Book on hepatitis from page 75 to 85

Book on hepatitis from page 75 to 85

HBV Virology  75
by capsid-assisted reverse transcription may result from a non-identical supply of
dNTPs inside individual nucleocapsids at the moment of their enclosure by the
dNTP impermeable envelope. This predicts that intracellular cores produced in the
absence of envelopment should contain further extended positive DNAs.
Alternatively, space restrictions in the capsid lumen could prevent plus-strand DNA
completion; in this view, further plus-strand elongation after infection of a new cell
might destabilize the nucleocapsid and thus be involved in genome uncoating (Beck
2007, Nassal 2008)
The final replication step, the assembly and release of HBV Dane particles, is also
not fully understood. The envelopment of the DNA-containing nucleocapsids
requires a balanced coexpression of the S and L proteins in order to recruit the
nucleocapsid to the site of budding. Although the role of the envelope proteins in
regulating the amplification of cccDNA in HBV is not well-characterised, recent
studies indicate that the lack of expression of the envelope proteins increased
cccDNA levels, while coexpression of the envelope proteins not only favours the
secretion of viral particles, but also limits the completion of the plus-strand (Lentz
2011).
Animal models of HBV infection
Because of the narrow host range and the lack of easily accessible and robust in
vitro infection systems the study of HBV biology has been limited. Consequently it
has been attempted by researchers all over the world to establish animal models and
cell culture systems that at least partially reproduce some stages of HBV infection
and can be used, e.g., for the preclinical testing of novel antiviral drugs.
Most of the progress in hepatitis B virus research are based on infection studies
performed with the two most used HBV-related animal viruses: DHBV, which
infects Peking ducks (Mason 1980) and WHV (Summers 1978), which infects the
Eastern American woodchuck (Marmota monax).
One of the major advantages of the DHBV model is that domestic Peking ducks
can be used under normal laboratory conditions and DHBV-permissive primary
hepatocytes from ducklings or embryos are easily accessible. Furthermore, ducks
show very high infectivity rates in vivo (Jilbert 1996) with high levels of DHBV
replication and antigen expression. However, in contrast to mammalian
hepadnaviruses, DHBV infection is cleared within a few days post-infection if the
virus is not transmitted vertically. The DHBV genome is also smaller than that of
the mammalian hepadnaviruses and  shares little primary nucleotide sequence
homology (40%) with HBV. Furthermore, DHBV infection is usually not associated
with liver disease and development of hepatocellular carcinoma (HCC).
Nevertheless, the duck model was widely used in preclinical trials (Zimmerman
2008, Reaiche 2010, Chayama 2011) and has contributed substantially to elucidate
the hepadnaviral replication scheme (Mason 1982, Summers 1988, Delmas 2002).
In vitro and in vivo studies with woodchuck hepatitis B virus (WHV) have been
fundamental in the preclinical evaluation of antiviral drugs now in use for treatment
of HBV infection (Moraleda 1997, Tennant 1998, Mason 1998, Block 1998, Dandri
2000, Korba 2004, Menne 2005). This is due to the fact that WHV is more similar
to HBV in terms of genomic organization than the avian hepadnaviruses.
Experimental infection of newborn woodchucks almost invariably leads to chronic
76  Hepatology 2012
infection, whereas most animals infected at older ages develop acute hepatitis that
results in an efficient immune response leading to viral clearance. Since acute and
chronic WHV infections in woodchucks show serological profiles similar to those
of HBV infection in humans, the woodchuck system has provided important
information about factors involved in the establishment of  virus infection,
replication and viral persistence (Lu 2001). Virtually all WHV chronic carrier
woodchucks succumb to HCC 2-4 years post infection. Like in human HCC,
regenerative hepatocellular nodules and hepatocellular adenomas are
characteristically observed in WHV-infected woodchuck livers (Korba 2004).
Proto-oncogene activation by WHV DNA integration has been observed frequently
and is thought to play an important role in driving hepatocarcinogenesis in
woodchucks, often activating a member of the myc family by various mechanisms
(Tennant 2004). Viral integration is commonly found in woodchucks even after
resolution of transient infection with WHV (Summers 2003), while its frequency
increases dramatically in chronically infected animals (Mason 2005). Interestingly,
WHV viral integration was used as a genetic marker to follow the fate of infected
hepatocytes during resolution of transient infection in woodchucks (Summers 2003)
and to estimate the amount of cell turnover occurring in the course of chronic
infection (Mason 2005).  Experimental infection studies in woodchucks also
demonstrated that WHV mutants that lacked the X gene were unable or severely
impaired to replicate  in vivo  (Chen 1993, Zoulim 1994, Zhang 2001). The
woodchuck model of viral-induced HCC has been used to test chemoprevention of
HCC using long-term antiviral nucleoside therapy and for the development of new
imaging agents for the detection of hepatic neoplasms by ultrasound and magnetic
resonance imaging (Tennant 2004). One main difference between human and rodent
hepatitis B resides in the absence of associated cirrhosis in woodchuck and squirrel
livers, even after prolonged viral infection (Buendia 1998). It is possible that the
rapid onset of hepatocyte proliferation following liver damage in rodents does
account for this discrepancy. In general, despite important advances achieved in
understanding the pathogenesis of WHV infection, one general disadvantage for
using woodchucks is that they are genetically heterogeneous animals, difficult to
breed in captivity and to handle in a laboratory setting.
Although HBV infects humans exclusively, it can be used to infect chimpanzees
experimentally and, to a certain extent, tupaia, the Asian tree shrew (Baumert
2005). Chimpanzees were the first animals found to be susceptible to HBV infection
(Barker 1973) and play an important role in the development of vaccines and in the
evaluation of the efficacy of therapeutic antibodies (Ogata 1999, Dagan 2003).
Though chimpanzees are not prone to develop  chronic liver disease (Gagneux
2004), they provide an ideal model for the analysis of early immunological events
of HBV acute infection and pathogenesis (Guidotti 1999). Infection experiments
with chimpanzees showed that the majority of viral DNA is eliminated from the
liver by non-cytolytic mechanisms that precedes the peek of T cell infiltration
(Guidotti 1999). T cell depletion studies in chimpanzees also indicate that the
absence of CD8-positive cells greatly delay the onset of viral clearance (Thimme
2003). Chimpanzees have been used for preclinical testing of preventive and
therapeutic vaccines (Will 1982, Guidotti 1999, Iwarson 1985, Kim 2008, Murray
2005). Nonetheless, the large size, the strong ethical constraints and the high costs
of chimpanzees severely limit their use for research purposes. 
HBV Virology  77
The tree shrew species Tupaia belangeri has been analyzed for the study of HBV
infection both in vitro and in vivo, taking advantage of the adaptability of these non-rodent mammals to the laboratory environment (Baumert 2005, von Weizsacker
2004). Inoculation of tree shrews with HBV-positive human serum was shown to
result in viral DNA replication in their livers, HBsAg secretion into the serum, and
production of antibodies to HBsAg and HBeAg (Walter 1996). Although
experimental infection of tree shrew with HBV infectious serum is not highly
efficient, productive HBV infection was successfully passed through five generations
of tree shrews and was specifically blocked by immunization with hepatitis B vaccine
(Yan 1996a). Interestingly, the development of hepatocellular carcinoma in tree
shrews exposed to hepatitis B virus and/or aflatoxin B1 was reported (Yan 1996b).
Whereas experimental infection of tree shrews causes only a mild, transient infection
with low viral titers in these animals, primary hepatocytes isolated from T. belangeri
turned out to be a valuable alternative source of HBV-permissive cells (von
Weizsacker 2004). More recently, the woolly monkey hepatitis B virus (WMHV)
was isolated from a woolly monkey (Lagothrix lagotricha), an endangered new world
primate (Lanford 1998). Interestingly, it has been shown that primary tupaia
hepatocytes are susceptible to infection with WMHBV (Kock 2001, Dandri 2005a),
providing a useful and more accessible alternative system for studying the early steps
of hepadnaviral infection in vitro (Schulze 2011) and in vivo (Petersen 2008).
Because of the different limitations encountered using chimpanzees and models
based on HBV-related viruses, it is not surprising that recent developments have
focused on using the natural target of HBV infection: the human hepatocyte.
However, primary human hepatocytes are not easy to handle, cannot be propagated
in vitro and their susceptibility to HBV infection is generally low and highly
variable. Furthermore, cultured cells may respond differently to the infection than
hepatocytes in the liver. The generation of mice harboring human chimeric livers
offered new possibilities to overcome some of these limitations. Two major models
are currently available: the urokinase-type plasminogen activator (uPA) transgenic
mouse (Rhim 1994) and the knockout fumarylacetoacetate hydrolase (FAH) mouse
(Azuma 2007). In both systems, the absence of adaptive immune responses permits
the engraftment of transplanted xenogenic hepatocytes, while the presence of
transgene-induced hepatocyte damage creates the space and the regenerative
stimulus necessary for the transplanted cells to repopulate the mouse liver. Both
models permit the establishment of HBV infection, which can then persist for the
life-span of the chimeric mouse (Dandri 2001, Bissig 2010).  While mouse
hepatocytes do not support HBV infection, human chimeric mice can be efficiently
infected by injecting infectious serum derived from either patients or chimeric mice.
