents a clear identification of viral strains. Genetic similarity within a short genomic region should therefore be interpreted with utmost caution for identifying genetic relatedness among viral strains. Analysis of extended genomic regions or the entire HEV genome should be considered for a reliable genetic characterization of HEV strains.

7. Potential human source of “zoonotic” HEV In northeast China, the overall prevalence of anti-HEV IgG in the general human population ranges from 32% among individuals with frequent contacts with swine to 21% among individuals with very rare contacts, while the overall prevalence of anti-HEV in swine older than three months is 82% (Yu et al., 2009). These data suggest the role for swine infections in contributing to the high prevalence among people via frequent direct exposure to animals. However, such exposure does not explain the large number of infections among people who have limited directed contact with swine. In the United States, a significant association of HEV seropositivity in humans with keeping pets in the home has been found

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(Kuniholm et al., 2009) but again these data do not necessarily indicate an exclusively zoonotic nature of HEV infections, and may rather reflect a greater exposure of pet owners to environmental contamination, which may occur from different sources including human waste. An alternative source of HEV infection may be presented by acutely infected patients, who develop viremia and shed virus in stool during infection. In some patients, HEV viremia and fecal shedding were observed to be protracted after the acute phase of infection (Chandra et al., 2010; Takahashi et al., 2007). Therefore, humans with acute infections, in propagating HEV, potentially contribute to the local viral pool. In addition, chronic HEV infection, as may occur in solid-organ transplant recipients (Gerolami et al., 2009; Haagsma et al., 2008; Kamar et al., 2008a; Kamar et al., 2008b), hematology patients on chemotherapy (le Coutre et al., 2009; Mansuy et al., 2009; Ollier et al., 2009; Peron et al., 2006; Tavitian et al., 2010), and human immunodeficiency virus-infected people (Colson et al., 2009; Dalton et al., 2009), increases the duration of HEV viremia and fecal shedding, and can be associated even more significantly with HEV transmission. Indeed, an increasing number of people in developed countries are immunocompromised owing to the ongoing epidemic with human immunodeficiency viruses, more widespread usage of immunomodulating drugs, and more instances of organ transplantation. When this group of people becomes infected by HEV, they disproportionately contribute to HEV shedding. However, the contribution of chronically infected patients to HEV dissemination, although conceivable, should be carefully examined. It has been recently shown that HEV recovered after a prolonged shedding in feces was not infectious in cell culture (Takahashi et al., 2007), suggesting that biological properties of HEV may vary during chronic infection.

8. The “true” number of human HEV infections Viral population size plays a significant role in viral evolution, and is strongly associated with the rate of HEV transmission. Therefore, the global and local number of infections is an important factor defining HEV evolution and affecting genetic heterogeneity of HEV strains. However, several distinctive features of HEV infection such as subclinical infections, duration of immune response, host range, rate of transmission among different hosts, varying modes of transmission and capacity to cause acute self-limiting or chronic infections considerably confound the estimates of the HEV population size. The number of subclinical human infections is at least two times greater than clinical infections among sporadic cases and during outbreaks in developing countries (Clayson et al., 1997; Clayson et al., 1998; Teshale et al., 2010). Recently, it was estimated that only 1–2% of HEV infections in China become symptomatic in adults (Wedemeyer and Pischke, 2011). It is known that development of asymptomatic HEV infection is dose-dependent in experimentally infected cynomolgus macaques (Aggarwal et al., 2001) and swine (Meng et al., 1998a; Meng et al., 1998b). Considering that the general human population in developed countries is exposed to low doses of HEV, it seems reasonable to assume that a proportion of HEV infections in these countries is not accompanied by any clinical symptoms and remains unrecognized. People with subclinical infections may induce a limited immune response against HEV but are, nevertheless, associated with viremia and fecal shedding (Aggarwal et al., 2001; Clayson et al., 1995; Husain et al., 2010; Nicand et al., 2001), thereby still being able to contribute to the HEV pool and transmission. Thus, it has been suggested that asymptomatic HEV-infected patients may also serve as a reservoir of hepatitis E (Nicand et al., 2001). Furthermore, HEV may be repeatedly passaged as a subclinical infection among humans resulting in

