DETECTION AND TYPING OF THE ZOONOTIC HEV - Istituto Superiore di Sanità Ilaria Di Bartolo - VISAVET
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DETECTION AND TYPING OF THE
ZOONOTIC HEV
Ilaria Di Bartolo
Istituto Superiore di Sanità
Department of Food Safety, Nutrition and Veterinary Public Health
HEV GENOME
RNA quasi-envelope, ss+RNA
Capsid 27-34 nm
5’ UTR 3’
AAAAA
ORF1: non structural protein; ORF2: capsid protein; ORF3: multifunctional protein
PATHOGENESIS IN PIGS
The incubation period varies from 3 to 8 weeks
Duration of viremia (as well as shedding in feces) is variable
The virus can be shed at high level in feces and bile (liver)
HEV shedded in feces for 7 to 8 weeks
Modified by https://www.pig333.com/public-health/
DETECTION OF HEV
• The techniques aimed at detecting different things
• serological evidence of prior infection (IgM)
• detection of virions (ELISA detects antigen) Diagnosis
• detection of virus nucleic acids
• detection of virus infectivity
Cell culture
Animal model (living animals)
ANIMAL MODEL
• Cynomolgus and rhesus monkeys HEV1 to 4. Model for infection in
humans
Genotype Origin of strain Infection
HEV3 and HEV4 pigs +
Human +
HEV ra rabbit +
HEV rat -
Food Thermal treat
HEV3 Pig liver 56°C for 1 hr + Feagins et al. 2008
HEV3 Patè (liver+fat+spices) 71°C for 5min + Barnaud et al. 2012
71°C for 20min -
NOVEL ANIMAL MODELS • Animal model not only to evaluate infectivity of HEV but also to study replicative cycle and pathogenesis…. • Infections (evaluated by HEV RNA in the stool and by viremia) with human (HEV3f) and chimpanzee (HEV1, Sar-55) stool suspensions succeeded (Allweiss et al., 2016; Geldof et al., 2016)
DETECTION OF HEV • The techniques can be arranged in four categories: • detection of virions (EM; ELISA; western blotting) • detection of virus nucleic acids • detection of virus infectivity • Cell culture • Animal model (living animals)
CELL CULTURE
First paper on cell cultivation Huang et al; 1992; 1995
human feces HEV4 A549 (human lung carcinoma cells) (Wei et al., 2000)
human feces HEV3 human hepatoma cell lines (PLC/PRF/5, A549) (Tanaka et al., 2007)
rabbit liver homogenate HEV3 ra human hepatoma cell lines (PLC/PRF/5, A549) (Jirintai et al., 2012)
12 swine and boar liver, feces, or HEV A549 and PLC/PRF/5 cells (Takahashi et al., 2012)
serum
swine feces HEV3 porcine stem cell line (PICM-19) porcine (Rogee et al., 2013;
hepatoma cell lines (HepaRG,) Talbot et al., 2013)
swine feces HEV3 primary cultured human hepatocytes (PHCs) (Oshiro et al., 2014)
HEV4
ISSUES
• initial inoculum (high viral load or MOI dependent)
• strain dependent (?)