Furthermore, genetically engineered viruses created in cell culture can be used to
investigate phenotype and in vivo fitness of distinct HBV genotypes and variants
(Tsuge 2005). Within the mouse liver human hepatocytes maintain a functional
innate immune system and respond to stimuli induced by exogenously applied
human IFN α.  The lack of an adaptive immune system and the undetectable
responsiveness of mouse liver cells to human IFN α make the model ideal to exploit
the capacities of HBV to interfere with pathways of the innate antiviral response in
human hepatocytes (Lutgehetmann 2011). Chimeric mice can be superinfected or
simultaneously infected with different human hepatotropic viruses to investigate the
78  Hepatology 2012
mechanisms of virus interference and response to antiviral treatment in the setting
of coinfection (Hiraga 2009).
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HCV Virology  85
6.  HCV Virology
Bernd Kupfer
History
Hepatitis C virus (HCV) is a major cause of progressive liver disease with
approximately 130-170 million people infected worldwide. HCV induces chronic
infection in up to 80% of infected individuals. The main complications of HCV
infection are severe liver fibrosis and cirrhosis, and 30-50% of individuals with
cirrhosis go on to develop hepatocellular carcinoma (Tong 1995, Poynard 1997).
Until 1975, only two hepatitis viruses had been identified, the “infectious hepatitis
virus” (hepatitis A virus, HAV) and the “serum hepatitis virus” (hepatitis B virus,
HBV). However, other viruses were excluded from being the cause of
approximately 65% of post-transfusion hepatitis. Therefore, these hepatitis cases
were termed “non-A, non-B hepatitis” (NANBH) (Feinstone 1975). Inoculation of
chimpanzees (Pan troglodytes) with blood products derived from humans with
NANB hepatitis led to persistent increases of serum alanine aminotransferase (ALT)
indicating that an infectious agent was the cause of the disease (Alter 1978,
Hollinger 1978). Subsequently, it was demonstrated that the NANBH agent could
be inactivated by chloroform (Feinstone 1983). Moreover, it was reported that the
infectious agent was able to pass through 80 nm membrane filters (Bradley 1985).
Taken together these findings suggested that the NANBH causing agent would be a
small virus with a lipid envelope. However, the lack of a suitable cell culture system
for cultivation of the NANBH agent and the limited availability of chimpanzees
prevented further characterization of the causative agent of NANBH for several
years. In 1989, using a newly developed cloning strategy for nucleic acids derived
from plasma of NANBH infected chimpanzees the genome of the major causative
agent for NANBH was characterized (Choo 1989). cDNA clone 5-1-1 encoded
immunological epitopes that interacted with sera from individuals with NANBH
(Choo 1989, Kuo 1989). The corresponding infectious virus causing the majority of
NANBH was subsequently termed hepatitis C virus (HCV).

Book on hepatitis from page 68 to 74

Book on hepatitis from page 68 to 74

68  Hepatology 2012
Figure 2. Genome organization and transcripts of the human hepatitis B virus. The
outer thin lines represent the viral transcripts that initiate at different sites, under the
control of distinct promoters, but are all terminated after a common polyadenylation site.
The RNA signal on the terminally redundant pgRNA is indicated as a hairpin. The thick
lines represent the rcDNA form of the genome as present in infectious virions. The 5’ end
of the minus-strand DNA is covalently linked to the terminal protein of the polymerase.
The 5´ end of the incomplete plus-strand DNA is constituted by an RNA oligo derived from
the 5’ end of pgRNA. DR1 and DR2 indicate the direct repeats. The inner arrows indicate
the open reading frames (adapted from Nassal 2008).
HBV structural and non-structural proteins
The three surface proteins (L, M, and S) are encoded from one open reading frame
(PreS/S) which contains three start codons (one for the large, one for the middle and
one for the small protein) but promotes the transcription of 2 mRNAs of 2.4 and 2.1
Kb, named preS and S RNAs (Glebe 2007). Notably, the preS/S ORF entirely
overlaps with the polymerase open reading frame (Lee 2004). The three HBV
envelope proteins share the C-terminal domain of the S-protein, while the M- and L-protein display progressive N-terminal extensions of 55 and, genotype-dependent,
107 or 118 amino acids (preS2 and preS1). The small envelope protein contains the
hepatitis B surface antigen (HBsAg). In virions the stoichiometric ratio of L, M and
S is about 1:1:4, while the more abundantly secreted non-infectious subviral
particles (SVPs) contain only traces of L-protein (Bruss 2007). The envelope
proteins are cotranslationally inserted into the ER membrane, where they aggregate,
bud into the ER lumen, and are secreted by the cell, either as 22 nm subviral
envelope particles (SVPs) or as 42 nm infectious virions (Dane particles), after
having enveloped the DNA-containing nucleocapsids. The surface proteins of
mammalian Hepadnaviridae  have been shown to be N-  and O-glycosylated
(Schildgen 2004, Schmitt 2004). These glycosylations have been shown to be
responsible for proper secretion of progeny viral particles. During synthesis, the
preS1 domain of L is myristoylated and translocated through the ER. This
modification and the integrity of the first 77 amino acids of preS1 have been shown
HBV Virology  69
to be essential for infectivity (Glebe 2005, Nassal 2008) (Schulze 2010). Both
spherical and filamentous SVPs are secreted into the blood of infected individuals in
a 10
3
-10
6
-fold excess relative to the infectious particles. The biological function of
the excess of SVPs in patients is not clear. It was suggested that SVPs might absorb
the neutralizing antibodies produced by the host and hence increase the ability of the
infectious particles to reach the hepatocytes. It has also been suggested that SVPs
contribute to create a state of immune tolerance, which is a precondition for highly
productive persistent infection.
In the cytoplasm, the core protein dimerises and self-assembles to form an
icosahedral nucleocapsid. The full-lengthcore protein is 183 amino acids in length
and consistsof an assembly domain and a nucleic acid-bindingdomain, which plays
an active role in binding and packaging of the pregenomic RNA together with the
viral polymerase, and thus enables the RT-polymerase/RNA complex to initiate
reverse transcription within the newly forming nucleocapsids (Kann 1994, Kann
2007, Kann 1999, Daub 2002). The core protein can be phosphorylated by several
kinases. This step along with the presence of the viral polymerase is important for
the specific packaging of the pgRNA (Kann 1999, Porterfield 2010).
The viral polymerase is the single enzyme encoded by the HBV genome and is an
RNA-dependent DNA polymerase with RNase H activity. The HBV polymerase
consists of three functional domains and a so-called spacer region; the terminal
protein (TP) is located at its N-terminal domain, and serves as a primer for reverse
transcription of the pgRNA into a negative-strand DNA (Zoulim 1994, Nassal
2008). The spacer domain separates the terminal protein from the polymerase
domains (Beck 2007)
Despite the occurrence of nucleotide mutations due to the lack of proofreading
capacity of the HBV polymerase, the peculiar genomic organization of HBV, where
most of the genes overlap, imposes stronger constraints on the amino acid sequence,
which significantly reduces the occurrence of mutations in the absence of strong
selective pressures. Nevertheless, it has been shown that antiviral therapy with
nucleoside analogs can promote the selection of nucleotide mutations within
conserved domains of the reverse transcriptase, which lead to mutations also on the
amino acid sequence of the envelope proteins. Changes on the HBsAg structure may
lead to reduced binding of anti-HBs antibodies, and hence, they may favour the
selection of antibody escape mutants (Harrison 2006).
HBV also produces distinct non-structural proteins whose exact functions are not
fully elucidated. Besides the production of large amounts of empty SVPs, HBV
produces and secretes a non-particulate form of the nucleoprotein, the precore
protein, or HBeAg, which is not required for viral infection or replication, but
appears to act as a decoy for the immune system, and hence, has tolerogenic
functions in promotingviral persistence in the neonates of viremic mothers (Chen
2005, Visvanathan 2006). Theprecore and core proteins are translated from 2
distinct RNAspecies that have different 5' initiation sites: the precore RNA and the
pgRNA. Indeed, the precoretranscript, which also contains the full core gene,
encodes a signal sequence that directs theprecore protein to the lumen of the
endoplasmic reticulum, where it is post-translationally processed. Here, the precore
protein undergoesN- and C-terminal cleavage to produce the mature HBeAg form
(p17), which is then secreted as a monomeric protein. Interestingly, 20 to 30% of
the mature protein is retained in the cytoplasm, where it may antagonise TLR
70  Hepatology 2012
signaling pathways and so contribute to the suppression of the host innate immune
responses (Lang 2011). As an important marker for active viral replication, the
HBeAg is widely used in molecular diagnostics (Chen 2005, Hadziyannis 2006).
The X protein is a multifunctional regulatory protein with transactivating and pro-apoptotic potential, which can modify several cellular pathways (Bouchard 2004)
and act as a carcinogenic cofactor (Kim 1991, Dandri 1996, Slagle 1996).
Numerous DNA transfection experiments have shown that over-expression of the X
protein (HBx) causes transactivation of a wide range of viral elements and cellular
promoters (Bouchard 2004). The evidence that HBx responsive enhancers/
promoters do not share any common DNA sequence and that HBx does not bind
double-stranded DNA suggested that HBx may exert its transactivating activity
through protein-protein interactions. In vitro studies have shown that HBx can affect
various cytoplasmic signal transduction pathways by activating the Src kinase,
Ras/Raf/MAP kinase, members of the protein kinase C, as well as Jak1/STAT.
Furthermore, in vitro binding studies show that HBx can regulate the proteasome
function, and thus, may control the degradation of cellular and viral proteins (Zhang
2004). It has also been reported that HBx can affect mitochondria function, by
altering its transmembrane potential, as well as that HBx can modulate calcium
homeostasis (Bouchard 2001, Nassal 2008, Yang 2011).
