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Hepatitis E Virus

20th IPFA/PEI Workshop Lanzarote, 13th-17th May 2013

Sally A. Baylis, Division of Virology, Paul-Ehrlich-Institut, Langen, Germany

Virology Division

Background to HEV

Blood and plasma issues

Transmission of HEV by S/D plasma, concentrates

Clearance of HEV

WHO International Standard for HEV for NAT

Update on introduction of HEV NAT for S/D plasma

Virology Division

HEV is a major cause of acute hepatitis and a public health concern in many developing countries

High mortality in certain patients – fulminant hepatitis Individuals with underlying liver disease Pregnant women

Primarily a water-borne disease – poor sanitation

New Delhi, India, winter 1955–1956, monsoon flooding of the Jamuna River - 30,000 cases of HEV “It was a perfect storm. For about a week, raw sewage was running

directly into the city’s water supply with no treatment at all.” Dr Robert Purcell, NIH

Hepatitis E Virus (HEV)

Virology Division

Examples of Large Reported Outbreaks of HEV

Teshale et al., Clin Infect Dis 2010

Virology Division

Ichalkaranji in Kolhapur, India

Most likely source of infection - consumption of contaminated water from the Panchganga river

Since 15 May 2012: 4085 infected (jaundice) >2096 patients treated and discharged 1250 patients hospitalized ~300 patients still under medical care 78 pregnant women infected 12 deaths, 3 pregnant women (as of 21st June 2012)

River used for bathing, washing, religious practices

HEV Outbreak in Kolhapur, India – May/June 2012

Water Quality Monitoring Panchganga River

Virology Division

Hepeviridae family

Humans, birds, pigs, wild boar, deer, rabbits, rats, ferrets, bats, fish

Small non-enveloped viruses 27-34 nm 7.2 kb RNA genome (5´ cap and 3´ poly A tail) ORF1 – non-structural protein ORF2 – virus capsid protein ORF3 – protein involved in morphogenesis (overlaps ORF2)

HEV strains infecting humans represent a single serotype

Hepeviridae Family of Viruses

Virology Division

HEV is an emerging (more recognized) infection in industrialised countries

Infection may be due to travel to endemic areas

More autochthonous cases of HEV are being recorded Tendency - older male patients

Testing important in patients where other causes of hepatitis have been excluded (global issue) Liver toxicity of drugs – some cases may be undiagnosed HEV

Chronic infections increasingly recognised Solid organ transplant recipients (Kamar et al., 2008) Patients with haematological disease HIV patients with low CD4 counts

HEV Infection in Industrialized Countries

Virology Division

4 main genotypes

Genotypes 1 and 2 - humans, epidemics, poor sanitation

Genotypes 3 and 4 - humans and range of animal species (pigs, wild boar, deer other animals) High sequence homology between locally circulating animal and

human strains, zoonotic transmission (food-borne, animal contact…)

The geographical distribution of HEV genotypes is complex

Approximately 74% nucleotide identity between genotypes

Genotype 3 is comprised of at least 10 sub-genotypes (sub-genotypes can vary by as much as 15% nucleotide identity)

HEV Infection of Man – Importance of Genotypes

Virology Division

Hepeviridae

Drexler et al., J Virol 2012

Virology Division

Geographic Distribution of HEV Genotypes

Pelosi and Clarke, Emerg. Health Threats J, 2008

Virology Division

Adapted from Hoofnagle et al., NEJM, 2012

Characteristic Genotypes 1 & 2 (Epidemic)

Genotypes 3 & 4 (Autochthonous)

Geographic distribution Developing countries only Both developing & developed countries

Pattern of spread Epidemic and sporadic Sporadic

Species specificity Human Swine, human (humans are accidental host)

Major mode of spread Fecal-oral, waterborne Foodborne

Rate of icteric illness High Low

Age distribution (disease rates)