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persistence similar to poliovirus in endemic regions of the world (Aggarwal et al., 2001). In developed countries, the seroprevalence of anti-HEV IgG was estimated to range from 7% to 21% (Buti et al., 2006; Dalton et al., 2007a; Ijaz et al., 2009; Kuniholm et al., 2009; Mansuy et al., 2008; Tanaka et al., 2005), and in China, the seroprevalence among blood donors was found to be as high as 33% (Guo et al., 2010), while that in a rural Bangladesh population was 23% (Labrique et al., 2009). It is remarkable that these relatively high antibody detection rates are achieved in spite of waning immunity and antibody decay following primary HEV infection. A recent report on hepatitis E antibody kinetics showed a 2-log decline in detectable anti-HEV IgG in infected patients over ∼400 days after acute illness (Myint et al., 2006). Other studies reported detection of hepatitis E antibodies in 47% of persons affected by icteric HEV infection 14 years after a hepatitis E epidemic, with the prevalence of antibodies falling to 25% among persons with history of HEV infection 30 years after the epidemic (Khuroo, 2010; Khuroo et al., 1993). Goldsmith et al. (1992) reported that HEV antibody declined to undetectable levels within 6–12 months of serologically confirmed infection in children. All these observations of subclinical infections, high seroprevalences, and waning immunity in infected patients suggest that the total number of HEV infections may be substantially greater than reported in seroprevalence studies. In the light of such a high HEV prevalence, however, it remains unclear what sustains human infections with “zoonotic” HEV genotypes in the USA, Europe, Japan and China. Is it possible that the introduction of HEV to human populations through food-borne transmission, and environmental exposure to animals and animal waste sufficiently account for the high seroprevalence? Existing epidemiological and molecular epidemiologic data do not always suggest direct zoonotic transmission (Borgen et al., 2008). Additionally, direct exposure to pigs in developed countries is not identified as a significant risk factor for acquisition of HEV infection (Meader et al., 2010). The role of zoonotic transmission therefore requires further evaluation. It can be hypothesized that environmental exposure to human and animal waste perpetuates genotype 3 and 4 HEV infections in human populations in developed countries. This hypothesis, if substantiated, has important implications for HEV evolution, as it challenges the notion of exclusive zoonotic transmission and more fully explains the wide diversity of genotype 3 and 4 HEV strains and the distinctiveness between human and animal strains.

9. Transmission rates and HEV genetic relatedness The basic reproduction ratio, R0 , for HEV transmission among pigs was estimated to range from 4.02 to 5.17 in one study (Satou and Nishiura, 2007) to up to 8.8 in another study (Bouwknegt et al., 2008), indicating that a single infected animal on average can cause from ∼4 to 9 secondary infections among susceptible animals. Such a high rate of secondary infections provides the potential for causing epidemics in the swine population. The force of infection among swine or the rate at which susceptible animals become infected was estimated at 2.68–3.45 × 10−2 per day (Satou and Nishiura, 2007). In another study, the rate of transmission among swine was estimated at 0.66 per day, thus giving the average exposure-time required for infection to be only 1.5 (0.7–3.1) days (Bouwknegt et al., 2009). By contrast, the average proportion of susceptible humans acquiring indigenous HEV infections in England was estimated to range from 0.2% to 1.74% per year (Ijaz et al., 2009). These estimates show the essential difference in the rate of transmission in swine and human populations. A rapid succession and high prevalence of HEV infections among farmed swine generates conditions for rapid HEV evolution in the

animal population. As a result, detectable genetic divergence is frequently observed among animal HEV strains despite the use of short genomic regions for molecular characterization of HEV strains. The large differences in the rates of HEV transmission among swine, from swine to humans and among humans should lead to corresponding differences in the rate of HEV evolution in swine and human populations. These disparities likely contribute to the genetic distinction between the local animal and human HEV strains. The extent of dissimilarity between human and swine HEV strains also depends on other factors such as the rate of HEV introduction to human populations from animal reservoirs, HEV transmissibility among human hosts, and the size of the animal and human reservoirs of HEV infections. Finally, homoplasy associated with replication in human or swine hosts is another important factor that may affect genetic similarity between the local swine and human HEV strains (Hall, 2007). In addition to the rate of transmission, HEV evolution in different epidemiological settings could be affected by the dominant mechanisms of transmission and the topology of contact networks among swine and human populations. The structure of host contact networks was shown to significantly affect the herd immunity threshold (Fu et al., 2010a), and pathogen diversity and strain structure (Buckee et al., 2004). The contact networks for indoor-farmed swine should have limited structural variations in different parts of the world (but should be different for free-ranging pigs), whereas networks of human contacts essential for transmission of HEV infections may vary significantly in different countries owing to differences in dietary preferences, health care, age structure, cultural traditions, infrastructure, geography, etc. The difference in contacts between swine and human populations and in the host contact networks of different human populations in conjunction with high prevalences of HEV infections may lead to separation of HEV strains over time, even when the same strains have been originally shared by these populations. The separation between swine and human HEV strains in central China (Zhang et al., 2009a) may represent an example of such genetic distinction and suggests different evolutionary paths for HEV strains found in swine and human populations. Lastly, specific modes of HEV transmission may significantly define the extent of HEV dissemination in different human populations. It was observed that contact exposure is a more efficient way for HEV transmission to susceptible pigs than direct oral inoculation (Kasorndorkbua et al., 2004; Meng et al., 1998a; Meng et al., 1998b), suggesting a greater efficiency of HEV transmission through continuous ingestion of contaminated material rather than a single ingested dose. If this mode of transmission applies to human infections, then in developing countries where contaminated sources of drinking water are frequently responsible for dissemination of HEV infection, the human population is continuously exposed to HEV and, therefore, efficiently infected; whereas in developed countries exposure to HEV infections is most probably sporadic and, therefore, less efficient in establishing HEV infections in susceptible hosts. Additionally, it was shown that the intravenous route is more efficient in causing HEV infection in pigs and primates than the oral route (Kasorndorkbua et al., 2004; Purcell and Emerson, 2001). Again, if these findings could be applicable to human infections, parenterally transmitted HEV may become more efficient in establishing infection than HEV per oral transmission. Variation of the aforementioned factors affecting transmission produces conditions for specific evolution of HEV in animal and human populations in different parts of the world and contributes to development of genetic distinctions between HEV strains infecting swine and human populations. Enteric transmission among human hosts in developed countries, if existent, would have frequently been disrupted owing to the high hygiene standards.