Although the exact role of HBx in the context of HBV infection has not been
clarified, several lines of evidence obtained first using the woodchuck model
(Zoulim 1994) and more recently using uPA/SCID mice (Tsuge 2010) and
HepaRG
TM
cells (Lucifora 2011), have convincingly shown that HBx is required to
initiate HBV replication and to maintain virion productivity. Notably, these studies
indicated that despite the establishment of comparable cccDNA amounts,
transcription of HBV RNAs was dramatically impaired in cells inoculated with
HBV X, indicating that HBx is essential for viral transcription. These findings are
also in agreement with data showing that HBx is recruited to the cccDNA
minichromosome, where it appears to be involved in epigenetic control of HBV
replication (Belloni 2009, Levrero 2009). In addition, HBx has been shown to
enhance encapsidation of the pgRNA by increasing phosphorylation of the core
protein (Melegari 2005), indicating that HBx may support virion productivity in
various steps of the HBV life cycle.
Most HBV-related HCC show the integration of HBV DNA sequences including
the X gene (Brechot 2004, Pollicino 2011, Lupberger 2007). Although HBV
integrated forms are frequently rearranged and hence not compatible with the
expression of functional proteins, HBx sequences deleted in the C-terminal portion
have been frequently detected in tumoral cells (Iavarone 2003). In virus-associated
cancers, viral proteins have been shown to participate in epigenetic alterations by
disturbing the host DNA methylation system. Interestingly, a study suggested that
the HBV regulatory X protein is a potent epigenetic modifying factor in the human
liver, which can modulate the transcription of DNA methyltransferases required for
normal levels of genomic methylation and maintenance of hypomethylation of
tumor suppressor genes (TSGs) (Park 2007). HBx-promoted hypermethylation of
TSGs suggests a novel mechanism by which this promiscuous transactivating
protein may accelerate hepatocarcinogenesis (Kekule 1993, Dandri 1996).
HBV Virology  71
The HBV replication cycle
During the last 30 years, the generation of various HBV–transfected human
hepatoma cell lines and the use of related HBV viruses, like the duck hepatitis B
virus (DHBV) and the woodchuck hepatitis virus (WHV) have  significantly
contributed to elucidate many steps of the hepadnavirus replication cycle (Schultz
2004, Roggendorf 1995, Roggendorf 2007). Nevertheless, the lack of efficient in
vitro infection systems and of easily accessible animal models has significantly
hindered the identification of mechanisms and cellular factors mediating viral entry
and uncoating in human hepatocytes. Although primary hepatocytes remain
permissive in vitro for only a short time after plating, the availability of primary
hepatocytes from tree-shrews (Tupaia belangeri) for infection studies with HBV
and the closely-related woolly monkey hepatitis B virus (WM-HBV) (Kock 2001),
and the discovery of a human hepatoma cell line (HepaRG) able to gain
susceptibility for HBV infection upon induction of differentiation in vitro (Gripon
2002), have lately expanded our possibilities to functionally dissect the HBV entry
process (Glebe 2007, Schulze 2010).
The first step in HBV infection appears to involve a non-cell-type specific
primary attachment to the cell-associated heparan sulfate proteoglycans (Schulze
2007). This first reversible attachment step is then followed by an irreversible
binding of the virus to a specific, but still unknown hepatocyte-specific receptor
(Urban 2010, Glebe 2007). This step probably requires activation of the virus,
resulting in exposure of the myristoylated N-terminus of the L-protein. Important
determinants for infectivity within the HBV envelope proteins were identified using
mutational analyses. These include 75 amino acids of the preS1 domain of the HBV
L‐protein, its myristoylation and the integrity of a region in the antigenic loop of the
S domain (Gripon 2005, Engelke 2006). Potential HBV receptor candidates have
been described in the past, but none of them has been confirmed in a functional
assay (Glebe 2007). Recent studies indicated that cell polarization, in addition to the
differentiation status of the hepatocytes, plays an important role in the infection
process (Schulze 2011).
Upon binding to the cell membrane, two possible entry pathways have been
proposed. Experimental evidence suggests that HBV can be either involved in an
endocytosis process, followed by the release of the nucleocapsid from endocytic
vesicles, or HBV may enter the hepatocytes after fusion of the viral envelope at the
plasma membrane. As soon as the viral nucleocapsids are released into the
cytoplasm, the viral relaxed circular partially double stranded DNA (rcDNA) with
its covalently linked polymerase needs to enter the cell nucleus in order to convert
the rcDNA genome into a covalently closed circular form (cccDNA) (Nassal 2008).
Previous studies indicated that the viral capsids are transported via microtubules to
the nuclear periphery (Rabe 2006). The accumulation of the capsids at the nuclear
envelope would then facilitate interactions with nuclear transport receptors and
adaptor proteins of the nuclear pore complex (Kann 2007). Although immature
capsids may remain trapped within the nuclear baskets by the pore complexes, the
mature capsids eventually disintegrate, permitting the release of both core capsid
subunits and of the polymerase-viral DNA complexes, which diffuse into the
nucleoplasm (Schmitz 2010).
72  Hepatology 2012
Within the infected nuclei the establishment of productive HBV infection requires
the removal of the covalently attached viral polymerase and completion of the
positive-strand by the cellular replicative machinery to form the supercoiled
cccDNA molecule, which is then incorporated into the host chromatin and serves as
the template of viral transcription and replication (Nassal 2008, Newbold 1995). For
the formation of the cccDNA, the terminal protein and one of the redundant
terminal repeats present on the rcDNA need to be removed. So far it is assumed that
cellular ligases and probably other enzymes involved in DNA repair mechanisms
become active and convey the relaxed circular form into the cccDNA (Seeger
2000). Unlike the provirus DNA of retroviruses, the cccDNA does not need to be
incorporated into the host genome. Nevertheless, integrations of HBV DNA
sequences do occur, particularly in the course of hepatocyte turnover and in the
presence of DNA damage, as has been shown in cell culture (Dandri 2002) and in
the woodchuck system (Petersen 1998, Summers 2004, Mason 2005).
Disguised as a stable non-integrated minichromosome (Bock 1994, Bock 2001),
the cccDNA utilizes the cellular transcriptional machinery to produce all viral
RNAs necessary for protein production and viral replication, which takes place in
the cytoplasm after reverse transcription of an over-length pregenomic RNA
(pgRNA) (Figure 3).
cccDNA
AAA
AAA
cccDNA
AAA
AAA
AAA
AAA
Figure 3. The HBV lifecycle. Upon hepatocyte infection the nucleocapsid is released into
the cytoplasm and the rcDNA transferred to the cell nucleus where it is converted into the
cccDNA minichromosome. After transcription of the viral RNAs, the pgRNA is
encapsidated and reverse-transcribed by the HBV polymerase. Through Golgi and ER
apparatus the core particles acquire the envelope and are secreted. Via viral entry and
retransporting of the newly synthesized HBV DNA into the cell nucleus, the cccDNA pool
can be amplified.
Experimental DHBV infection studies indicate that the cccDNA can be formed
not only from incoming virions, but also from newly synthesized nucleocapsids,
which instead of being enveloped and secreted into the blood, are rather transported
into the nucleus to ensure accumulation, and later maintenance, of the cccDNA pool
(Zoulim 2005b, Nassal 2008). According to this scenario, multiple rounds of
infection are not needed to establish a cccDNA pool in infected duck hepatocytes.
HBV Virology  73
Moreover, expression of the DHBV viral large surface (LS) protein was shown to
induce a negative-feedback mechanism, whereby the accumulation of the LS protein
would be fundamental to shut off the cccDNA amplification pathway and redirect
the newly synthesized rcDNA-containing nucleocapsids to envelopment and
extracellular secretion (Kock 2010). Although this peculiar nuclear reentry
mechanism has been clearly demonstrated for the duck HBV (Summers 1991,
Nassal 2008, Wu 1990) and a high copy number of cccDNA molecules is generally
detected in chronically infected ducks and woodchucks (1 to 50 copies/cell) (Zhang
2003, Dandri 2000), lower cccDNA intrahepatic loads are generally determined in
human liver biopsies obtained from chronically HBV -infected patients (median 0.1
to 1 cccDNA copy/cell) (Werle-Lapostolle 2004, Wong 2004, Laras 2006, Volz
2007, Wursthorn 2006, Lutgehetmann 2008) and in chronically HBV-infected
human-liver chimeric uPA/SCID mice (Petersen 2008, Lutgehetmann 2011a,
Lutgehetmann 2011b, Lutgehetmann 2010), suggesting that different viral and host
mechanisms may control cccDNA dynamics and cccDNA pool size in human
infected hepatocytes (Levrero 2009). A recent study elegantly showed that HBV
converted the rcDNA into cccDNA less efficiently than DHBV in the same human
cell background (Kock 2010).
Although the formation of the cccDNA minichromosome is essential to establish
productive infection, recent studies performed in uPA/SCID mice indicate that this
step is achieved, initially, only in a minority of human hepatocytes. Indeed, three
weeks post-infection, the intrahepatic cccDNA load is very low (ca. 1 copy/50
human hepatocytes) and only sporadic cells stain HBcAg-positive, while within 8
weeks the majority of human hepatocytes become infected. Thus, several weeks
appear to be necessary for HBV to spread among human hepatocytes in vivo, even
in the absence of adaptive immune responses (Dandri 2011).
