Journal of Viral Hepatitis, 2016, 23, 6879
12445
REVIEW
Hepatitis E: an emerging global disease from discovery
towards control and cure
Mehnaaz S. Khuroo1 and Mohammad S. Khuroo2,3
Kashmir, India; Gastroenterology and Chairman Dept. Medicine, Sher-i-Kashmir Institute of Medical Sciences, Kashmir, India and 3Digestive
2
Diseases Centre, Dr. Khuroos Medical Clinic, Kashmir, India
Received July 2015; accepted for publication July 2015
SUMMARY. Hepatitis E is a systemic disease affecting the
liver predominantly and caused by infection with the
hepatitis E virus (HEV). HEV has marked genetic hetero-
geneity and is known to infect several animal species
including pigs, boar, deer, mongoose, rabbit, camel,
chicken, rats, ferret, bats and cutthroat trout. HEV is the
sole member of the family Hepeviridae and has been
divided into 2 genera: Orthohepevirus (mammalian and
avian HEV) and Piscihepevirus (trout HEV). Human HEVs
included within the genus Orthohepevirus are designated
Orthohepevirus A (isolates from human, pig, wild boar,
deer, mongoose, rabbit and camel). Hepatitis E is an
important public health concern, and an estimated one-
third of the world population has been infected with
HEV. In recent years, autochthonous hepatitis E is recog-
nized as a clinical problem in industrialized countries.
Several animal species especially domestic swine, wild
Hepatitis E is a systemic disease affecting the liver predomi-
nantly and caused by infection with the hepatitis E virus
(HEV) [1]. Hepatitis E has a major global impact. It is esti-
mated that one-third of the world population has been
exposed to the agent. In India alone, around 2.2 million
cases of hepatitis E are thought to occur. In 2005, around
20 million cases of incident HEV infections were estimated
to occur in nine endemic zones causing estimated 3.4 mil-
lion cases, 70 000 deaths and 3000 stillbirths [2]. These
calculated numbers are a gross underestimate of the actual
disease load in developing countries and need to be revised
Abbreviations: AFLP, acute fatty liver of pregnancy; ALF, acute
liver failure; HEV, hepatitis E virus; LCHAD, long-chain 3-hydroxy-
acyl-CoA dehydrogenase; tMRCAs, times to the most recent com-
mon ancestors.
Correspondence: Prof. Mohammad Sultan Khuroo, MD, DM, FRCP
(Edin), FACP, MACP, Director, Digestive Diseases Centre,
Dr. Khuroos Medical Clinic, Sector 1, Qamarwari, SK Colony,
Srinagar, Kashmir, J&K 190010, India.
E-mail: khuroo@yahoo.com
doi:10.1111/jvh.
1
Department of Pathology, Govt: Medical College Srinagar,
boar and wild deer are reservoirs of genotype HEV-3 and
HEV-4 in these countries. Human infections occur
through intake of uncooked or undercooked meat of the
infected animals and pig livers or sausages made from
these livers and sold in supermarkets. HEV can be trans-
mitted through blood and blood component transfusions,
and donor screening for HEV is under serious considera-
tion. Chronic hepatitis E resulting in rapidly progressive
liver cirrhosis and end-stage liver disease has been
described in organ transplant patients. Ribavirin
monotherapy attains sustained virological response in
most patients. HEV 239 vaccine has been marketed in
China and its long-term efficacy over four and a half
years reported.
Keywords: communicable diseases, discovery, hepatitis E,
hepatitis E virus, vaccine, zoonosis.
based on the following: (i) occurrence of repeated epi-
demics of hepatitis E in the same population on periodic
intervals, (ii) disease load with estimated HEV incidence of
6%, (iii) dynamics of antibody response to HEV infection
and (iv) occurrence of reinfections with altered immune
response [36]. Hepatitis E is being recognized as an
important clinical problem in many industrialized countries
(Fig. 1; [7]). Most of these infections are autochthonous
rather than travelling to endemic zones. Data acquired
from seroprevalence studies point to the fact that these
numbers constitute only a tip of the iceberg and most
infections occur as asymptomatic events or are not recog-
nized or reported alternatively as drug-induced liver injury
[8].
Discovery of hepatitis E is a remarkable human-interest
story related to the complexities, the missteps, the near
misses and the ups and downs as with many similar events
in history ([9], www.drkhuroo.com). The story is fascinat-
ing as it originated from one of most remote region of the
world with very hard weather conditions and primitive
healthcare and investigative facilities (Fig. 2). Thus, the
2015 John Wiley & Sons Ltd