• origin of inoculum (liver, feces)
• time of growing
• final titer
• visualization of growing (replication of HEV did not
cause any cytopathic changes in the cell line)
Okamoto et al., 2011 HEV3 humans
CELL CULTURE IS THE MOST PROMISING TEST
TO EVALUATE INFECTIVITY (FOOD)DETECTION OF HEV
• The techniques aimed at detecting different things
• serological evidence of prior infection (IgM)
• detection of virions (EM; ELISA detects antigen)
• detection of virus nucleic acids
• detection of virus infectivity
Cell culture
Animal model (living animals)DETECTION OF VIRAL NUCLEIC ACID
Humans: from plasma or faeces
Animals: liver, faeces, blood, bile
HEV genome
detection
RNA extractionRNA EXTRACTION • Food of animal and non animal origin No ISO available for RNA extraction from food either AO or NAO. Berries and shellfish (ISO/TS 15216-1:2013) e.g. Mesquita et al., 2016 >10%
RNA EXTRACTION FROM FOOD • Starting materials (30-100mg liver; 5gr meat, salami, 2 gr for liver sausages (Szabo et al., 2015; Moor et al., 2018 ) • Sample preparation: homogenize samples by Stomacher, Tissue lyser (zyrconia beads); lysis buffer (Vital project; Di Bartolo et al., 2012; Rose at al., 2011), water (Martin-Latil et al., 2014) or Trizol (Szabo et al., 2015) additional step with PEG (Martin-Latil et al., 2014) • Magnetic beads with silica • LOD 5.3 x 104 GE / 2 g; 1.56x 103 GE / g (liver sausage); • 2.9 x 10³ GE / 5 g (salami), 1.56x 102 GE / g (raw meat sausage) (Szabo et al., 2015; Moor et al., 2017)
HEV RNA DETECTION • Real-time RT-PCR protocol by Jothikumar et al., 2006 Cited 304 • HEV1-4 • TaqMan® chemistry (4 copies for reaction) • Performed using one or two steps (c-DNA followed by Real-time PCR), different kits, including IC • WHO international standard for HEV RNA Quantification in GE does not correspond to viable virus (1 up to 4 log difference with virus units)
RNA detection
RNA extraction End-point RT-PCR
Phylogenetic analyses Sequencing
Subtype
Strain identificationTYPING OF HEV
Only 1 serotype has been described
Typing by molecular method: sequencing and comparisons
From Ricci et al., 2017 EFSA
Opinion on the public
health risks
associated with hepatitis E
virus (HEV) as a food-borne
pathogen. EFSA Journal
2017;15(7):4886,
Full genome alignment and p-distance calculationFULL GENOME SEQUENCING
• NGS Sample preparation: removal of bacteria; treatment with
Benzonase (removal of RNA and DNA);
Sequence-Independent, Single-Primer Amplification (SISPA)
Ion Torrent PGM
Matrices Subtype GE Reads HEV contings
Wild boar liver HEV3i 4.4 x 106 1,596,275 Full genome
Wild boar liver HEV3NA 1.7 x 108 1,243,513 6,900
Swine feces L75 HEV3l 2.8 × 105 1,206,199 2 contigs in ORF1 and 2 in ORF2 (300-900 bp)
Swine feces R13 HEV3l 7.1 × 105 4,884,086 No contig
Different approach: use of specific HEV-primers (limit the use of PCR amplification)ORTHOHEPEVIRUS A
• Orthohepevirus A: 7 genotypes (8 recently
proposed)
• p-distance threshold between genotypes of
0.088 for amino acid distances of
concatenated ORF1 and ORF2 (lacking
hypervariable regions) (Smith et al., 2014)
Lisa J. Krain et al. 2014TYPING OF ZOONOTIC HEV
a
b
c
h
• Genotypes are classified in subtypes (letters)
• Smith et al., 2016: set of reference sequences for each subtype
• HEV4: 10 reference strains; 9 subtypes and 1 strain not assigned
• HEV3: 20 reference strains; 2 major clades with six subtypes (3a, e
f
3b, 3c, 3h, 3i and 3j) and with the three subtypes (3e-3g). Two g
recently proposed novel subtypes
• HEVra not assigned, separate clade
Smith et al., 2016NOVEL SUBTYPE
FR-SHEV3c-like strain
• No threshold established for subtype
• By Smith et al. 2016, if at least three complete genome
sequences not related to any of the subtypes described and
that were epidemiologically unrelated (strains from different
studies or localities)NOVEL SUBTYPE
• 2 Italian swine HEV3 not epidemiologically correlated
• Full genome sequences
• Alignment using the reference set
The two Italian strains cluster together with the FR-SHEV3c-like strain
(JQ953664) (not assigned)
p-distance value of 0.129 with
the major clade represented by -3c, -3i, -3h prototype strains and some
not classified strains
Proposed novel subtype HEV3-l(De Sabato et al., 2018)NOVEL SUBTYPE
WHAT IS THE MEANING OF SUBTYPES? • silent mutations • some subtypes are more frequently observed • geographical distribution (pigs movement) Pigs experimental infections with subtypes 3c, 3e, and 3f (Rogee et al., 2015) the replication efficacy of the three different strains was similar 61 proteins differentially expressed during hepatitis E virus infection.
• The sequences databases: web-based typing platform • strains from humans animals, food and environment
GRAZIE! Dr. Luca De Sabato Dr.ssa Marina Monini Dr. Giovanni Ianiro Edoardo Vignolo Dr. Gabriele Vaccari Dr. Franco Maria Ruggeri Dr.ssa Giorgia Angeloni Dr.ssa Eleonora Ponterio University of Bologna Prof. Fabio Ostanello
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