HBV polymerase inhibitors do not directly affect cccDNA activity and various in
vitro and in vivo studies support the notion that the cccDNA minichromosome is
very stable in quiescent hepatocytes (Moraleda 1997, Dandri 2000, Dandri 2005,
Lutgehetmann 2010). Thus, the significant decrease in cccDNA levels
(approximately 1 log10 reduction) generally determined after 1 year of therapy with
polymerase inhibitors (Werle-Lapostolle 2004) is imagined to derive from the lack
of sufficient recycling of viral nucleocapsids to the nucleus, due to the strong
inhibition of viral DNA synthesis in the cytoplasm, and less incoming viruses from
the blood. Nevertheless, cccDNA depletion is expected to require many years of
nucleos(t)ide drug administration. Thus, despite the absence of detectable viremia,
the persistence of the cccDNA minichromosome within the infected liver is
responsible for the failure of viral clearance and the relapse of viral activity after
cessation of antiviral therapy with polymerase inhibitors in chronically infected
individuals.  Furthermore, if viral suppression is not complete, the selection of
resistant variants escaping antiviral therapy is likely to occur (Zoulim 2005a,
Zoulim 2005b, Zoulim 2009). Resistant HBV genomes can be archived in infected
hepatocytes when nucleocapsids produced in the cytoplasm by reverse transcription
and containing resistant mutants are transported into the nucleus and added to the
cccDNA pool. Under antiviral pressure, these variants will coexist with wild-type
cccDNA molecules and function as templates for the production and possibly
further selection of replication-competent resistant mutants, which will spread to
74  Hepatology 2012
other hepatocytes and, eventually may even replace the wild-type cccDNA
molecules in the liver (Zoulim 2006, Zoulim 2009).
During chronic HBV infection immune-mediated cell injury and compensatory
hepatocyte proliferation may favour cccDNA decline and selection of cccDNA-free
cells (Mason 2005, Zhang 2003, Thermet 2008). Notably, studies with the duck
model show that antiviral therapy with polymerase inhibitors induce a greater
cccDNA reduction in animals displaying higher hepatocyte proliferation rates
(Addison 2002). cccDNA decrease was also determined in chronically WHV-infected woodchuck hepatocytes when cell turnover was induced  in vitro  by
addition of cellular growth factors and viral replication was suppressed by adefovir
treatment (Dandri 2000). Furthermore, the identification of uninfected cccDNA-negative cell clones containing “traces” of the infection in form of viral integrations
indicate that cccDNA clearance without cell destruction can occur in chronically
infected woodchucks  (Mason 2005). Thus, in chronic infection, killing of
hepatocytes may be instrumental not only to eliminate infected cells but also to
induce hepatocyte proliferation which, in turn, may favour cccDNA loss (Dandri
2005, Lutgehetmann 2010). On the other hand, studies have shown that very low
levels of cccDNA can persist indefinitely, possibly explaining lifelong immune
responses to HBV despite clinical resolution of HBV infection (Rehermann 1996).
As mentioned previously, the cccDNA acts chemically and structurally as an
episomal DNA with a plasmid-like structure (Bock 1994, Bock 2001, Newbold
1995), which is organized as a minichromosome by histone and non-histone
proteins. Hence its function is regulated, similarly to the cellular chromatin, by the
activity of various nuclear transcription factors, including transcriptional
coactivators, repressors and chromatin modifying enzymes (Levrero 2009).
Congruent with the fact that HBV infects hepatocytes, nearly all elements regulating
viral transcription have binding sites for liver-specific transcription factors (Levrero
2009, Quasdorff 2008). Nevertheless, although a number of factors regulating viral
transcription are known, the exact molecular mechanisms regulating HBV
transcription are still poorly defined. Both messenger and pregenomic RNAs are
transported into the cytoplasm, where they are respectively translated or used as the
template for progeny genome production. Thus, the transcription of the pgRNA is
the critical step for genome amplification and determines the rate of HBV
replication. Identification of the factors affecting stability and transcriptional
activity of the cccDNA in the course of infection and under antiviral therapy may
assist in the design of new therapeutic strategies aimed at silencing and eventually
depleting the cccDNA reservoir.
The next crucial step in HBV replication is the specific packaging of pgRNA, plus
the reverse transcriptase, into newly forming capsids. The pgRNA bears a secondary
structure – named the ε structure - that is present at both the 5’ and the 3’ ends. The
ε hairpin loops at the 5’ end are first recognized by the viral polymerase and act as
the initial packaging signal (Bartenschlager 1992). Binding of polymerase to the
RNA stem-loop structure ε initiates packaging of one pgRNA molecule and its
reverse transcription. The first product is single-stranded (ss) DNA of minus
polarity; due to its unique protein priming mechanism, its 5′ end remains covalently
linked to the polymerase. The pgRNA is concomitantly degraded, except for its 5′
terminal (~15–18 nucleotides which serve as primer for plus-strand DNA synthesis),
resulting in rcDNA. The heterogeneous lengths of the plus-strand DNAs generated

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60  Hepatology 2012
Acute and chronic HEV infections in organ
transplant recipients
Chronic courses of HEV infections have been described in European liver or kidney
transplant recipients since 2008 (Gerolami 2008, Haagsma 2009, Kamar 2008,
Pischke 2010c). 14 cases of acute hepatitis E were initially reported in kidney- and
liver-transplanted patients from southwest France (Kamar 2008). Eight of them
developed a chronic course leading to persistently elevated ALT levels, significant
histological activity and fibrosis after a follow-up of more than 12 months (range 10
to 18). Subsequently, additional cases of chronic HEV infections have been reported
in transplant patients by several groups (Pischke and Wedemeyer 2010), clearly
demonstrating that chronic hepatitis E can be associated with progressive liver
disease in patients after organ transplantation (Kamar 2011c).
A study from Germany examined 226 liver-transplanted patients and 129 patients
with chronic liver disease to evaluate the frequency of chronic HEV infections in
liver transplant recipients in a low endemic country (Pischke 2010c). All patients
were tested for HEV RNA and anti-HEV IgG. Two cases of chronic HEV infections
in liver transplanted patients were identified showing different courses. One of them
developed significant liver fibrosis (ISHAK F3) within less than 2 years. Both
patients were infected with HEV genotype 3. The possibility of reverse zoonotic
transmission was experimentally confirmed by infecting pigs with the patient’s
blood. HEV RNA was detectable in various organs of the pigs including muscle.
Thus, these findings further support the recommendations that eating uncooked
meat should be avoided by organ transplant recipients as this may represent a source
for acquiring HEV infection.
A recent study summarized retrospective data on hepatitis E in transplant
recipients in 17 centres. Overall, 85 cases of HEV infections were described and 56
(66%) patients developed chronic hepatitis E. Of note, chronicity was associated
with the use of tacrolimus and with low platelet count (Kamar 2011c). However it
has to be considered that the vast majority of patients had been recruited by one
center (Toulouse) and experiences from other regions and transplant centres need to
be reported.
Chronic courses of HEV infection have also been reported in heart transplant
recipients (de Man 2011, Pischke 2011b). Overall, all recipients of solid organ
transplant with elevated liver enzymes should be tested for HEV RNA unless other
obvious reasons already explain the hepatitis. In immunosuppressed patients testing
for HEV RNA should be applied as antibody testing may lack sensitivity. 
Hepatitis E in patients with HIV infection
Chronic hepatitis E was described for the first time in a patient with underlying HIV
infection in 2009 (Dalton 2009). This patient had a CD4 T cell count of less than
200 cells and high HIV RNA levels (>100,000 copies/ml). However, subsequent
studies from Spain (n=93) (Madejon 2009), Germany (n=123) (Pischke 2010a) and
England (n=138) (Keane 2012) could not identify cases of chronic hepatitis in HIV-infected individuals. HEV RNA was detected for more than 10 months in only one
out of 184 HIV-positive individuals in France (Kaba 2010). This patient had
particularly low CD4 counts (<50 cells/mm) while two additional patients with
Hepatitis E: an underestimated problem?  61
higher CD4 levels were able to clear HEV spontaneously. Thus, persistent HEV
infection is rarely observed in HIV-infected patients and only subjects with strongly
impaired immune system seem to be at risk for chronic hepatitis E.
Extrahepatic manifestations of hepatitis E
There is some evidence that HEV infections maybe associated with extrahepatic
manifestations. One case report described muscular weakness and a pyramidal
syndrome in a kidney transplant recipient with persistent HEV infection (Kamar
2011b). Moreover, neurological disorders including polyradiculopathy, Guillain-Barre syndrome, bilateral brachial neuritis, encephalitis or proximal myopathy, have
been reported in patients with acute and chronic HEV infections (Kamar 2011b).
The underlying mechanisms and the clinical relevance of this association require
further investigation.
Treatment of chronic hepatitis E
Treatment options for chronic hepatitis include reduction of immunosuppression,
administration of pegylated-interferon  α  with ribavirin. The first step in the
treatment of chronic HEV infection should be to evaluate if it is possible to reduce
the immunosuppressive medication (Pischke and Wedemeyer 2010). Reduction of
immunosupression in 16 solid organ transplant recipients with chronic hepatitis E
led to clearance of HEV in 4 cases (25%) (Kamar 2011a). A second possible
treatment option is the use of pegylated-interferon  α  (Haagsma 2010, Kamar
2010a). Treatment durations varied between 3 and 12 months. Overall, 4 out 5
patients were successfully treated with sustained clearance of HEV RNA. However,
the use of interferon can be associated with significant side effects and may cause
rejection in organ transplant recipients. Interferon α is therefore not recommended
in heart or kidney  transplant recipients. The antiviral efficacy of ribavirin
monotherapy has been evaluated by two French groups (Kamar 2010b, Mallet
2010). A sustained virological response was observed in 2/2 and 4/6 treated
patients, respectively. Ribavirin has also been used in a not-transplanted patient with
severe acute hepatitis E who showed rapid improvement of symptoms and liver
function tests during treatment (Gerolami 2011).