Fig. 1 Reported cases of hepatitis E virus infection from
industrialized countries. Data source: references 7, 8 and
39.
story also focuses on the fact that discoveries do not neces-
sarily require high-tech laboratories or institutions with
cutting-edge research facilities but can be accomplished in
very primitive situations as well, an example in focus. The
1978 epidemic had caused colossal human suffering and
loss of life, and an estimated 52 000 patients had icteric
disease with around 1700 deaths [10,11]. Through an
ingenious house-to-house survey protocol extending for
over 14 years, we identified an enterically transmitted
non-A, non-B hepatitis with many unique features, namely
(i) repeated occurrence of large-scale waterborne epidemics
in same geographic region; (ii) disease affecting young
adults and selectively sparing children; (iii) exceptional
increased incidence and severity in pregnant women; (iv)
propensity towards vertical transmission with high foetal
and perinatal mortality; (vi) self-limiting disease; and (vii)
loss of antibodies in a substantial proportion of infected
subjects over time [5,1214]. A similar disease was
reported constituting over half of endemic hepatitis in the
Fig. 2 Epidemic region Kashmir 1978.
Drinking water is collected from a
canal in which public latrine sewage
flows, garbage of the whole locality is
dumped, utensils and linen are washed,
children swim and locals buy fish. The
hard weather conditions and primitive
healthcare and investigative facilities
are depicted.
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Hepatitis E: an emerging global disease 69
same environment [15]. While the hepatitis E story has
moved forward over 35 years, it is a matter of concern
that we have not been able to furnish answers for many of
the unique epidemiological and clinical features of this
remarkable pathogen.
Is hepatitis E an emerging disease that has appeared in a
population for the first time, or did it exist previously and
now rapidly increased in incidence or geographic range?
Recently, the evolutionary history of mammalian HEV was
reported [16]. The times to the most recent common
ancestors (tMRCAs) for all four HEV genotypes were calcu-
lated using sequences from the nonoverlapped region of
ORF2 in a Bayesian analysis. This finding showed that the
most common recent ancestor for modern mammalian
HEV existed between 536 and 1344 years ago. This pro-
genitor appears to have given rise to anthropotropic and
enzootic variants of HEV, which evolved into genotypes
HEV-1 and HEV-2 and genotypes HEV-3 and HEV-4,
respectively. The discovery of a genetically distinct avian
HEV indicates a very long evolutionary history for the HEV
group of viruses. In other studies, indigenization and
spread of HEV in Japan and China were associated with
the popularization of eating pork.
Hepatitis E virion is a 27- to 32-nm, spherical particle, is
icosahedral in symmetry and has spikes on the surface
[17]. Virus is resistant to heating at 56 C for 1 h; how-
ever, it is susceptible to boiling and frying for 5 min and to
chlorination. The HEV genome is a single-stranded RNA of
~7.2 kb that is positive-sense, with a 7-methylguanine cap
(m7G) at its 50 end and a poly (A) at its 30 end. HEV RNA
replicons express genomic RNA and only one bicistronic
2.2-kb subgenomic RNA. The genome contains three par-
tially overlapping open reading frames. ORF1 encodes a
nonstructural polyprotein of 1693 amino acids. ORF2
encodes the major viral capsid protein of 660 amino acids.
ORF3 is a small phosphoprotein of 114 amino acids
(Fig. 3).
70 M. S. Khuroo & M. S. Khuroo
The life cycle of HEV is poorly understood, largely
because of the nonavailability of efficient in vitro culture
methods or small animal models of infection [18]. The
viral particles are concentrated on the surface of hepato-
cytes, bind a specific yet uncharacterized receptor and are
internalized. The virus then uncoats to release genomic
RNA that is translated in the cytoplasm into nonstructural
proteins. RNA-dependent RNA polymerases replicate the
positive-sense genomic RNA into negative-sense tran-
scripts; the latter then act as templates for the synthesis of
a 2.2-kb subgenomic RNA as well as full-length positive-
sense transcripts. The positive-sense subgenomic RNA is
translated into ORF2 and ORF3 proteins. The ORF2 protein
packages the genomic RNA to assemble new virions, while
the ORF3 protein may optimize the host cell environment
for viral replication. The ORF3 protein is also associated
with endomembranes or plasma membranes and may aid
in viral egress. Recent studies suggest that mature virions
excreted from the liver into the circulation are enveloped
by the ORF3 protein and lipids, which are subsequently
removed through a process that is not understood at pre-
sent, to resume a fresh infection cycle (Fig. 4).
HEV has remarkable heterogeneity with many groups
and genotypes and subtypes but with one serotype. Classi-
fying this agent has been a huge hassle, and as of today, a
recent consensus has classified the agent in one family of
Hepeviridae which has been broadly divided into 2 genera
Fig. 3 (a) The hepatitis E virus
genome, (b) genomic RNA and
bicistronic subgenomic RNA, (c) open
reading frames (ORFs) and (d) 3
encoded proteins (pORF1, pORF2 and
pORF3). For details, see text about
hepatitis E virus.
namely Orthohepevirus and Piscihepevirus (Table 1; [19]).
Orthohepevirus is further divided into 4 species, namely A
to D. Orthohepevirus A is the species infecting humans
and swine and other animals with possible human spread/
transmission, Orthohepevirus B includes all 3 avian HEV
strains, Orthohepevirus C includes 2 species one from rat
(HEV C1) and another one from ferret (HEV C2) and Ortho-
hepevirus D includes bat HEV. Piscihepevirus includes 2
trout HEV strains within a single species. Orthohepevirus A
comprises of isolates from human, pigs, boar, deer, mon-
goose, rabbit and camel. These include two genotypes iso-
lated from humans alone (HEV-1 and HEV-2), two
genotypes reported in both humans and different animal
species and associated with the zoonotic cases (HEV-3 and
HEV-4), two isolates from wild boar in Japan (genotype
HEV-5 & HEV-6) and a single isolate from dromedary
camel in Dubai (genotype HEV-7). Rabbit HEV and closely
related human isolate has been placed as a distant member
in HEV-3. The moose virus appears to cluster closely to
genotype HEV-3, HEV isolates from mink to ferret virus
(HEV C2) and HEV isolates from fox to rat virus (HEV C1).
These viruses have not been placed in any specific geno-
type and need complete genomic sequences for a definite
taxonomic classification.
The course of acute hepatitis E infection has been well
studied (Fig. 5; [20]). Incubation period is roughly
46 weeks; however, it may range from 9 days to
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 Hepatitis E: an emerging global disease 71

