Book on hepatitis from page 60 to 67

Book on hepatitis from page 60 to 67

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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64  Hepatology 2012
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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