Highest among adolescents & young adults

Highest among older adults

Sex distribution Similar disease rates among men & women

Higher disease rates among men

Mortality High among pregnant women High among older adults

Extrahepatic features Few Neurological complications

Chronic infection None Common in immunosuppressed

Clinical & Epidemiological Characteristics - HEV Genotypes

Virology Division

HEV Transmission

Adapted from Kamar et al., Lancet 2012

Transplantation

Virology Division

HEV Infection

Kamar et al., Lancet, 2012

Virology Division

Serology – detection of anti-HEV Anti-IgM – acute infection Anti-IgG – acute infection and past infection

Wide variability in sensitivity, specificity, and interassay agreement among anti-HEV-IgM immunoassays Drobeniuc et al., Clin Infect Dis. 2010

Commercial assays for anti-IgG show different sensitivities, WHO reference reagent Bendall et al., J Med Virol. 2010 Underestimation of HEV seroprevalence - many published studies

Detection of HEV RNA in blood and stool samples Limited mainly to in-house assays, not standardized

Diagnosis of HEV Infection

Virology Division

HEV 239 - recombinant vaccine HEV virus-like particles (E. coli)

Zhu et al., Lancet 2010 Efficacy and safety of a recombinant hepatitis E vaccine in

healthy adults: a large-scale, randomized, double-blind placebo-controlled, phase 3 trial

Manufactured by Xiamen Innovax Biotech Co., China

HEV 239 has recently been approved for use in China

Other vaccine candidates are in development

HEV Vaccine

Virology Division

HEV - Blood and Plasma Issues

Virology Division

Several cases of documented cases of TT HEV

Iran, Khuroo et al., J Gastroenterol Hepatol. 2004

Retrospective IgM anti-HEV and HEV RNA detected in a significantly higher number of multiple transfused patients (13 of 145)

Post-transfusion HEV infection developed in three of 22 susceptible (IgG anti-HEV negative) transfused patients

UK, Boxall et al., Transfus. Med. 2006

Donor, 40 yr old male, post-donation illness and jaundice Platelets (plasma removed) and red cells Red cell recipient (lymphoma) developed elevated alanine

transaminase (ALT)/mild jaundice Genotype 3 virus identified in donor and recipient

Transfusion Transmission of HEV

Virology Division

France, Colson et al., EID, 2007

Donor, 24 yr old male, later seroconverted 7 yr old boy with a kidney tumour, received erythrocytes, platelets Elevated ALT (~800 IU/L), jaundice, hepatitis Genotype 3f virus identified in donor and recipient

Series of cases in Japan:

Matsubayashi et al., Transfusion 2004 Mitsui et al., J Med Virol. 2004 (haemodialysis - gt 3) Tamura et al., Hepatol Res. 2007

Transfusion Transmission of HEV contd.

Virology Division

Japan, Matsubayashi et al., Transfusion 2008

Platelet donor, 39 yr old male, subsequent donation showed ↑ ALT

Lookback study showed previous donation was HEV RNA positive ~3 log copies/ml, gt 4

HEV contaminated platelets were transfused to a 64 yr old patient with non-Hodgkin's lymphoma, developed acute hepatitis

The donor and 13 relatives ate pork liver and intestines at a barbecue restaurant 23 days prior to donation

Father of the donor died - fulminant hepatitis 6 other family members showed serum markers of HEV infection

Demonstration that donor was infected by the zoonotic food- borne route

Transfusion Transmission of HEV contd.

Virology Division

France, Haïm-Boukobza et al., J Hepatol 2012

Initial diagnosis was drug induced liver toxicity - cyclosporine treatment in the patient

Patient didn‘t eat pork for religious reasons Patient received several transfusions; one donation was part of a

platelet concentrate (4.2 log10 IU/ml) Identical genotype 3f virus sequences detected in donor and

recipient

Transfusion Transmission of HEV contd.