Fig. 4 Proposed replication of hepatitis

E virus. For details, see text about
hepatitis E replication.

Table 1 Hepatitis E virus: taxonomy history and classification

Ref/year Family Genus Species Host Genotypes

6th ICTV [57] Caliciviridae Calicivirus Hepatitis E Virus

7th ICTV [58] Unassigned Hepatitis E-like viruses Hepatitis E Virus
8th ICTV [59] Unassigned Hepevirus Hepatitis E Virus
9th ICTV [60] Hepeviridae Hepevirus Hepatitis E virus Human HEV-1
HEV-2
Human/Pig* HEV-3
HEV-4
Unassigned Avian HEV Chicken
Smith et al. [19] Hepeviridae Orthohepevirus Orthohepevirus A Human HEV-1
(ICTV [61]) HEV-2
Human/pig* HEV-3
HEV-4
Boar HEV-5
HEV-6
Camel HEV-7
Orthohepevirus B Chicken
Orthohepevirus C Rat HEV-C1
Ferret HEV-C2
Orthohepevirus D Bat
Piscihepevirus Piscihepevirus A Trout

ICTV, International Committee for Taxonomy of viruses.

Moose (HEV-3), Mink (HEV-C2), Fox (HEV-C1).
*In addition to pigs, several other animals including wild boar, deer and rabbit are reservoirs of hepatitis E virus.

2 months. Disease is present if there were symptoms such as levels. Symptoms may persist for few weeks to a month or
fever, anorexia, vomiting and jaundice. The symptoms coin- more. ALT levels return to normal during convalescence.
cide with a sharp rise in serum alanine transaminase (ALT) HEV RNA may be detected in both serum and stool early in

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72 M. S. Khuroo & M. S. Khuroo

HEV disease has significant morbidity and mortality in

Fig. 5 Clinical, biochemical and serological profile of
hepatitis E virus infection. Based on data from Pinglina
epidemic, Kashmir 1992.
the course of infection, but serum viraemia may be difficult
to detect by the time cases come to clinical attention. Anti-
HEV IgM titres increase rapidly and then wane over the
weeks following infection, while anti-HEV IgG antibody titres
continue to rise more gradually during the convalescence
period, and detectable anti-HEV IgG may persist for months
to years.
developing countries [11]. The disease is multifaceted and
in its manifestations keeps multiple medical specialists on
their toes to face and fight this python (Table 2). Repeated
large-scale epidemics hit each region on periodic intervals,
causing panic and tsunami-like phenomenon (Table 3;
[1,3,10]). Most of these epidemics are not reported for
political pressures or not studied for lack of medical infras-
tructure. Endemic HEV disease is the commonest cause of
acute sporadic hepatitis and has been estimated to cause
around whooping 2.2 million infections per year in India
alone [15]. Acute liver failure is a common occurrence in
developing countries although there are no actual esti-
mates. HEV is the dominant aetiological cause of acute
liver failure [21]. One of the most intriguing manifestations
of hepatitis E in developing countries is increased incidence
and severity in pregnant women. HEV strikes pregnant
women so much that it is difficult to find a pregnant
mother in a community recently hit by the epidemic
[12,22]. HEV in developing countries is a major threat to
patients with stable chronic liver disease. Around 21% of
patients with cirrhosis get HEV superinfection and develop
rapidly progressive liver disease with around 34% short-
term mortality [23]. Continuing the catastrophes caused
by HEV in developing countries, foetal and neonatal deaths
are estimated to cause 3000 deaths per year and post-