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However, the sheer strength of the transmission vector from swine to human hosts, the efficacy of which has presumably increased about 50 years ago (Ijaz et al., 2009), may be responsible for the current high HEV prevalence among humans in these countries. This high prevalence in turn may generate conditions for more efficient dissemination of HEV through routes that hitherto have a limited capacity to contribute to the viral pool, such as parenteral transmission. 10. Conclusion The diversity and varying efficacy of the potential modes of transmission between animal and human populations in different parts of the world coupled with the high prevalence of HEV infection generate conditions for a very complex HEV evolution that has resulted in significant heterogeneity among HEV strains. Unfortunately, the study of this multifaceted evolution is considerably hampered with the almost universal use of short disparate HEV genomic regions in genetic analysis. Further advancements in molecular epidemiology of HEV infection require a more extensive sampling of HEV variants in different epidemiological settings and improvement in accurate identification of genetic relatedness among HEV strains using longer or even whole-genome sequences. Acknowledgements The authors would like to thank Dr. C.G. Teo for his insightful comments and suggested modifications. We would also like to thank in-house reviewers and one external reviewer who read this paper and made helpful suggestions, which have improved this paper. References Aggarwal, R., Kamili, S., Spelbring, J., Krawczynski, K., 2001. Experimental studies on subclinical hepatitis E virus infection in cynomolgus macaques. J. Infect. Dis. 184 (11), 1380–1385. Arankalle, V.A., Paranjape, S., et al., 1999. Phylogenetic analysis of hepatitis E virus isolates from India (1976–1993). J. Gen. Virol. 80 (7), 1691–1700. Arankalle, V.A., Chobe, L.P., Joshi, M.V., Chadha, M.S., Kundu, B., Walimbe, A.M., 2002. Human and swine hepatitis E viruses from Western India belong to different genotypes. J. Hepatol. 36 (3), 417–425. Aye, T.T., Uchida, T., et al., 1992. Complete nucleotide sequence of a hepatitis E virus isolated from the Xinjiang epidemic (1986-1988) of China. Nuc. Acids Res. 20 (13), 3512. Balayan, M.S., 1997. Epidemiology of hepatitis E virus infection. J. Viral Hepat. 4 (3), 155–165. Bilic, I., Jaskulska, B., Basic, A., Morrow, C.J., Hess, M., 2009. Sequence analysis and comparison of avian hepatitis E viruses from Australia and Europe indicate the existence of different genotypes. J. Gen. Virol. 90 (4), 863–873. Borgen, K., Herremans, T., Duizer, E., Vennema, H., Rutjes, S., Bosman, A., de Roda Husman, A.M., Koopmans, M., 2008. Non-travel related Hepatitis E virus genotype 3 infections in the Netherlands; a case series 2004–2006. BMC Infect. Dis. 8, 61. Bouwknegt, M., Frankena, K., Rutjes, S.A., Wellenberg, G.J., de Roda Husman, A.M., van der Poel, W.H., de Jong, M.C., 2008. Estimation of hepatitis E virus transmission among pigs due to contact-exposure. Vet. Res. 39 (5), 40. Bouwknegt, M., Rutjes, S.A., Reusken, C.B., Stockhofe-Zurwieden, N., Frankena, K., de Jong, M.C., de Roda Husman, A.M., Poel, W.H., 2009. The course of hepatitis E virus infection in pigs after contact-infection and intravenous inoculation. BMC Vet. Res. 5, 7. Boxall, E., Herborn, A., Kochethu, G., Pratt, G., Adams, D., Ijaz, S., Teo, C.G., 2006. Transfusion-transmitted hepatitis E in a “nonhyperendemic” country. Transfus. Med. 16, 79–83. Buckee, C.O., Koelle, K., Mustard, M.J., Gupta, S., 2004. The effects of host contact network structure on pathogen diversity and strain structure. Proc. Natl. Acad. Sci. U.S.A. 101 (29), 10839–10844. Buti, M., Dominguez, A., Plans, P., Jardi, R., Schaper, M., Espunes, J., Cardenosa, N., Rodriguez-Frias, F., Esteban, R., Plasencia, A., Salleras, L., 2006. Community-based seroepidemiological survey of hepatitis E virus infection in Catalonia, Spain. Clin. Vaccine Immunol. 13 (12), 1328–1332. Chandra, V., Taneja, S., Kalia, M., Jameel, S., 2008. Molecular biology and pathogenesis of hepatitis E virus. J. Biosci. 33 (4), 451–464.

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