Vaccination
No commercial HEV vaccine is currently available. A vaccine developed by GSK
and the Walter Reed Army Institute that was successfully tested in a Phase II study
(Shrestha 2007). However, this vaccine has not been further developed. A group
from China reported data recently from a very large successful Phase III vaccine
trial (Zhu 2010). This trial included almost 110,000 individuals who received either
a recombinant HEV vaccine (“HEV 239”) or placebo. The vaccine efficacy after
three doses was 100%. It is currently not known if and when this vaccine will
become available in China and other countries. Moreover, the efficacy of this
vaccine needs to be evaluated in special risks groups such as patients with end-stage
liver disease or immunosuppressed individuals. It is also unknown if HEV-239 also
protects from HEV genotype 3 infection (Wedemeyer and Pischke 2011). 
62  Hepatology 2012
Conclusions/Recommendations
−  The prevalence of chronic HEV infections in liver transplant recipients
depends on the general prevalence in the population and is low in most
industrialized countries. However, chronic hepatitis E occurs and needs to be
considered in the differential diagnosis of graft hepatitis as persistent HEV
infection can be associated with progressive graft hepatitis and the
development of liver cirrhosis. Currently all reported cases of chronic HEV
infections in transplant recipients are caused by HEV genotype 3. It is not
known if chronic hepatitis E can also be caused by the other genotypes.
−  The diagnosis of HEV infection should not be based on serological assays
alone in organ transplant recipients as these assays may lack sensitivity.
Detection of HEV RNA by PCR in serum or stool represents the gold standard
to determine the diagnosis of HEV infection.
−  Organ transplant recipients and other immunocompromised individuals should
avoid eating uncooked meats to avoid infection with HEV.
−  Additional studies investigating the use of ribavirin for treatment of chronic
hepatitis E are necessary.
−  The relevance of extrahepatic manifestations associated with acute or chronic
HEV infections needs further examination.
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HBV Virology  65
5.  HBV Virology
Maura Dandri, Jörg Petersen
Introduction
The human hepatitis B virus (HBV) is a small enveloped DNA virus causing acute
and chronic hepatitis. Although a safe and effective vaccine has been available for
the last two decades, HBV infection still represents a major global health burden,
with about 350 million people chronically infected worldwide and more than 1
million deaths per year due to HBV-associated liver pathologies (Block 2007).
Many epidemiological and molecular studies have shown that chronic HBV
infection represents the main risk factor for hepatocellular carcinoma development
(Shepard 2006, Lok 2004, Pollicino 2011). The rate for chronicity is approximately
5% in adult infections, but it reaches 90% in neonatal infections. HBV transmission
occurs vertically and horizontally via exchange of body fluids. In serum, up to 10
12
HBV genome equivalents per ml serum can be found. Although HBV does not
induce direct cytopathic effects under normal infection conditions (Wieland 2004,
Thimme 2003), liver damage (fibrosis, cirrhosis, and eventually hepatocellular
carcinoma) is believed to be induced by the ongoing immune reaction and a
consistent inflammation of the liver (McMahon 2009, Chisari 2007).
HBV is the prototype member of the Hepadnaviridae family, which are the
smallest DNA-containing, enveloped animal viruses known. Characteristic of HBV
is its high tissue- and species-specificity, as well as a unique genomic organization
with asymmetric mechanism of replication (Nassal 2008). Since all hepadnaviruses
use a reverse transcriptase to replicate their genome, they are considered distantly
related to retroviruses. Despite decades of research and significant progresses in
understanding of the molecular virology of HBV, important steps of the infection,
such as the mechanism and cellular receptor(s) mediating viral entry, have not yet
been  clarified (Glebe 2007). Only recently, innovative infection models and
molecular techniques have opened new possibilities to investigate specific steps of
the lifecycle, as well as the organization and the activity of the covalently closed
circular DNA (cccDNA), the viral minichromosome serving as the template of HBV
transcription in the nucleus of the infected hepatocytes, which enables maintenance
of chronic HBV infection (Levrero 2009). 
66  Hepatology 2012
Taxonomic classification and genotypes
The Hepadnaviridae  form their own taxonomic group, since their biological
characteristics are not observed in any other viral family. Based on host and
phylogenetic differences, the family of Hepadnaviridae contains two genera: the
orthohepadnaviruses  infecting mammals, and the  avihepadnaviruses  that infect
birds. To date, orthohepadnaviruses have been found in human (HBV), woodchuck
(WHV) (Korba 1989), ground squirrel (GSHV), arctic squirrel (ASHV) and woolly
monkey (WMHBV) (Lanford 1998). Avihepadnaviruses  include duck HBV
(DHBV) (Mason 1980), heron HBV (HHBV) (Sprengel 1988), Ross’s goose HBV,
snow goose HBV (SGHBV), stork HBV (STHBV) (Pult 2001) and crane HBV
(CHBV) (Roggendorf 2007, Funk 2007, Dandri 2005b, Schaefer 2007).
Due to the lack of proofreading activity of the viral polymerase, misincorporation
of nucleotide mutations occurs during viral replication. This has led to the
emergence of eight HBV genotypes, A-H, which differ in more than 8% of the
genome, as well as different subgenotypes, which differ by at least 4% (Fung and
Lok 2004, Guirgis 2010). The HBV genotypes have different geographic
distribution (Liaw 2010), with predominance of genotype A in northwestern
Europe, North and South America, genotype B and C in Asia and genotype D in
eastern Europe and in the Mediterranean basin. The less diffuse remaining
genotypes are mostly found in West and South Africa (genotype E), in Central and
South America (genotypes F and H), while genotype G has been detected in France
and in the US (Pujol 2009). The phylogenetic tree of HBV genomes is reviewed
elsewhere (Schaefer 2007)
HBV structure and genomic organization
Three types of viral particles can be visualized in the infectious serum by electron
microscopy: the infectious virions and the subviral particles (SVPs). The infectious
virus particles are the so-called Dane particles (Dane 1970), have a spherical,
double-shelled structure of 42-44 nm containing a single copy of the viral DNA
genome, covalently linked to the terminal protein of the virus. A hallmark of HBV
infection is the presence of two additional types of particles, the spheres and the
filaments, which are exclusively composed of hepatitis B surface proteins and host-derived lipids (Glebe and Urban 2007). Since they do not contain viral nucleic
acids, the subviral particles are non-infectious. The spherical structures measure
around 22 nm in diameter, while the filaments are similarly width, but display
variable lengths (Figure 1).
The viral membrane contains three viral surface proteins and is acquired by the
virus  during budding into the endoplasmic reticulum (ER), whereas the viral
particles are transported via the secretory pathways through the ER and Golgi. The
surface proteins are named, according to their size, the preS1 (or large), the preS2
(or middle) and the S (or small), which corresponds to the HBsAg. As with nearly
all enveloped viruses, the HBV particle also contains proteins of host origin (Glebe
2007, Urban 2010).
The HBV genome consists of a partially double-stranded relaxed circular DNA of
approximately 3200 nucleotides in length, varying slightly from genotype to
genotype, that in concert with the core protein (HBcAg) forms the nucleocapsids
HBV Virology  67
(Nassal 2008). Within the Dane particle the negative strand of the viral DNA is
present in full-length, thus carrying the complete genetic information. In contrast,
the positive strand spans only ~ 2/3 of the genome in length, whilst its 3’ end is
variable in size (Summers 1988, Lutwick 1977). The viral polymerase is covalently
bound to the negative strand by a phosphotyrosine bond. At the 5’ end of the
positive strand a short RNA oligomer originating from the pre-genomic (pg) RNA
residually remains bound covalently after the viral DNA synthesis. The negative
strand also contains a small redundancy of 8-9 nucleotides in length on both the 5’
end and the 3’ end, named the R region. These redundant structures are essential for
viral replication (Seeger 1986, Seeger 2000, Nassal 2008, Lee 2004).
Figure 1. Schematic representation of the HBV virion and non-infectious empty
subviral particles (filaments and spheres). Within the nucleocapsid (HBcAg, shown in
black) is depicted the partial double-stranded viral genome (rcDNA) covalently linked to
the terminal protein of reverse transcriptase. The presence and distribution of the three
surface proteins L (preS1 or large), M (preS2 or middle) and S (small) are shown both on
HBV and subviral particles (adapted from Glebe 2007).
The HBV genome displays four major open reading frames (ORFs) that are
organized in a unique and highly condensed way (Block 2007). As shown in Figure
2, all ORFs are in an identical orientation, partially overlap and are encoded by the
negative strand. On the genome, 6 start codons, four promoters and two
transcription-enhancing elements have been identified. The four major ORFs are: I)
the preS/S, encoding the three viral surface proteins; II) the precore/core, encoding
both the core protein, essential for the formation of the nucleocapsid, and the non-structural pre-core protein, also known as the secreted e-antigen (HBeAg); III) the
pol ORF of the viral polymerase, which possesses reverse transcriptase, DNA
polymerase and RNase H activities, and the terminal protein; and IV) the X ORF,
coding for the small regulatory X protein, which has been shown to be essential in
vivo  for viral replication (Zoulim 1994, Lucifora 2011) and is capable of
transactivating numerous cellular and viral genes. Characteristic of the 4 major
HBV ORFs is that they utilize a single common polyadenylation signal motif
(Nassal 2008). Thus, all RNA transcripts are polyadenylated and capped.