Table 2 Hepatitis E: Global Epidemiology and clinical profile

Developing countries Developed countries

Genotypes HEV-1 HEV-2 HEV-3 HEV-4

Distribution Asia, Africa, Latin America Mexico, West Africa Worldwide China, East Asia, Central Europe
Disease pattern Epidemic, Endemic Autochthonous, sporadic, case clusters
Attack rate ~1 in 2 6798% asymptomatic
Seasonality Yes No
Reservoir Human Animals (pig, boar, deer)
Transmission Water, person-to-person, vertical Zoonotic foodborne, vocational, infected
water
Transfusion-associated Reported Yes (well-studied)
Seroprevalence Low (<15 year), rapid increase Steady increase throughout age groups;
(1530 year), plateau at 3040% varies 721%
Seroincidence 64/1000 years 30 (south France), 2 (UK)
7 (USA)/1000 years
Age (year) 1540 >50
Sex 2:1 >3:1
Clinical outcome Self-limiting in most Self-limiting in most
Risk factors Pregnancy, Cirrhosis Cirrhosis, LTx, HIV
Deaths in pregnancy High (25%) Not reported
HEV superinfections Common, poor outcome Reported, poor outcome
Extrahepatic disease Yes Yes
Chronic infection Not reported HEV-3; SOT, HIV, hem NP
Burden 3.4 million cases/year, 70 000 deaths, 3000 still Unknown
births

LTx, Liver transplant; SOT, Solid organ transplants; hem NP, hematological neoplasms.

