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HepatitisE:Anemergingglobaldisease-fromdiscoverytowardscontrolandcure

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Hepatitis E: an emerging global disease – from discoverytowards control and cureMehnaaz S. Khuroo1 and Mohammad S. Khuroo2,3 1Department of Pathology, Govt: Medical College Srinagar,

Kashmir, India; 2Gastroenterology and Chairman Dept. Medicine, Sher-i-Kashmir Institute of Medical Sciences, Kashmir, India and 3Digestive

Diseases Centre, Dr. Khuroo’s 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

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.

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

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 [3–6]. 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

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. Khuroo’s Medical Clinic, Sector 1, Qamarwari, SK Colony,

Srinagar, Kashmir, J&K 190010, India.

E-mail: khuroo@yahoo.com

© 2015 John Wiley & Sons Ltd

Journal of Viral Hepatitis, 2015 doi:10.1111/jvh.12445

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 [5,12–14]. A similar disease was

reported constituting over half of endemic hepatitis in the

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).

Fig. 1 Reported cases of hepatitis E virus infection from

industrialized countries. Data source: references 7, 8 and

39.

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.

© 2015 John Wiley & Sons Ltd

2 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

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

4–6 weeks; however, it may range from 9 days to

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.

© 2015 John Wiley & Sons Ltd

Hepatitis E: an emerging global disease 3

2 months. Disease is present if there were symptoms such as

fever, anorexia, vomiting and jaundice. The symptoms coin-

cide with a sharp rise in serum alanine transaminase (ALT)

levels. Symptoms may persist for few weeks to a month or

more. ALT levels return to normal during convalescence.

HEV RNA may be detected in both serum and stool early in

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]

(ICTV [61])

Hepeviridae Orthohepevirus Orthohepevirus A Human HEV-1

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.

© 2015 John Wiley & Sons Ltd

4 M. S. Khuroo & M. S. Khuroo

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.

HEV disease has significant morbidity and mortality in

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-

Fig. 5 Clinical, biochemical and serological profile of

hepatitis E virus infection. Based on data from Pinglina

epidemic, Kashmir 1992.

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 67–98% 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

(15–30 year), plateau at 30–40%Steady increase throughout age groups;

varies 7–21%Seroincidence 64/1000 years 30 (south France), 2 (UK)

7 (USA)/1000 years

Age (year) 15–40 >50Sex 2:1 >3:1Clinical 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

births

Unknown

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

© 2015 John Wiley & Sons Ltd

Hepatitis E: an emerging global disease 5

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

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

Site Year Icteric cases Mortality pregnancy (%)

Indian subcontinent

Kashmir 1978–82 52 000 22

Delhi 1955–56 29 000 10

Kanpur 1991 78 000 NA

Burma 1976–77 20 000 18

Central Asia

Kirgiz 1956–56 10 000 18

China 1987 120 000 18

Africa

Sudan 1985 2000 NA

Somalia 1985–86 2000 NA

Uganda 2008 10 196 6.9

Data source: references [1,11]. NA, data not available.

Table 3 Major reported epidemics

© 2015 John Wiley & Sons Ltd

6 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

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 donor–patient 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;

[33–36]). 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 80 000–100 000

Table 4 HEV RNA in healthy donors

References Region HEV RNA (%) HEV+ donations rate

Arankalle et al. [51] Pune India 3/200 (1.5) 1:67

Khuroo et al. [29] Kashmir India 4/107 (3.7) 1:27

Gotanda et al. [52] Japan 9/6700 (0.13) 1:745

Vollmer et al. [35] Germany 13/16 125 (0.08) 1:1240

Juhl et al. [53] Germany 35/23 500 (0.14) 1:671

Ren et al. [54] China 6/10 741 (0.06) 1:1790

Hewitt et al. [32] England 79/225 000 (0.04) 1:2848

Hogema et al. [55] Netherlands 20/35 220 (0.06) 1:1761

Fischer et al. [56] Austria 7/58 915 (0.01) 1:8416

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 [29–38].