Virology Division

Adlhoch et al., Vox Sang. 2009

A regular plasma donor was diagnosed with acute hepatitis and elevated levels of ALT

Negative for HAV, HBV, HCV, EBV, CMV and adenovirus Tested for anti-HEV - IgM positive

Robert Koch-Institut was notified and a look-back study was performed

A genotype 3f virus was identified - donor worked in a slaughter house

HEV Infection in a German Plasma Donor

Virology Division

The HEV Window Period

Adlhoch et al., Vox Sang 2009

Virology Division

Canada, Andonov et al., ISBT July 2012

Retrospective study of thrombotic thrombocytopenic purpura patients (TTP) treated with large volumes of pooled plasma Solvent/detergent (S/D)-treated plasma (2500 donors) Cryosupernatant plasma (150 donors)

38 patients received 20-40 litres of plasma 17 - S/D plasma 19 - cryosupernatant plasma 2 - FFP and Pentaspan or albumin

Samples taken at 0, 1 and 6 months post-treatment

HEV and Plasma Products – TTP Patients

Virology Division

No clinical signs of viral hepatitis in any of the patients

No serological evidence of HEV infection at 0 and 1 month post-treatment in any patients

4 out of 17 patients treated with S/D plasma seroconverted at 6 months for anti-HEV IgG, 2 also anti-HEV IgM +ve

The 2 patients positive for anti-HEV IgM and IgG were HEV RNA +ve at 1 month post-treatment - gt 3a

Indirect evidence of HEV transmission by pooled plasma autochthonous HEV in Canada, 4 cases in last 5 years

Annual incidence of HEV infection in German blood donors 0.34% (Juhl et al., Transfusion, Epub ahead of print)

HEV and Plasma Products – TTP Patients Contd.

Virology Division

Japan, Toyoda et al., Intervirology, 2008 Evaluation of haemophiliacs, haemodialysis patients vs.

blood donors

16.3% of haemophiliacs were anti-HEV positive vs.

blood donors (3.7%) Parental transmission of HEV likely in recipients of non-

virally inactivated concentrates

HEV and Plasma Products - Haemophiliacs

Virology Division

HEV small non-enveloped virus

Yunoki et al., Vox Sang. 2008

Investigation of: Liquid heat treatment - 25% albumin, 60°C Dry heat treatment - 60°C, 80°C Virus filtration

Viruses used for spiking studies – swine faeces (gt 3 or 4)

A549 cells (lung carcinoma) used for virus titration or detection of HEV RNA genome by qRT-PCR

Inactivation/Removal of HEV

Virology Division

Virus titres determined by infectivity assays

Residual virus was detected after heat treatment in the presence of albumin

HEV Reduction Factors (log10) - Liquid-Heat Treatment

Heat treatment 3JP± swJB-N2

3US swJB-M5

3SP swJB-E10

4JP swJB-H1

Liquid heating, 60°C, 30 min

e 2.7

e 3.7

e 3.7

e 2.4

Liquid heating, 60°C, 5h, 25% albumin

2.0

1.0

2.0

e 2.2

Yunoki et al., Vox Sang. 2008

Virology Division

Virus titres determined by infectivity assays

Residual moisture was < 0.3%

Matrix may affect sensitivity to inactivation method

HEV Reduction Factors (log10) - Dry-Heat Treatment

Heat treatment 3US swJB-M5

3SP swJB-E10

Dry-heat, 80°C, 24h, fibrinogen

e 4.0

e 4.0

Dry-heat, 60°C, 72h, fibrinogen

2.0

3.0

Yunoki et al., Vox Sang. 2008

Virology Division

Virus spiked into PBS ’ 0.2 µm filtration/75 nm filtration

Dead end filtration using Planova 35N, 20N and 15N virus filters

Virus titres determined by qRT-PCR

HEV Reduction Factors (log10) - Virus Filtration

Filter 3JP± swJB-N2

3US swJB-M5

3SP swJB-E10

3SP cultured HEV

4JP swJB-H1

35N 1.3 e 3.6 2.6 e 2.8 1.1

20N e 3.8 e 3.6 e 3.2 e 2.8 e 2.6

15N e 3.8 e 3.6 e 3.2 e 2.8 e 2.6

Yunoki et al., Vox Sang. 2008

Virology Division

Intermediates spiked HEV derived from swine faeces or human plasma

Evaluation using real-time PCR

Partitioning of HEV (different spike preparations) during ethanol fractionation is variable - ? lipid, Ab effects