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Table 3 Major reported epidemics Site
Indian subcontinent
Kashmir
Delhi
Kanpur
Burma
Central Asia
Kirgiz
China
Africa
Sudan
Somalia
Uganda
Data source: references [1,11]. NA, data not available.
transfusion HEV infections lead to undefined disease load
in such communities [14,24].
How does HEV cause such major catastrophes in such
regions? Gross faecal contamination of drinking water sup-
plies occurs through a panorama of mechanisms [25,26].
Around 2.4 billion people, one-third of the world popula-
tion, will remain without access to improved sanitation by
2015. Globally, an estimated 1.8 billion shall drink pol-
luted/faecally contaminated water. Over 300 million Indi-
ans still defecate in the open. India accounts for about
60% of the world population without toilets, with human
excrement that goes into a field polluting groundwater,
crops and waterways. That is at a time when more people
shall have a mobile phone in hand to make a call than a
toilet to defecate. The situation in India shall continue to
remain a matter of great concern.
Sanitation and sewage disposal in India and other devel-
oping countries is the ultimate among so many practices
which shall infuse major impact to economy; stabilize
health care; give dignity, respect and safety to women; and
improve children health statistics [25]. In addition, a com-
mon Indian shall have a safe glass of water to drink and
help prevent many waterborne diseases including HEV
infection. It is heartening to know that Govt of India has
started Clean India Campaign in October 2014. An esti-
mated US$ 10 billion will be spent on the mission over the
next 5 years. Targets identified include cleaning the envi-
ronment, construction of public and school latrines and
implementing hygienic practices especially hand hygiene.
By 2019, around 1.2 billion residents shall have access to
toilets. With all this, I yet believe it shall take a long time
for a common Indian to have access to portable clean
water. It is painful to know that many regions hit by the
epidemics lack basic healthcare facilities and need an emer-
gency makeshift health delivery system for surveillance,
health education and identifying high-risk patients for
referral to tertiary healthcare centres. Mass chlorination of
water resources and its use at domestic level is regularly
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Hepatitis E: an emerging global disease 73
Year Icteric cases Mortality pregnancy (%)
197882 52 000 22
195556 29 000 10
1991 78 000 NA
197677 20 000 18
195656 10 000 18
1987 120 000 18
1985 2000 NA
198586 2000 NA
2008 10 196 6.9
being practised to control spread of disease. All what is
done is equal to a drop in the ocean for what is needed to
face and fight this dangerous python. All of us have eyes
on the availability of a cheap, effective and safe HEV
vaccine. Earlier it is made available is better than never.
Why large-scale epidemics occur on a periodic basis in
such regions and what triggers an epidemic while there is
constant pressure from faecally contaminated water sup-
plies? Is it a phenomenon of reinfections in population with
HEV antibodies, or loss of HEV antibodies over time in a
substantial proportion of people weakening herd immunity
and susceptibility to new epidemic, or is it an influenza-like
phenomenon where new strains of HEV are introduced in
the community causing repeat epidemics? Over the last 3
decades, we have repeatedly estimated IgG anti-HEV preva-
lence in our population following epidemics and second
epidemics and defined a temporal pattern of seroprevalence
in our community. IgG anti-HEV prevalence is around 4%
reaching a plateau of around 10% in 3rd and 4th decades.
Following an epidemic, around 20% population of all ages
test seropositive to HEV. Subsequent to this, there is a
gradual decline in seropositivity of IgG anti-HEV over the
next 2 decades and poor sero-exposure in the new cohort
population during the interepidemic period. Repeat out-
breaks occur when overall seropositivity reaches around
4%, low in first 2 decades (new cohort) and around 10%
in 3rd and 4th decades of life [1,3,5]. We believe gross fae-
cal contamination is constantly present due to poor sanita-
tion and improper sewage disposal system in such
hyperendemic zones [25].
HEV-related acute liver failure (ALF) in pregnancy is an
explosive disease with rapid progression of symptoms: high
occurrence of cerebral oedema, sepsis and disseminated
intravascular coagulation [12,22]. Why should pregnant
women with hepatitis E have such a devastating clinical
course? We and several other groups have compared
demographic, nutritional, and obstetric and biochemical
features of mothers with ALF to those with nonfulminant
74 M. S. Khuroo & M. S. Khuroo
disease and found no significant difference between the
two groups. All these patients are infected with HEV-1 and
viral load, and duration of viraemia has been similar in
these two groups. Thus, there seems to be no obvious risk
factor in host or agent which could enhance liver injury in
pregnancy. A shift of the TH1-TH2 balance towards a shift
to TH2 response in pregnant women with HEV infection
but not in nonpregnant women with HEV infection has
been documented and may point towards a primary
immunologic cause of sever disease in pregnancy. How-
ever, as the mechanisms of liver cell injury in hepatitis E
remain unknown, it is difficult to ascribe the severity of
liver injury in these patients to this immune phenomenon.
Also hormones of pregnancy especially estrogens and pro-
gesterone might impair cellular immunity by triggering
adapter protein (ORF3) which could facilitate viral replica-
tion and lead to release of cytokines and liver cell apoptosis
[27].
Recently, we wanted to study a provocative hypothesis
Could Severe fetal HEV infection be the possible cause of
increased severity of HEV infection in the mother, which
may represent another example of mirror syndrome [28].
First, we found a close relationship between severity of
HEV infection in the mother and HEV infection in babies.
Second, we and others found that mothers who delivered
babies early on expectant basis survived and had rapid
clinical recovery from ALF. This pointed to dangers of long
gestational period after onset of coma. In addition, DIC was
limited to mothers who delivered babies with ALF and not