HBV virion
“Dane
particle”
Subviral particles

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46  Hepatology 2012
Organ transplantation
Transplant recipients who receive organs from HCV-positive donors have a high
risk of acquiring HCV infection. Transmission rates in different cohorts vary from
30 to 80% (Pereira 1991, Roth 1994). Therefore, most transplant organisations have
developed strategies for screening and selective utilization of organs from anti-HCV
positive donors.
Sexual or household contact
Usual household contacts do not pose a risk of HCV transmission.
The efficiency of HCV transmission by sexual contact is very low. However,
there is no doubt that sexual transmission of hepatitis C is possible.
The exact risk of HCV transmission in monogamous heterosexual relationships
has been difficult to determine. It appears that the risk in long-term partnerships is
very low. In prospective cohorts of monogamous, heterosexual couples, there was a
long-term transmission risk of 0.01% or lower (Vandelli 2004). Factors that may
increase the risk of HCV infection include greater numbers of sex partners, history
of sexually transmitted diseases, and not using a condom. Whether underlying HIV
infection increases the risk of heterosexual HCV transmission to an uninfected
partner is unclear. Very often it is difficult to rule out the possibility that
transmission results from risk factors other than sexual exposure.
Outbreaks of cases of acute hepatitis C in several cities in Europe and the United
States among men who have sex with men (MSM) have focused attention on sexual
transmission of HCV (Boesecke 2011). There is clear evidence unprotected sex can
account for the transmission of HCV. Unprotected anal sex, fisting, having many
sex partners in a short time period, a concomitant sexually transmitted disease
including HIV and use of recreational drugs were identified as risk factors (Danta
2007, Schmidt 2011). It appears that mucosal damage is a prerequisite for HCV
transmission. According to these observations, the seroprevalence of HCV in MSM
ranges from about 4 to 8%, which is higher than the HCV prevalence reported for
general European populations.
Patients with acute or chronic HCV infection should be advised that transmission
to sexual contacts is a possibility, although the risk is extremely low in heterosexual
relationships. It is likely that the use of condoms will lower the risk of sexual
transmission further. However, in most countries there are no firm
recommendations to use barrier precautions in stable monogamous sexual
partnerships. The transmission risk in MSM is considerably higher so that – in
conjunction with the risk of other sexually transmitted diseases – safer sex practices
are advised for this group.
Perinatal transmission
The risk of perinatal transmission of HCV in HCV RNA-positive mothers is
estimated to be 5% or less (Ohto 1994). In mothers coinfected with HIV this risk
correlates with immunosuppression and has been described in up to 20%. Today,
there are no specific recommendations for prevention of perinatal transmission
(Pembrey 2005). Cesarean section has not been shown to reduce the transmission
risk. There is no evidence that breastfeeding is a risk for infection among infants
born to HCV-infected women. Early diagnosis of infection in newborns requires
Hepatitis C  47
HCV RNA testing since anti-HCV antibodies are passively transferred from the
mother.
Hemodialysis
Patients who participate in chronic hemodialysis programs are at increased risk for
hepatitis C. The prevalence of HCV antibodies in such patients reaches 15%,
although it has declined in recent years (Fissell 2004). A number of risk factors have
been identified for HCV infection among dialysis patients. These include blood
transfusions, duration of hemodialysis, prevalence of HCV infection in the dialysis
unit, and type of dialysis. The risk is higher with in-hospital hemodialysis as
opposed to peritoneal dialysis. The best strategy to prevent hemodialysis-associated
HCV transmission is subject to debate.
Other rare transmission routes
Rare sources of percutaneous transmission of HCV are contaminated equipment
used during medical procedures, procedures involved in traditional medicine (e.g.,
scarification, cupping), tattooing, and body piercing (Haley 2001). All these routes
bear the potential of transmitting HCV. However, in most instances it is not clear if
the risk is due to the procedure itself, or whether there are possible contacts with
persons involved who are HCV-positive. In addition, transmission via these routes
is so rare that persons with exposure are not at increased risk for acquiring hepatitis
C.
Needlestick injury
There is some risk of HCV transmission for health care workers after unintentional
needlestick injury or exposure to other sharp objects. The incidence of
seroconversion after exposure to an HCV-positive source is generally estimated to
be less than 2% (MMWR 2001). However, data are divergent and figures ranging
from 0 to 10% can be found (Mitsui 1992). Exposure of HCV to intact skin has not
been associated with HCV transmission.
Clinical manifestations
The spectrum of clinical manifestations of HCV infection varies in acute versus
chronic disease. Acute infection with HCV is most often asymptomatic (Vogel
2009) and leads to chronic infection in about 80% of cases. The manifestations of
chronic HCV range from an asymptomatic state to cirrhosis and hepatocellular
carcinoma. HCV infection usually is slowly progressive. Thus, it may not result in
clinically apparent liver disease in many patients if the infection is acquired later in
life. Approximately 20-30% of chronically infected individuals develop cirrhosis
over a 20-30 year period of time.
Acute hepatitis
After inoculation of HCV, there is a variable incubation period. HCV RNA in blood
(or liver) can be detected by PCR within several days to eight weeks.
Aminotransferases become elevated approximately 6-12 weeks after exposure
(range 1-26 weeks). The elevation of aminotransferases varies considerably among
individuals, but tends to be more than 10-30 times the upper limit of normal
48  Hepatology 2012
(typically around 800 U/l). HCV antibodies can be found for the first time around 8
weeks after exposure although in some patients it may take several months before
HCV antibodies can be detected by ELISA testing.
However, the majority of newly-infected patients will be asymptomatic and have
a clinically non-apparent or mild course. Jaundice as a clinical feature of acute
hepatitis C will be present in less than 25% of infected patients. Therefore, acute
hepatitis C will not be noticed in most patients (Vogel 2009). Periodic screening for
infection may be warranted in certain groups of patients who are at high risk for
infection, e.g., homosexually-active patients with HIV infection.
Other symptoms that may occur are similar to those in other forms of acute viral
hepatitis, including malaise, nausea, and right upper quadrant pain. In patients who
experience such symptoms of acute hepatitis, the illness typically lasts for 2-12
weeks. Along with clinical resolution of symptoms, aminotransferases levels will
normalize in about 40% of patients. Loss of HCV RNA, which indicates cure from
hepatitis C, occurs in fewer than 20% of patients regardless of normalisation of
aminotransferases.
Fulminant hepatic failure due to acute HCV infection is very rare. It may be more
common in patients with underlying chronic hepatitis B virus infection (Chu 1999).
Chronic hepatitis C
The risk of chronic HCV infection is high. 80-100% of patients remain HCV RNA
positive after acute hepatitis C (Alter 1999, Vogel 2009). Most of these will have
persistently elevated liver enzymes in further follow-up. By definition, hepatitis C is
regarded to be chronic after persistence of more than six months. Once chronic
infection is established, there is a very low rate of spontaneous clearance.
It is unclear why infection with HCV results in chronic infection in most cases.
Genetic diversity of the virus and its tendency toward rapid mutation may allow
HCV to constantly escape immune recognition. Host factors may also be involved
in the ability to spontaneously clear the virus. Factors that have been associated with
successful HCV clearance are HCV-specific CD4 T cell responses, high titers of
neutralising antibodies against HCV structural proteins, IL28B gene polymorphisms
and specific HLA-DRB1 and -DQB1 alleles (Lauer 2001, Thomas 2009, Rauch
2010). Infection with HCV during childhood appears to be associated with a lower
risk of chronic infection, approximately 50-60% (Vogt 1999). Finally, there seem to
be ethnic differences, with lower risk of chronicity in certain populations.
Most patients with chronic infection are asymptomatic or have only mild
nonspecific symptoms as long as cirrhosis is not present (Merican 1993, Lauer
2001). The most frequent complaint is fatigue. Less common manifestations are
nausea, weakness, myalgia, arthralgia, and weight loss. HCV infection has also been
associated with cognitive impairment. All these symptoms are non-specific and do
not reflect disease activity or severity (Merican 1993). Very often symptoms may be
caused by underlying diseases (e.g., depression), and it can be difficult to
distinguish between different diseases. Fatigue as the most common symptom may
be present in many other situations (including healthy control groups within clinical
studies). Hepatitis C is rarely incapacitating.
Aminotransferase levels can vary considerably over the natural history of chronic
hepatitis C. Most patients have only slight elevations of transaminases. Up to one
third of patients have a normal serum ALT (Martinot-Peignoux 2001, Puoti 2002).
Hepatitis C  49
About 25% of patients have a serum ALT concentration of more than twice normal,
but usually less than 5 times above the upper limit of normal. Elevations of 10 times
the upper limit of normal are very seldomly seen.
There is a poor correlation between concentrations of aminotransferases and liver
histology. Even patients with normal serum ALT show histologic evidence of
chronic inflammation in the majority of cases (Mathurin 1998). The degree of injury
is typically minimal or mild in these patients. Accordingly, normalisation of
aminotransferases after interferon therapy does not necessarily reflect histologic
improvement.
Extrahepatic manifestations
Around 30 to 40% of patients with chronic hepatitis C have an extrahepatic
manifestation of HCV (Zignego 2008). There is a wide variety of extrahepatic
manifestations described as being associated with HCV:
−  Hematologic manifestations (essential mixed cryoglobulinemia, lymphoma)
−  Autoimmune disorders (thyroiditis, presence of various autoantibodies)
−  Renal disease (membranoproliferative glomerulonephritis)
−  Dermatologic disease (porphyria cutanea tarda, lichen planus)
−  Diabetes mellitus
For further details refer to Chapter 16.
