Proposal to Prepare Reference Panels & Standards for Hepatitis E Virus Sally Baylis Viral Safety, Paul-Ehrlich-Institut SoGAT XXI, Brussels, 28th-29th May 2009

Hepatitis E Virus (HEV) Water borne, spread via faeces, causes acute hepatitis, with major outbreaks Mortality rates amongst pregnant women are ~20%; overall mortality 0.5-4%, higher in those with chronic liver disease Worldwide occurrence, but particular problem in developing countries. Endemic in Africa, SE Asia, Middle East, Central America – poor sanitation Emerging infection in industrialized countries. Better awareness, better diagnostics or a genuine rise in infections?

Slide courtesy of R. Purcell

HEV - Characteristics of the Virus Small non-enveloped RNA virus (~32-34 nm), Hepeviridae family Five virus genotypes; types 1 and 2 infect humans; types 3 & 4 infect pigs, boar & deer; type 5 is a distantly related avian virus; 23-31% divergence Genotypes 3 & 4 are zoonotic & infect humans. High seroprevalence in pig herds, virus detected in meat products (Wichmann et al., JID, 2008) HEV shed in faeces, but also causes a viraemia ~107 copies/ml plasma

Slide courtesy of R. Purcell

Slide courtesy of R. Purcell

Slide courtesy of R. Purcell

Slide courtesy of R. Purcell

HEV – Transplantation & Transfusion Normally causes acute hepatitis, however emerging as a cause of chronic liver disease in organ transplant recipients; may remain neg. for anti-HEV (e.g. Kamar et al., NEJM 2008) Transmitted by transfusion with cases reported in endemic & non-endemic areas Saudi Arabia, Japan, UK & France Platelets, red cells Elevated levels of ALT, some develop acute hepatitis, or may become persistently infected Genotypes 3 & 4 identified in TTI cases Seroprevalence in blood donors e.g. Bavarian Red Cross ~10% seropositive, France ~3.2% Swedish pig farmers ~13%

HEV - Blood Donation Blood donors with elevated levels of alanine aminotransferase (ALT) can be infected with HEV Hokkaido, Japan ALT > 500 IU/l HEV RNA was detected in ~20% of samples Blood donors in Hokkaido (1 in 8,300 donors) Nationwide Japan ALT > 200 IU/l HEV RNA 1.1% (gt 3 and 4) anti-HEV IgM 1.0% anti-HEV IgG 3.2% (Sakata et al., 2008, Transfusion) HEV RNA positive plasma samples may be ALT negative

HEV and Plasma Derivatives Haemophiliacs have high level of anti-HEV (16% vs 3% in blood donors in Japan), patients < 20 years old did not have antibodies and had received virus-inactivated coagulation factors (Toyoda et al., 2008) Pasteurization of albumin at 60ºC, 5 h, slow inactivation kinetics (similar to CPV), reduction factor (Rf) of ≤2 log10 (Yunoki et al., Vox Sang. 2008) Lypohilized fibrinogen preparations, slight difference observed Rfs dependent upon stabilizer composition. After 24 h, 80ºC Rf ≥4.0 log10 HEV is completely removed by 20 nm filters

Issues Concerning Blood Components and Plasma Derivatives Possible concern for blood for transfusion, ALT testing may remove HEV positive units Possible concern for S/D treated plasma (analagous to HAV NAT and S/D plasma. Ph. Eur. 01/2009:1646, declining levels of anti-HAV in start pools) The availability of NAT standards for HEV could facilitate the introduction of screening as required

HEV and Plasma Fractionation Pools Plasma pools from different manufacturers worldwide were tested for HEV RNA by PCR Plasma was extracted using the Roche AmpliPrep (TNAI kit) HEV RNA was detected by conventional and real-time PCR using primers and probes targeting conserved regions of ORF2 and ORF3 (modified Jothikumar et al., 2006; Gyarmati et al., 2008)

Analysis of Plasma Pools for HEV RNA Source of Pools No. positive/no. analysed W Europe/N America 3/25 E Europe 2/20 Middle East 0/11 South East Asia 4/27 Overall 9/83

Analysis of Plasma Pools for HEV RNA Approximately 10% of plasma pools tested are positive for HEV RNA Genotype 3 and genotype 4 viruses have been identified, sequence analysis is on-going Viral loads of positive pools identified to date are below 1000 copies/ml plasma

Proposal for an HEV RNA Standard Clinical use: where patients present with hepatitis, but the cause can not be ascertained; specialist reference laboratories Blood screening use: plasma donations for use in S/D treated plasma, analogous to HAV RNA (manufacturers & associated control laboratories) Vaccine studies, monitoring viral loads Development of assays EQA providers, HEV serology already in place

Candidate Materials Viraemic plasma of genotypes 3 or 4 Bile from swine; high titre genotype 3 HEV samples, work well in several different conventional and real-time PCR assays targeting conserved regions of the HEV genome Viral loads in two particular samples are > 9 log10 copies/ml bile and > 10 log10 copies per ml of bile Confirmed by 2 different qPCR assays, and end-point dilution (Thomas Gärtner, Octapharma)

Proposed Study Sally Baylis baysa@pei.de It is proposed to perform an initial study using dilutional panels, of blinded samples Determine the most suitable virus strain for further development as a standard Provide information on assay performance Laboratories wishing to participate in this study should contact: Sally Baylis baysa@pei.de