in those who delivered normal or babies with nonfulmi-
nant HEV infection. This led us to believe that foetuses
with severe hepatitis caused by vertically transmitted HEV
infection may be related to outcome of ALF in the mothers.
This relationship may be akin to what happens in mirror
syndrome in which mother develops generalized anasarca
subsequent to foetal and/or placental oedema. The possible
mechanism of increased severity of liver disease in the
mother may be due to the production of toxic metabolites
in the foetus with ALF. Such toxins can cross over to
Table 4 HEV RNA in healthy donors
References Region
Arankalle et al. [51] Pune India
Khuroo et al. [29] Kashmir India
Gotanda et al. [52] Japan
Vollmer et al. [35] Germany
Juhl et al. [53] Germany
Ren et al. [54] China
Hewitt et al. [32] England
Hogema et al. [55] Netherlands
Fischer et al. [56] Austria
Infectious dose required for HEV infection seems to be low. Duration of viraemia in asymptomatic donors lasts for up to
45 days. Data source: references [2938].
maternal blood and precipitate the onset of hepatic
encephalopathy and possibly DIC in the mother with HEV
infection. Such a mechanism has been established for the
pathogenesis of acute fatty liver of pregnancy (AFLP) in
which foetus is homozygous for long-chain 3-hydroxyacyl-
CoA dehydrogenase (LCHAD) deficiency and produces large
amounts of toxic omega fatty acids, which cross over to
the maternal blood. This hypothesis needs further studies.
In 2004, we were the first to report on transfusion-asso-
ciated hepatitis E from Kashmir, India [29]. Thirteen of the
145 multiply transfused subjects had HEV infection as
detected by IgM anti-HEV and HEV RNA as against 2 of
the 250 control subjects. In a prospective study, we traced
3 transfusion-associated HEV infections to 4 HEV RNA-pos-
itive donors. All infections were caused by HEV-1. Around
same time, a Japanese patient on transfusion-associated
HEV infection with complete donorpatient sequence iden-
tity was reported [30]. Several cases of transfusion-associ-
ated HEV infection have come to light over the years [31].
Data from England showed 18 (42%) of the 42 HEV-posi-
tive transfusion recipients developed HEV-3 infection.
Three cleared infection with ribavirin. Ten developed pro-
longed or persistent infection [32].
Several groups including ours from India had shown
that a significant percentage of healthy donors have short-
lasting circulating HEV RNA of HEV-1 and HEV-positive
donation rate is substantially high reaching 1:27 in hyper-
endemic zones [29,33]. Now, data on HEV RNA in healthy
blood donors are available from several countries (Table 4;
[3336]). HEV RNA was uniformly detected in variable
proportion in all countries. It varied from 0.01 to 0.14%.
Accordingly, HEV RNA-positive donation rate varied from
1:672 as in Germany to 1:8416 as in Austria. Two obser-
vations that infectious dose required for HEV infection was
as low as to the limit of detection by RT-PCR and duration
of viraemia can extend up to 45 days are of concern. How
do these data explode into load of HEV infections in these
countries? Based on HEV infection rate of 0.04% and dura-
tion of viraemia of 8 weeks, around 80 000100 000
HEV RNA (%) HEV+ donations rate
3/200 (1.5) 1:67
4/107 (3.7) 1:27
9/6700 (0.13) 1:745
13/16 125 (0.08) 1:1240
35/23 500 (0.14) 1:671
6/10 741 (0.06) 1:1790
79/225 000 (0.04) 1:2848
20/35 220 (0.06) 1:1761
7/58 915 (0.01) 1:8416
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HEV infections are likely to have occurred in England dur-
ing the year 2013. A total of 7.4 million blood products
were administered in Germany in 2013, and between
1600 and 5900 HEV RNA-positive blood donations could
be occurring in Germany per year. In the Netherlands, one
HEV-positive donation per day has been reported, implying
that transmission by transfusion is a likely event in this
country. HEV infection in pregnant women, patients with
underlying liver disease, solid organ transplant patients
and immunosuppressed and those with haematological
neoplasm can run a severe course and/or lead to rapidly
progressive chronic liver disease. Such patients often need
blood or blood products. Based on magnitude of problem
and possible severe consequences especially in high-risk
groups, screening for HEV RNA is an urgent need espe-
cially in countries with high HEV prevalence [37].
It is now well accepted that HEV-3 infection can induce
chronic hepatitis, and cirrhosis in solid organ transplant
(SOT) patients, in patients with HIV positive and in those
with haematological diseases [38,39]. Diagnosis of chronic
hepatitis is acceptable if HEV RNA persists for 3 months or
longer. The prevalence of HEV RNA in SOT varies from
0.9 to 3.5% in Europe [40]. Most of the chronic HEV infec-
tions in SOT are asymptomatic. Others have constitutional
symptoms and modest enzyme elevation. Extrahepatic dis-
ease can occur. Liver fibrosis progresses rapidly in a sub-
group of patients ending up in cirrhosis in 23 years. One
large follow-up study of 85 SOT patients with HEV infec-
tion showed that 29 cleared the virus, 56 had chronic
HEV infection and 9 patients ended up to develop cirrhosis.
Predictors for the development of chronic hepatitis in SOT
patients include the use of tacrolimus as an immunosup-
pressant and low platelet in SOT patients and low CD4 in
HIV-infected patients [41].
While industrialized countries have controlled spread of
hepatitis E by ensuring clean portable water, HEV-3 and
HEV-4 spread through ingenious foodborne transmission
Table 5 Diagnosis of hepatitis E virus infection
Test Method Uses
IgM anti-HEV ELISA Acute infection
ICT (POCT)
IgG anti-HEV ELISA Seroprevalence
ICT (POCT) Acute infection
Natural protection
Vaccine efficacy
HEV RNA NAT Acute infection
Confirm chronicity
Antiviral response
Donor screening
HEV antigen EIA Acute infection
ICT, immunochromatographic test; POCT, point of care test; NAT, nucleic acid test; EIA, enzyme immunoassay.
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Hepatitis E: an emerging global disease 75
[42]. Domestic pigs, wild boar and sika deer show cross-
transmission of hepatitis E, and consuming raw or under-
cooked meat or liver (a luxury in such countries) can
cause outbreaks of hepatitis E. More serious is the practice
of visiting supermarkets and buying raw liver or Corsican
Figatelli sausage and eating it undercooked or raw. Signifi-