HEV Partitioning During Ethanol Fractionation

M. Yunoki, Pers. Comm. 2013

Virology Division

Anti-HEV may not always effectively neutralize virus

Possible to propagate infectious HEV in culture using viraemic serum containing anti-HEV – Takahashi et al., J Clin Microbiol 2010

Vaccine studies – protective levels of anti-rHEV immunoglobulin of at least 20 WR U/ml (~ 2.5 WHO units/ml) Shrestha et al., NEJM 2008

Transfusion Transmission of HEV - Japan

M. Yunoki, Pers. Comm. 2013

Year HEV Markers in Donor Plasma HEV RNA HEV IgG HEV IgM

Hepatitis E severity in recipient

2005 + - - -

2005 + - - -

2008 + - - -

2008 + + + -

2008 + - - -

Virology Division

Genotypes 3 and 4 indentified in donors

HEV RNA Positive Japanese Donations

M. Yunoki, Pers. Comm. 2013

Area Dates HEV Positive Ratio

Hokkaido (Japanese Red Cross)

01/2005 - 12/2012

254 / 2,207,772

(1 / 8,692)

Tokyo (Japanese Red Cross)

05/2006 - 07/2006

3 / 44,332

(1 / 14,777)

Japan excl. Hokkaido (Benesis – source plasma)

07/2007 – 03/2013

24 / 378,718

(1 / 15,800)

Virology Division

Analysis of HEV RNA in Indian and Chinese Blood Donors

Country Rate Reference

India 3: 200 Arankalle & Chobe, 1999 J Viral Hepat

China Detection of gt 1 & gt 4 in anti-HEV IgM positive donations

Guo et al., 2010 J Clin Microbiol

Virology Division

Analysis of HEV RNA in Europe and the US

Country Rate Reference

England 1: 7,040 Ijaz et al. Vox Sang 2012 – extrapolated*

Germany 1: 4,415 Baylis et al.,

Vox Sang 2012** Sweden 1: 8,278

USA <1: 50,456

* Blood donors ** S/D plasma donors

Similar data in Germany - Vollmer et al., J Clin Micro, 2012 - Hourfar et al., ISBT abstract, 2012 - Corman et al., Vox Sang, 2013

Virology Division

HEV RNA Positive German & Swedish Donations

Viraemic titers usually ~2 to >5 log10 IU/ml However, viraemic titers may exceed 7 log10 IU/ml Only 3 of 12 viraemic donations were ALT positive All viruses genotype 3 (3a, 3c, 3e, 3f etc.)

Virology Division

HEV in Plasma Pools for Fractionation

Source of Pools No. Positive / No. Analysed

Europe 3/34

Europe/North America 0/3

North America 1/4

Middle East 0/11

Asia 4/23

Overall 8/75

RNA concentration: d 1000 copies/ml Low antibody levels in fractionation pools (except Asia) Genotype 4 HEV – Asia; genotype 3 Europe and USA

Virology Division

HEV Strains Developed into Reference Materials for NAT-Based Assays

Genotype Virus strain HEV RNA

(copies/ml)

Anti-HEV

IgM/IgG

ALT (IU/L) Reference

preparation

3a

HRC-HE104

1.6 x 107

-/-

36

WHO

International

Standard

3b

JRC-HE3

2.5 x 107

+/-

398

Japanese

National

Standard

Virology Division

Nominal concentration (log10 copies/ml)

6.2 5.2 4.2 3.2 2.2 1.2

Lab no. 1 + + + +/- - - 2 a + + + + + - 2 b + + + + +/- - 3 + + + + + - 4 + + + + - +/- 5 + + + + + - 6 + + + + - - 7 + + + + - - 8 + + + - + - 9 + + + + - -

10 + + + - +/- - 11 a + + - - - - 11 b + + +/- - - - 12 + + + + + + 13† + + + + - - 14 + + + + + +

15 a + + + + - - 15 b + + + + - - 16 + + + + - - 17 + + + + - -

18 a + + + - - - 18 b + + + + - - 19 - - - - - - 20 + + + - +/- -

Total number of tests 24 24 24 24 24 24 Percentage positive 96 96 92/88 75/67 38/25 13/8

Example - Qualitative Analysis of HRC-HE104 (Genotype 3a)

Virology Division

Quantitative assays (white - copies/ml); qualitative assays (blue - NAT-detectable /ml).