© 2015 John Wiley & Sons Ltd

Hepatitis E: an emerging global disease 7

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 2–3 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

[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-

Table 5 Diagnosis of hepatitis E virus infection

Test Method Uses Comments

IgM anti-HEV ELISA

ICT (POCT)

Acute infection Assays vary in performance, issue of genotype applicability

and poor performance in immune disorders, and are

cross-reactive with other viral infections

IgG anti-HEV ELISA

ICT (POCT)

Seroprevalence

Acute infection

Natural protection

Vaccine efficacy

Assays vary in performance

HEV RNA NAT Acute infection

Confirm chronicity

Antiviral response

Donor screening

Viraemia short-lasting, in-house assays vary in performance,

advantage immune disorders

HEV antigen EIA Acute infection 81% concordance with HEV RNA

ICT, immunochromatographic test; POCT, point of care test; NAT, nucleic acid test; EIA, enzyme immunoassay.

© 2015 John Wiley & Sons Ltd

8 M. S. Khuroo & M. S. Khuroo

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-

Fig. 6 Hepatitis E virus ORF2 proteins expressed in Baculovirus expression system and Escherichia coli. For details, see

details about hepatitis E ORF2 expression.

© 2015 John Wiley & Sons Ltd

Hepatitis E: an emerging global disease 9

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 368–606 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. Khuroo’s 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.

REFERENCES

1 Khuroo MS. Discovery of hepatitis E:

the epidemic non-A, non-B hepatitis

30 years down the memory lane.

Virus Res 2011; 161(1): 3–14.2 Rein DB, Stevens GA, Theaker J,

Wittenborn JS, Wiersma ST. The

global burden of hepatitis E virus

genotypes 1 and 2 in 2005. Hepatol-

ogy 2012; 55(4): 988–997.

3 Khuroo MS, Khuroo MS. Seroepi-

demiology of a second epidemic of

hepatitis E in a population that had

recorded first epidemic 30 years

before and has been under surveil-

lance since then. Hepatol Int 2010;

4(2): 494–499. Erratum at: Hepatol

Int 2010;4(2):536

4 Labrique AB, Zaman K, Hossain Z

et al. Epidemiology and risk factors

of incident hepatitis E virus infection

in rural Bangladesh. Am J Epidemiol

2010; 172: 952–961.5 Khuroo MS, Kamili S, Dar MY,

Moecklii R, Jameel S. Hepatitis E

and long term antibody status. Lan-

cet 1993; 341: 355.

© 2015 John Wiley & Sons Ltd

10 M. S. Khuroo & M. S. Khuroo

6 Seriwatana J, Shrestha MP, Scott RM

et al. Clinical and epidemiological rel-

evance of quantitating hepatitis E

virus-specific immunoglobulin M.

Clin Diagn Lab Immunol 2002; 9(5):

1072–1078.7 Pischke S, Behrendt P, Bock CT, Jilg

W, Manns MP, Wedemeyer H.

Hepatitis E in Germany–an under-re-

ported infectious disease. Dtsch Arz-

tebl Int 2014; 111(35-36): 577–583.8 Beale MA, Tettmar K, Szypulska R,

Tedder RS, Ijaz S. Is there evidence

of recent hepatitis E virus infection

in English and North Welsh blood

donors? Vox Sang 2011; 100(3):

340–342.9 Khuroo MS. Discovery of hepatitis E:

the untold story. JK-Pract 2004; 11

(4): 291–294. [video at www.

drkhuroo.com]

10 Khuroo MS. Study of an epidemic of

non-A, non-B hepatitis: possibility

of another human hepatitis virus

distinct from post transfusion non-

A, non-B type. Am J Med 1980; 68:

818–823.11 Khuroo MS. Hepatitis E: the enteri-

cally transmitted non-A, non-B hep-

atitis. Ind J Gastroenterol 1991; 10

(3): 96–100.12 Khuroo MS, Teli MR, Skidmore S,

Sofi MA, Khuroo M. Incidence and

severity of viral hepatitis in preg-

nancy. Am J Med 1981; 70: 252–255.

13 Khuroo MS, Saleem M, Teli MR,

Sofi MA. Failure to detect chronic

liver disease after epidemic non-A,

non-B hepatitis. Lancet 1980; 2

(8185): 97–98.14 Khuroo MS, Kamili S, Jameel S. Verti-

cal transmission of hepatitis E virus.

Lancet 1995; 345: 1025–1028.15 Khuroo MS, Duermeyer W, Zargar

SA, Ahanger MA, Shah MA. Acute

sporadic Non-A, non-B hepatitis in

India. Am J Epidemiol 1983; 118:

360–364.16 Purdy MA, Khudyakov YE. Evolu-

tionary history and population

dynamics of hepatitis E virus. PLoS

ONE 2010; 5(12): e14376.