cant percentages of such livers and sausages contain live
HEV. Sewage from pig in such countries can flow to water-
ways, and visiting sea beaches or consuming infected mol-
luscs has led to outbreaks of HEV.
Chronic HEV infection in SOT patients is a substantial
clinical problem in many European countries and needs to
be controlled [43]. Apart from advice against intake of pos-
sible infected swine meat, liver or sausage, antiviral ther-
apy for HEV RNA patients prior to transplant has been
recommended for fear of chronic HEV infection. Some have
recommended expanding explant testing for HEV RNA to
prevent explant-related HEV infection. As SOT patients are
at high risk for contracting HEV in HEV-3 endemic zones,
HEV vaccine in such patients is an option in future if vac-
cine is found safe and efficacious. Treatment algorithm for
HEV RNA-positive patients in SOT patients is being devel-
oped. It is advisable to reduce immunosuppression which
helps to clear the virus in a large proportion of patients. If
HEV RNA persists for 3 months and beyond, ribavirin ther-
apy 600 mg per day is advisable. Persistent HEV infection
can be managed with pegylated IFN selectively in liver
transplant patients. A subgroup of patients may have
rapidly progressive liver failure and may be considered for
liver transplant or retransplant.
The development of accurate diagnostic assays for the
detection of serological markers of HEV infection remains
challenging (Table 5; [44]). The antigen structure of HEV
protein has been characterized, and highly immunoreactive
diagnostic antigens have been engineered and efficient
diagnostic tests devised. However, many outstanding issues
related to sensitivity and specificity of these assays in clini-
Comments
Assays vary in performance, issue of genotype applicability
and poor performance in immune disorders, and are
cross-reactive with other viral infections
Assays vary in performance
Viraemia short-lasting, in-house assays vary in performance,
advantage immune disorders
81% concordance with HEV RNA
76 M. S. Khuroo & M. S. Khuroo
Fig. 6 Hepatitis E virus ORF2 proteins expressed in Baculovirus expression system and Escherichia coli. For details, see
details about hepatitis E ORF2 expression.
cal and epidemiological settings remain to be resolved.
Some of these assays have shown issues of genotype appli-
cability and poor performance in immune disorders and
are cross-reactive with other viral infections. There are two
major factors that potentially affect the detection of anti-
HEV in human sera samples. These include diagnostic prop-
erties of the antigen and, secondly, variable nature of the
specific HEV-antibody responses. It is essential that assays
should be developed and evaluated for analytical sensitivity
against WHO reference reagent for hepatitis E and not
against sera obtained from patients with recent infection.
Broadly, 2 formats, namely indirect ELISA and class cap-
ture ELISA, have been employed to develop these assays. In
the latter, immobilized antibodies against mu chain of IgM
to capture this class of antibodies are employed.
IgM anti-HEV is a marker of recent or current HEV
infection [45]. Overall, >90% of patients infected with HEV
have detectable IgM anti-HEV in the first 2 weeks after the
onset of illness and lasts for up to 5 months. IgM anti-HEV
is recommended as the first-line diagnostic assay for acute
infection in immunocompetent host. In immunocompro-
mised host, additional HEV RNA testing may be needed
due to impaired immune responses and poor performance
of IgM assays for this population. Many in-house and com-
mercial assays are available. A recent evaluation of perfor-
mance of these assays has shown considerable variability
in their sensitivity and specificity. A commercially available
assay based on improved mu capture (based on mu chain
of IgM) has been developed and marketed by Beijing Wan-
tai Biological Pharmacy. An immunochromatographic
method (rapid test) for the detection of IgM anti-HEV has
been developed by Genelabs Diagnostics, Singapore
(ASSURETM). The test has sensitivity of 93% and specificity
of 99.7%. IgG anti-HEV assays have utility in seropreva-
lence studies and in diagnosis of acute HEV infection. The
determination of the anti-HEV IgG concentration could be
useful for determining the level of antibodies that reliably
prevents infection after natural infection or vaccine admin-
istration in vaccine trials. A vaccine study suggests that an
antibody concentration of 2.5 WHO units/ml was protec-
tive. HEV RNA detection in serum and stools can be
applied to the diagnosis of acute HEV infection especially
in patients with immune disorders and to document
chronicity and evaluate antiviral response in chronic hep-
atitis E. HEV RNA testing may be extended for screening of
blood donors in future. Viraemia during acute HEV infection
is short-lasting and peaks during the incubation period and
early symptomatic phase. There have been issues with some
in-house assays, and recently, a WHO standard (genotype
HEV-3a) has been established for HEV RNA detection and
accurate quantification. Detection and quantification of HEV
RNA in blood and other components are based on real-time
PCR with primers targeting conserved regions between HEV
genotypes. Recently, another nucleic acid amplification tech-
nique, the loop-mediated isothermal amplification (LAMP)
assay, has been developed based on a set of six primers that
recognize eight distinct regions of the target sequence. This
is a one-step, single-tube isothermal amplification of HEV
RNA. The assay is quicker, needs no special equipment and
is suitable for resource limited areas.
When expressed in insect cells by baculovirus expression
system, full-length and truncated ORF2 genes can generate
a number of capsid proteins with various molecular
weights (Fig. 6; [46]). However, only two HEV capsid pro-
teins self-assemble into virus-like particles (VLPs), namely
VLP/T = 1 (a 23-nm empty particle) and VLP/T3 (a 42-
nm particle with a 2-kb RNA). When recombinant ORF2
was expressed in E. coli, three proteins of HEV capsid pro-
tein are expressed. These occur as homodimers which
model dominant antigenic determinants of HEV. p239 has
the capacity to form a 23-nm empty particle. VLPs
expressed in Baculovirus and E coli display similar proper-
2015 John Wiley & Sons Ltd
 Hepatitis E: an emerging global disease 77