Natural history
The risk of developing cirrhosis within 20 years is estimated to be around 10 to
20%, with some studies showing estimates up to 50% (Poynard 1997, Wiese 2000,
Sangiovanni 2006, de Ledinghen 2007). Due to the long course of hepatitis C the
exact risk is very difficult to determine, and figures are divergent for different
studies and populations. In fact, chronic hepatitis C is not necessarily progressive in
all affected patients. In several cohorts it has been shown that a substantial number
of patients will not develop cirrhosis over a given time. It is estimated that about
30% of patients will not develop cirrhosis for at least 50 years (Poynard 1997).
Therefore, studies with short observation periods sometimes fail to show an
increase in mortality. In addition, survival is generally not impaired until cirrhosis
has developed. On the other hand, there is no doubt that patients with chronic
hepatitis C have a high risk of cirrhosis, decompensation, and hepatocellular
carcinoma in long-term follow-up. For example, in a cohort of patients with post-transfusion hepatitis C evaluated more than 20 years after transfusion 23% had
chronic active hepatitis, 51% cirrhosis, and 5% hepatocellular carcinoma (Tong
1995). It is not completely understood why there are such differences in disease
progression. An influence of host and viral factors has to be assumed.
Cirrhosis and hepatic decompensation
Complications of hepatitis C occur almost exclusively in patients who have
developed cirrhosis. Interestingly, non-liver related mortality is higher in cirrhotic
patients as well. However, cirrhosis may be very difficult to diagnose clinically, as
most cirrhotic patients will be asymptomatic as long as hepatic decompensation
does not occur. Findings that can be associated with cirrhosis are hepatomegaly
50  Hepatology 2012
and/or splenomegaly on physical examination, elevated serum bilirubin
concentration,  hyperalbuminemia, or low platelets. Other clinical findings
associated with chronic liver disease may be found such as spider angiomata, caput
medusae, palmar erythema, testicular atrophy, or gynaecomastia. Most of these
findings are found in less than half of cirrhotic patients, and therefore none is
sufficient to establish a diagnosis of cirrhosis.
Hepatic decompensation can occur in several forms. Most common is ascites,
followed by variceal bleeding, encephalopathy and jaundice. As mentioned earlier,
hepatic decompensation will develop only in cirrhotic patients. However, not all
patients with cirrhosis actually show signs of decompensation over time. The risk
for decompensation is estimated to be close to 5% per year in cirrhotics (Poynard
1997). Once decompensation has developed the 5-year survival rate is roughly 50%
(Planas 2004). For this group of patients liver transplantation is the only effective
therapy.
Similar to decompensation, hepatocellular carcinoma (HCC) develops solely in
patients with cirrhosis (in contrast to chronic hepatitis B). The risk for HCC has
been estimated to be less than 3% per year once cirrhosis has developed (Di
Bisceglie 1997, Fattovich 1997). However, HCV-associated HCC has significant
impact on survival (see Chapter 21).
Elevated concentrations of α-fetoprotein (AFP) do not necessarily indicate HCC.
AFP may be mildly elevated in chronic HCV infection (i.e., 10 to 100 ng/mL).
Levels above 400 ng/mL as well as a continuous rise in AFP over time are
suggestive of HCC.
Disease progression
Chronic hepatitis C has different courses among individuals. It is not completely
understood why there are differences in disease progression. Several factors have
been identified that may be associated with such differences. However, other factors
not yet identified may also be important.
Age and gender: Acquisition of HCV infection after the age of 40 to 55 may be
associated with a more rapid progression of liver injury, as well as male gender
(Svirtlih 2007). On the contrary, children appear to have a relatively low risk of
disease progression (Child 1964). In one cohort, for example, only 1 of 37 patients
with HCV RNA in serum had elevated levels of serum aminotransferases, and only
3 of 17 (18%) who had liver biopsies approximately 20 years after exposure had
histologic signs of progressive liver disease.
Ethnic background: Disease progression appears to be slower and changes in
liver histology less severe in African-Americans (Sterling 2004).
HCV-specific cellular immune response: The severity of liver injury is
influenced by the cellular immune response to HCV-specific targets. Inflammatory
responses are regulated by complex mechanisms and probably depend on genetic
determinants such as HLA expression (Hraber 2007). Whether this determines
progression of liver disease is not clear.
Alcohol intake: Alcohol increases HCV replication, enhances the progression of
chronic HCV, and accelerates liver injury (Gitto 2009). Even moderate amounts of
alcohol appear to increase the risk of fibrosis. Accordingly, in alcoholic patients
with cirrhosis and liver failure a high prevalence of anti-HCV antibodies has been
Hepatitis C  51
described. Alcohol intake should be avoided in all patients with chronic hepatitis C.
There is no clear amount of safe alcohol intake.
Daily use of marijuana: Daily use of marijuana has been associated with more
rapid fibrosis progression, possibly through stimulation of endogenous hepatic
cannabinoid receptors.
Other host factors: Genetic polymorphisms of certain genes might influence the
fibrosis progression rate (Jonsson 2008). For example, transforming growth factor
B1 (TGF B1) phenotype or PNPLA3 (adiponutrin) are correlated with fibrosis stage
(Zimmer 2011). Patients with moderate to severe steatosis are at higher risk for
developing hepatic fibrosis.
Viral coinfection: Progression of hepatitis C clearly is accelerated in HIV-infected patients (see section on coinfection). Acute hepatitis B in a patient with
chronic hepatitis C may be more severe. Chronic hepatitis B may be associated with
decreased HCV replication as opposed to HCV monoinfected patients, although
HCV usually predominates. Nevertheless, liver damage is usually worse and
progression faster in patients with dual HBV/HCV infections. Around one third of
patients coinfected with HBV and HCV lack markers of HBV infection (i.e.,
HBsAg) although HBV DNA is detectable.
Geography and environmental factors: There are some obvious geographic
differences (Lim 2008). For example, hepatocellular carcinoma is observed more
often in Japan than in the United States. The reason for this is not clear.
Use of steroids: It is well known that use of steroids increases the HCV viral
load, while the effect on aminotransferases is variable. They tend to decrease in
most patients, although increases in transaminases and bilirubin have also been
described. Reducing dosage of corticosteroids returns HCV viral load to baseline.
However, the clinical consequences of corticosteroid use are largely unknown. It
seems to  be reasonable to assume that short-term use of corticosteroids is not
associated with significant changes in long-term prognosis.
Viral factors: The influence of viral factors on disease progression is unclear.
Overall, there seems to be no significant role of different genotypes and
quasispecies on fibrosis progression or outcome. However, coinfection with several
genotypes may have a worse outcome as compared to monoinfection.
It is very difficult to predict the individual course of hepatitis C due to the many
factors influencing disease progression. Today, assessment of liver fibrosis by non-invasive techniques such as transient elastography, AFRI or by liver biopsy is the
best predictor of disease progression (Gebo 2002). The grade of inflammation and
stage of fibrosis are useful in predicting further clinical course. In patients with
severe inflammation or bridging fibrosis virtually all patients will develop cirrhosis
within ten years. In contrast, patients with mild inflammation and no fibrosis have
an annual progression risk to cirrhosis of around 1%.
Several predictive models of disease progression that include clinical parameters
(e.g., hepatic decompensation) and laboratory parameters (e.g., bilirubin, INR) have
been evaluated, but none of these models is routinely used in the clinic at present. In
patients with cirrhosis, the MELD score (Model for End-Stage Liver Disease) and
the Child score (Table 1) are used to stage disease and to describe the prognosis (see
Chapters 22 & 23). The MELD Score is used especially to estimate relative disease
severity and likely survival of patients awaiting liver transplant. It is calculated as:
MELD Score = 10 x ((0.957 x ln(Creatinine)) + (0.378 x ln(Bilirubin)) + (1.12 x
52  Hepatology 2012
ln(INR))) + 6.43. An online calculator and further information can be found at the
website of The United Network for Organ Sharing (UNOS) (http://www.unos.org).
Table 1. Child-Pugh classification of severity of liver disease (Child 1964).*
Points assigned
1  2  3
Ascites  Absent  Slight  Moderate
Bilirubin, mg/dL  <2  2-3  >3
Albumin, g/dL  >3.5  2.8-3.5  <2.8
Prothrombin time  
Seconds over control  <4  4-6  >6
INR  <1.7  1.7-2.3  >2.3
Encephalopathy  None  Grade 1-2  Grade 3-4
* A total score of 5-6 is considered stage A (well-compensated disease); 7-9 is stage B
(significant functional compromise); and 10-15 is stage C (decompensated disease). These
grades correlate with one- and two-year patient survival (stage A: 100 and 85 percent; stage B:
80 and 60 percent; stage C: 45 and 35 percent).
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Hepatitis E: an underestimated problem?  55
4.  Hepatitis E: an underestimated problem?
Sven Pischke and Heiner Wedemeyer
Introduction
Hepatitis E is an inflammatory liver disease caused by the hepatitis E virus (HEV),
which is endemic in many tropical countries. Hepatitis E has been considered to be
a travel-associated, acute, self-limiting liver disease that only causes fulminant
hepatic failure in specific, high-risk groups. It has recently been estimated that HEV
infection causes approximately 70,000 deaths each year worldwide (Rein 2011). In
recent years sporadic cases of HEV infections have emerged also in industrialized
countries, mostly caused by HEV genotype 3, for which zoonotic transmission has
been described (Pischke 2010b).