17 Ahmad I, Holla RP, Jameel S.

Molecular virology of hepatitis E

virus. Virus Res 2011; 161: 47–58.18 Cao D, Meng X-J. Molecular biology

and replication of hepatitis E virus.

Emerg Microbes Infect 2012; 1: e17.

19 Smith DB, Simmonds P, Jameel S

et al. Consensus proposals for classi-

fication of the family Hepeviridae. J

Gen Virol 2014; 95: 2223–2232.20 Khuroo MS, Rustgi VK, Dawson GJ

et al. Spectrum of hepatitis E infec-

tion in India. J Med Virol 1994; 43:

281–286.21 Khuroo MS, Kamili S. Etiology and

prognostic factors of acute liver fail-

ure in India. J Viral Hepat 2003;

10: 224–231.22 Khuroo MS, Kamili S. Etiology, clin-

ical course and outcome of sporadic

viral hepatitis in pregnancy. J Viral

Hepat 2003; 10: 61–69.23 Kumar A, Saraswat VA. Hepatitis E

and acute-on-chronic liver failure. J

Clin Exp Hepatol 2013; 3(3): 225–230.24 Khuroo MS, Kamili S, Khuroo MS.

Clinical course and duration of vire-

mia in vertically transmitted hepati-

tis E virus (HEV) infection in babies

born to HEV-infected mothers.

J Viral Hepat 2009; 16: 519–523.25 Khuroo MS, Khuroo MS. Sanitation

and sewage disposal in India. JK-

Pract 2015; 20(1-2): 43–48.26 Khuroo MS, Dar MY. Hepatitis E: evi-

dence for Person-to-Person transmis-

sion and inability of low dose

immune serum globulin from an

Indian source to prevent it. Ind J Gas-

troenterol 1992; 11(3): 113–116.27 Navaneethan U, Al Mohajer M,

Shata MT. Hepatitis E and preg-

nancy-understanding the pathogen-

esis. Liver Int 2008; 28(9): 1190–1199.

28 Khuroo MS, Kamili S. Association

of severity of hepatitis E virus infec-

tion in the mother and vertically

transmitted infection in the fetus.

JK-Pract 2006; 13(2): 70–74.29 Khuroo MS, Kamili S, Yattoo GN.

Hepatitis E virus infection may be

transmitted through blood transfu-

sion in an endemic area. J Gastroen-

terol Hepatol 2004; 19: 778–784.30 Matsubayashi K, Nagaoka Y, Sakata

H et al. Transfusion-transmitted

hepatitis E caused by apparently

indigenous hepatitis E virus strain

in Hokkaido, Japan. Transfusion

2004; 44: 934–940.31 Boxall E, Herborn A, Kochethu G

et al. Transfusion-transmitted hep-

atitis E in ‘nonhyperendemic’ coun-

try. Transfus Med 2006; 16: 79–83.

32 Hewitt PE, Ijaz S, Brailsford SR et al.

Hepatitis E virus in blood compo-

nents: a prevalence and transmission

study in southeast England. Lancet

2014; 384(9956): 1766–1773.33 Bajpai M, Gupta E. Transfusion

transmitted hepatitis E: is screening

warranted? Indian J Med Microbiol

2011; 29: 353–358.34 Gallian P, Piquet Y, Assal A et al.

Hepatitis E virus: blood transfusion

implications. Transfus Clin Biol

2014; 21(45): 173–177.35 Vollmer T, Diekmann J, Johne R,

Eberhardt M, Knabbe C, Dreiera J.

Novel approach for detection of

hepatitis E virus infection in Ger-

man blood donors. J Clin Microbiol

2012; 50: 2708–2713.36 Bayliss SA, Gartner T, Nick S et al.

Occurrence of hepatitis E virus RNA

in plasma donations from Sweden,

Germany and the United States.

Vox Sang 2012; 103: 89–90.37 Pawlotsky JM. Hepatitis E screening

for blood donations: an urgent

need? Lancet 2014; 384(9956):

1729–1730.38 Kamar N, Dalton HR, Abravanel F,

Izopet J.HepatitisEvirus infection.Clin

MicrobiolRev2014;27:116–138.39 Khuroo MS, Khuroo MS. Hepatitis E

virus. Curr Opin Infect Dis 2008; 21:

539–543.40 Haagsma EB, Niesters HG, van den

Berg AP et al. Prevalence of hepati-

tis E virus infection in liver trans-

plant recipients. Liver Transpl 2009;

15(10): 1225–1228.41 Kamar N, Garrouste C, Haagsma EB

et al. Factors associated with

chronic hepatitis in patients with

hepatitis E virus infection who have

received solid organ transplants.