ties to native particles in terms of antigenity and surface
structure and have been exploited to study a three-dimen-
sional structure of the native virus. Also 2 of these VLPs
have markedly enhancing immunogenic properties and
form the basic units for 2 HEV vaccines, which have com-
pleted phase III trials. p239 vaccine is commercially avail-
able as Hecolin in Chinese market.
HEV 239 is a particulate vaccine expressed in E. coli, con-
sisting of 368606 aa of ORF2 from HEV-1 Chinese strain.
HEV 239 has 2 epitopes between 533 and 552 aa and
induces a vigorous T cell-dependent antibody response. HEV
239 has successfully completed phase II and phase III trials
in China and is commercially available in China (Hecolin) in
30-lg doses to be administered at 0-, 1- and 6-month regi-
mens [47]. At 4.5 years, vaccine efficacy was 86.8%; 87%
of vaccine group maintained antibodies and 9% control
group developed antibodies [48]. HEV 239 gave cross-pro-
tective efficacy as HEV-4 is the predominant genotype in the
region of study. Despite several limitations as of today on
global use, the successful phase III trial of HEV 239 vaccine
is a major leap forward in the path to control hepatitis E.
Hepatitis E vaccine is available at present only in Chi-
nese market. To make it available, there are several impor-
tant issues to be sorted out for its global launch. First,
available safety data on this vaccine from phase 1, II and
III clinical trials in healthy subjects age group 16 to
65 years are reassuring. However, there are no safety data
in paediatric and elderly, persons with underlying diseases
namely chronic liver diseases, solid organ transplant, HIV-
infected and other immunosuppressed conditions. Safety of
p239 vaccine given concomitantly with other vaccines also
needs to be studied. There are limited data on safety of this
vaccine with regard to maternal and foetal outcomes fol-
lowing administration during pregnancy, and these need
to be extended [49]. Postmarketing phase IV study can be
conducted once vaccine is available globally. The cost-ef-
fectiveness of the vaccine programme to control massive
epidemics needs to be measured. A preliminary study using
Uganda epidemic data found the cost of US$ 875 per dis-
ability-adjusted life years, although this estimate is sensi-
tive to changes in the assumptions used. HEV 239 vaccine
was found efficacious in a context of lesser endemicity
(background HEV attack rate 0.03%) and virulence, and it
is important to determine whether this vaccine shall main-
tain efficacy in Indian subcontinent where the disease is
hyperendemic, with high background attack rate of around
~7.36% and highly virulent pathogen. Also vaccine effi-
cacy needs to be determined in the setting of indigenous
HEV-3 disease in industrialized countries [50].
Let us summarize what are our challenges in control
and cure of this global human pathogen. We need better
and more accurate diagnostic tools which should be avail-
able and extendable to regions of the world with primitive
healthcare facilities and even those with political unstable
and disturbed regions. We need to get a better understand-
ing of many perplexing issues about the epidemiology of
hepatitis E both in resource poor countries and in industri-
alized world. We need to develop treatment strategies for
HEV infection and management plans for those with severe
infections especially liver failure. Finally, we need to
develop and implement effective preventive strategies, espe-
cially HEV vaccine [1].
Hepatitis E story started 35 years back with my extreme
belief that epidemics of jaundice and resultant mortality in
pregnant women in our community had a hidden saga
and to uncover it needed hard work, persistence, belief in
oneself and honesty of purpose. It needed courage to stand
on feet to fight the sceptics and listen to the wise. It meant
spending days in snowbound roads of those villages and
feeling the pain and anguish of the sufferers. I needed
nights to spend awake with pen and paper to write what
one believed is true and to uncover. While this journey
was treacherous, hard, full of failures and some successes,
the end has been pleasant and rewarding. For discoveries
do not come without price to be paid.
ACKNOWLEDGEMENTS
This work was supported by Dr. Khuroos Medical Trust,
a nonprofit organization which supports academic activi-
ties, disseminates medical education and helps poor
patients for medical treatment.
DISCLOSURES
Both authors have nothing to disclose.

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