Histograms of Participants Results

Virology Division

Quantitative assays (white); qualitative assays (blue).

Potency relative to candidate IS = difference in estimated log10 units/ml + assigned value of candidate IS (5.39 log10 IU/ml)

Potencies Expressed Relative to Sample 1

Virology Division

1st WHO International Standard (IS) for Hepatitis E Virus RNA was established in October 2011 Japanese NIID – simultaneously establishing a national

standard

The IS contains a blood donor-derived genotype 3a HEV strain, diluted in plasma, and lyophilized

The IS has a unitage of 250,000 International Units/ml

The IS is available from the PEI (code # 6329/10)

WHO/BS/2011.2175

Establishment of the 1st WHO IS for HEV RNA

Virology Division

The WHO Expert Committee on Biological Standardization endorsed the proposal to prepare an HEV RNA genotype panel at the annual meeting in October 2011 (WHO/BS/2011.2179)

The panel is intended to contain representative of all genotypes and important sub-genotypes

Candidate samples for the preparation of the panel include materials evaluated in the original collaborative study, strains detected in blood/plasma donors & clinical isolates

Future, re-evaluate the WHO anti-HEV IRR

HEV Reference Panels

Virology Division

b

Analysis based upon partial RdRp sequence

Virology Division

The proposal is to amend monograph 1646 - Human plasma (pooled and treated for virus inactivation) Not plasma for fractionation

Plasma for fractionation undergo further processing including steps for virus inactivation/removal

No inactivation/removal step for non-enveloped viruses, such as HEV, during the production of S/D plasma

HEV detected in respective European plasma donations

Amendment would see the introduction of HEV NAT

Proposal to Amend the Ph. Eur. Monograph 1646

Virology Division

The Hepatitis E virus RNA: The plasma pool is tested using a validated nucleic acid amplification technique (2.6.21). A positive control with 2.5 log10 IU of hepatitis E virus RNA per mililitre and, to test for inhibitors, an internal control prepared by addition of a suitable marker to a sample of the plasma pool are included in the test. The test is invalid if the positive control indicates the presence of inhibitors. The pool complies with the test if it is found non-reactive for hepatitis E virus RNA.

Proposed Text

Virology Division

The proposal was discussed at the group 6B (Human Blood and Blood Products) meeting at EDQM - March 2012 Jan-Mar 2013 Pharmeuropa public consultation Mar-May 2013 Pharmeuropa National Authority comments Pharmeuropa 25.1.: http://pharmeuropa.edqm.eu, until 31/05/2013

Oct 2013 Discussion of comments by Group 6B Nov 2013 Proposal to Ph. Eur. Commission 1 Jul 2014 Publication 1 Jan 2015 Implementation

Biological Standardisation Programme project (BSP127) Biological Reference Preparation HEV RNA for NAT

Proposal to Amend the Ph. Eur. Monograph 1646

Virology Division

Acknowledgments

JRCS Keiji Matsubayashi Hidekatsu Sakata

JBPO Mikihiro Yunoki

NIID, Japan Saeko Mizusawa Yoshiaki Okada

Thomas Gärtner

Anton Andonov

WHO Ana Padilla and Collaborative Study Participants

PEI Johannes Blümel Kay-Martin Hanschmann Roswitha Kleiber Sigrid Nick Micha Nübling Gudrun Winskowsky

Institute of Virology, Bonn Felix Drexler Victor Corman

UK Harry Dalton Linda Scobie/Claire Crossan