In immunocompetent individuals infection with HEV usually leads to a clinically
silent seroconversion or to an acute self-limited inflammation of the liver. In
pregnant women and patients with pre-existing chronic liver diseases cases of
fulminant liver failure by HEV infection are reported (Pischke 2010b).
Moreover, cases of chronic HEV infection associated with progressive liver
disease have been described in several cohorts of immunocompromised individuals.
In this context, diagnosis of HEV infection should rely on detection of HEV RNA,
as testing for HEV-specific antibodies may lack sensitivity (Pischke 2010c).
Therapeutic options for chronic hepatitis E include reduction of
immunosuppressive medication (Kamar 2011a), treatment with  α-interferon
(Haagsma 2010, Kamar 2010a) or therapy with ribavirin (Kamar 2010b, Mallet
2010).
Recently, results of a large Phase III study were presented investigating a novel
recombinant HEV vaccine in China. The vaccine had an efficacy to prevent acute
symptomatic hepatitis E of >90% (Zhu 2010). It is unknown yet if and when this
vaccine might become available for other countries.
HEV: genetic characteristics of the virus
The hepatitis E virus is a non-enveloped, single-stranded RNA virus classified into
the family of Hepeviridae and its own genus Hepevirus (Pischke and Wedemeyer
2010). There are 5 known genotypes. The HEV genome includes two short non-coding regions surrounding three open reading frames (ORF1 to 3). These ORFs
contain the genetic information for various proteins that are necessary for capsid
56  Hepatology 2012
formation, virus replication and infectivity of HEV. Various HEV isolates have
been differentiated by phylogenetic analysis based on a hypervariable region within
ORF1 (Meng 1999). Four of five HEV genotypes are able to infect humans, while
genotype 5, called “avian HEV”, has only been detected in birds.
HEV genotype 1 is responsible for endemic and epidemic infections by HEV in
Asia, while genotype 2 is endemic in Africa and Mexico (Figure 1). These
genotypes are usually transmitted orally-faecally by contaminated drinking water
under conditions of poor sanitation. There is no known animal reservoir for these
genotypes (Pischke 2010b).
HEV genotype 3 can be found in humans and animals in Europe, the US and Asia
(Pischke 2010b). For this genotype zoonotic transmission, foodborne or by contact
with infected animals has been described. HEV genotype 3 has been identified in
pigs, wild boars, shellfish, deer, oysters, cats, rats and various rodents (Pischke
2010b). Genotype 4 has also been detected in both humans and pigs in Asia (Geng
2009) and Europe (Hakze-van der Honing 2011).
Foodborne transmission can be avoided by cooking meat above 60°C, which
inactivates the virus (Emerson 2005).
Figure 1. Worldwide distribution of HEV genotypes.
Diagnosis of hepatitis E
In immmunocompetent patients the diagnosis of hepatitis E is based on the
detection of HEV-specific antibodies. While IgG antibodies indicate acute and past
HEV infections, IgM antibodies can only be found in patients with acute infections
(Pischke and Wedemeyer 2010). There are different commercial assays available for
detection of HEV-specific antibodies. Comparison of six of these assays revealed a
wide variation of diagnostic sensitivities and specificities as well as interassay
disagreements (Drobeniuc 2010). Thus, some of the remarkable discrepancies in
HEV seroprevalence rates reported in different studies may be explained by varying
sensitivities of the respective assays.
HEV-specific IgG antibodies can be detected in patients with previous contact
with HEV. They do not differentiate between ongoing HEV infection and past
contact with the virus. To prove current infection the detection of HEV RNA by
PCR has been established. Numerous assays using different primers have been
Hepatitis E: an underestimated problem?  57
developed (Meng 1999, Zhao 2007). Furthermore, few quantitative PCR assays
have been described (Ahn 2006, Enouf 2006).
In immunocompromised individuals, diagnosis of HEV infection may only be
based on the detection of HEV RNA as seroassays lack sensitivity especially in the
early phase of infection (Pischke 2010c). HEV RNA can not only be detected in
serum samples but also in stool (Pischke 2010b) and thus infectivity of HEV
infected persons can be determined by investigating stool for HEV RNA.
Worldwide distribution of HEV infections
In the last few years an increasing frequency of diagnosed cases of HEV infections
has been reported from various industrialised countries (Pischke 2010b). The
presence of HEV RNA in urban sewage samples from Spain, the US and France has
been shown, suggesting that HEV may be more prevalent in industrialised countries
than previously assumed (Clemente-Casares 2003). In each of these three countries
it was possible to discover HEV contamination in sewage samples in a notably high
frequency. These findings may partially explain the huge gap between
seroprevalence rates and the rather low numbers of diagnosed and reported cases of
acute hepatitis E in Western countries. For example, Germany has a seroprevalence
rate of 2% in a population of 80 million individuals (representing 1.6 million
persons with possible previous HEV infection) but only about 200 cases of hepatitis
E are diagnosed and reported each year (Pischke 2011a, Pischke 2010b). The
mismatch between high seroprevalence rates and the low number of symptomatic
cases has also been investigated in a recent study from Egypt. 919 anti-HEV
seronegative individuals from rural Egypt were followed and, interestingly, 3.7%
(n=34) of these individuals seroconverted to anti-HEV within 11 months of follow
up (Stoszek 2006). However, none of these 34 individuals suffered from
symptomatic hepatitis E. This finding corresponds with data from a recently
published large vaccine study performed in China where very few of the patients in
the placebo group who seroconverted during a follow-up period developed
symptomatic acute hepatitis E (Zhu 2010). Overall, these data suggest that far less
than 5% of all contacts with HEV lead to symptomatic hepatitis E (Wedemeyer and
Pischke 2011).
Even so, a rapid increase in reported HEV infections has been recognized in
several industrialized countries over the last 10 years. To investigate the potential
underlying reasons for this phenomenon, we analyzed the time trend of the anti-HEV seroprevalence in healthy German individuals versus the number of reported
cases of acute hepatitis E. Even though the number of reported cases increased more
than 5-fold over the last ten years (Figure 1), the anti-HEV IgG seroprevalence rate
remained rather stable over the last 15 years (Pischke 2011a). In contrast, the
number of scientific articles on HEV infections published in PubMed increased
sharply during the same period (Figure 1). These findings could indicate that the
increase of reported HEV cases in Germany and other industrialized countries is
based on an increased awareness associated with more frequent diagnosis of
hepatitis E but not a true increase in incidence rates (Pischke 2011a).
58  Hepatology 2012
Figure 2. Number of reported HEV infections in Germany over the last decade (Figure 2a)
and number of publications on HEV over the same time period (Figure 2b).
Transmission of HEV
The vast majority of HEV infections worldwide happens via the faecal-oral route
(Figure 2). Patient-to-patient transmission is very rare but has been described from a
large outbreak in Northern Uganda (Teshale 2011) and from hematology wards in
Europe (Pischke 2010b). Bloodborne transmission of HEV has been suggested in
the late nineties (Fainboim 1999). Subsequent studies from Hong Kong, Japan,
Great Britain and France confirmed blood transfusions as a possible source of HEV
transmission (Pischke 2010b). A single case of HEV  transmission by
transplantation of a liver graft from a patient with occult hepatitis E has been
reported (Schlosser 2011).
Zoonotic transmission of HEV has recently been assumed to be the main source
of HEV infections in industrialized countries (Figure 3). Both direct contact with
HEV-infected domestic animals and foodborne transmission are possible (Pischke
2010b). Commercial food products such as pig meat may be contaminated with
HEV as shown in studies from the Netherlands, France and Germany (Colson 2010,
Melenhorst 2007, Wenzel 2011). Meat should be heated to over 70°C to prevent
foodborne HEV infections (Emerson 2005). 
Hepatitis E: an underestimated problem?  59
Figure 3. Possible sources of HEV infection.
Acute hepatitis E in immunocompetent individuals
In the vast majority of cases, contact with HEV takes an asymptomatic course
(Stoszek 2006, Wedemeyer and Pischke 2011), especially if the contact happens
during childhood (Buti 2008). Immunocompetent individuals should be able to clear
the virus spontaneously. In symptomatic cases the incubation period of HEV
infections ranges from three to eight weeks with a mean of 40 days (Pischke 2010b).
The peak of HEV viremia can be detected in the early phase of infection while the
peak of ALT elevations usually occurs around 6 weeks after infection (Pischke
2010b).
Initial symptoms in acute hepatitis E are typically unspecific and can include flu-like myalgia, arthralgia, weakness and vomiting. In some patients jaundice, itching,
uncoloured stool and darkened urine occur accompanied by elevation of liver
transaminases, bilirubin, alkaline phosphatase and gamma-glutamyltransferase.
HEV infection can lead to more severe acute liver disease in pregnant women or
patients with underlying chronic liver diseases progressing to fulminant hepatic
failure in individual cases (Pischke 2010b). Possible explanations for the severe
courses in pregnant women are hormonal and immunological changes during
pregnancy (Navaneethan 2008). Recently an association between reduced
expression of the progesterone receptor and fatal outcome of hepatitis E in pregnant
women has been reported (Bose 2011).
Single cases of prolonged courses of HEV infection  in immunocompetent
individuals with up to two years of viremia have been described in the US (Mallet
2010), Spain (Gonzalez Tallon 2011) and China (Liu and Liu 2011). However, no
case of HEV-associated liver cirrhosis or development of hepatocellular carcinoma
has been reported in immunocompetent individuals.