Gastroenterology 2011; 140(5):

1481–1489.42 Yugo DM, Meng X-J. Hepatitis E

virus: foodborne, waterborne and

zoonotic transmission. Int J Environ

Res Public Health 2013; 10(10):

4507–4533.43 Kamar N, Izopet J, Dalton HR.

Chronic hepatitis E virus infection

and treatment. J Clin Exp Hepatol

2013; 3: 134–140.44 Khudyakov Y, Kamili S. Serological

diagnosis of hepatitis E virus infec-

tion. Virus Res 2011; 161: 84–92.

© 2015 John Wiley & Sons Ltd

Hepatitis E: an emerging global disease 11

45 Mirazo S, Ramos N, Mainardi V,

Gerona S, Arbiza J. Transmission,

diagnosis, and management of hep-

atitis E: an update. Hepat Med Evid

Res 2014; 6: 45–59.46 Kamili S. Towards the development

of a hepatitis E vaccine. Virus Res

2011; 161: 93–100.47 Zhu FC, Zhang J, Zhang XF et al.

Efficacy and safety of a recombi-

nant hepatitis E vaccine in

healthy adults:a largescale, random-

ized, double-blind placebo-controlled

phase 3 trial. Lancet 2010; 376: 895–902.

48 Zhang J, Zhang XF, Huang SJ et al.

Long term efficacy of a hepatitis E

vaccine. N Engl J Med 2015; 372:

914–922.49 Wu T, Zhu FC, Huang SJ et al.

Safety of the hepatitis E vaccine for

pregnant women: a preliminary

analysis. Hepatology 2012; 55:

2038.

50 WHO Geneva. Weekly Epidemiologi-

cal Record 1st May 2015. Hepatitis

E vaccine. Geneva: WHO, 2015.

position paper No. 18. 90;185-200.

http://www.who.int/wer

51 Arankalle VA, Chobe LP. Retrospec-

tive analysis of blood transfusion

recipients: evidence for post-transfu-

sion hepatitis E. Vox Sang 2000; 79

(2): 72–74.52 Gotanda Y, Iwata A, Ohnuma H

et al. Ongoing subclinical infection

of hepatitis E virus among blood

donors with an elevated alanine

aminotransferase level in Japan. J

Med Virol 2007; 79(6): 734–742.53 Juhl D, Baylis SA, Blumel J, et al.

Seroprevalence and incidence of

hepatitis E virus infection in Ger-

man blood donors. Transfusion

2013; doi: 10.1111/trf.12121.

54 Ren F, Zhao C, Wang L, et al. Hepa-

titis E virus seroprevalence and

molecular study among blood

donors in China. Transfusion 2014;

54(3 Pt 2): 910–917.55 Hogema BM, Molier M, Slot E, Zaai-

jer HL. Past and present of hepatitis

E in the Netherlands. Transfusion

2014; 54(12): 3092–3096.

56 Fischer C, Hofmann M, Danzer M,

Hofer K, Kaar J, Gabriel C. Seropre-

valence and Incidence of hepatitis E

in blood donors in Upper Austria.

PLoS One 2015; 10(3): e0119576.

57 International Committee on Taxon-

omy of Viruses; Hepatitis E virus.

1995. http://www.ictvonline.org/

virusTaxInfo.asp

58 International Committee on Taxon-

omy of Viruses; Hepatitis E virus.

1998. http://www.ictvonline.org/

virusTaxInfo.asp

59 International Committee on Taxon-

omy of Viruses; Hepatitis E virus.

2004. http://www.ictvonline.org/

virusTaxInfo.asp

60 International Committee on Taxon-

omy of Viruses; Hepatitis E virus.

2009. http://www.ictvonline.org/

virusTaxInfo.asp

61 International Committee on Taxon-

omy of Viruses; Hepatitis E virus.

2015. http://www.ictvonline.org/

virusTaxInfo.asp

© 2015 John Wiley & Sons Ltd

12 M. S. Khuroo & M. S. Khuroo