- Research article
- Open Access
Detection of serum antibodies to hepatitis E virus in domestic pigs in Italy using a recombinant swine HEV capsid protein
BMC Veterinary Research volume 10, Article number: 133 (2014)
The hepatitis E virus (HEV) has been detected in both humans and animals, particularly pigs, worldwide. Several evidences, including human infection following consumption of raw contaminated meat, suggest a zoonotic transmission of HEV. In Italy, large circulation of genotype 3 HEV has been reported in swine, and recent studies have confirmed the involvement of this genotype in autochthonous human cases.
In this study 111 sera collected from healthy pigs in two Italian regions were tested for anti-HEV IgG antibodies. For specific HEV antibody detection in swine, we developed ELISA and Western blotting methods, using a truncated capsid (ORF2) protein lacking the first 111 amino acids of a swine HEV genotype 3 strain. The ORF2-based ELISA revealed anti-HEV antibodies in 104 out of 111 pigs compared with 102 detected with a commercial ELISA kit. A lower number of sera reacted with the recombinant ORF2 protein in a Western blotting format (81/111). Using a Latent class analysis (LCA), the estimated sensitivities for ELISA-ORF2 and ELISA-kit tests were 0.961 and 0.936, respectively, whereas specificities were 0.599 and 0.475. The estimated sensitivity of Western blotting was 0.775, and the specificity was 0.944.
The overall results confirm the high prevalence of HEV seropositive healthy pigs in Italy. Through comparisons with a commercial ELISA test, the swine genotype 3 HEV antigen produced in this study was proven suitable to detect anti-HEV antibodies in pig sera by both ELISA and Western Blotting.
Hepatitis E virus (HEV) has long been recognized as an endemic pathogen in developing countries, involved in large waterborne outbreaks. An increasing number of autochthonous cases of hepatitis E have been recently reported also in industrialized areas [1–4].
HEV is a small non enveloped RNA virus, belonging to the Hepeviridae. The genome is a single-stranded RNA of approximately 7.3 kb, containing three open reading frames (ORFs). ORF1 encodes non-structural proteins, ORF2 the viral capsid protein, and ORF3 a cytoskeleton-associated phosphoprotein . Mammalian HEV strains have been classified into four genotypes designated genotype 1 through 4 . More recently, new and genetically distant viral strains have been detected in rats , ferrets , foxes  and bats , but the current HEV classification has not been modified yet. Up to date, despite a further classification of genotypes in subtypes, a single serotype has been confirmed .
The first HEV strains infecting animals were identified in swine , and later in humans , and were classified as genotype 3. This HEV genotype is now recognized as the most common one in both humans and swine in industrialized countries [2, 3, 14–16]. Several evidences support the zoonotic transmission of HEV from domestic pigs, wild boar, and deer. Furthermore, the strict correlation between animal and human strains from the same geographical areas and the numerous reports of hepatitis E cases in humans correlated with consumption of undercooked or raw meat from deer, wild boar and pig contaminated with HEV support the zoonotic transmission [4, 17–20].
Usually HEV viremia and shedding have short duration in man [21–23], whereas HEV-specific serum IgG are detectable for years. The same is thought to occur in animals, and determination of HEV-specific serum antibodies can help assess the extent of past exposure to HEV in both individual herds and/or the overall animal population in a country . Serological HEV studies on swine are normally conducted using commercial kits based on human HEV antigens, but use of swine virus antigens was proposed to increase testing sensitivity [24, 25].
Several studies have been conducted in swine yielding different HEV seroprevalence rates, but none of the tests used was fully validated due to the absence of proper “gold standards” [24–26].
In this study, we implemented a reliable ELISA test for detection of anti-HEV antibodies in swine sera, using a genotype 3 swine HEV capsid protein expressed by a recombinant baculovirus in insect cells as coating antigen. We assessed the in house ELISA by a Latent class analysis (LCA), which permits test validation in the absence of gold standards . The test was employed to evaluate the presence of HEV antibodies in 111 swine sera collected from different farms (No. 65) and a slaughterhouse (No. 46) in Italy. Results obtained with the ELISA based on recombinant swine HEV genotype 3 rΔ111ORF2 ORF2 capsid protein (ELISA-ORF2) were compared with a Western blotting (WB) test using the same antigen and with a commercial ELISA kit.
Expression of swine HEV genotype 3 rΔ111ORF2 capsid protein in Sf9 cells
Sf9 cells infected with the baculovirus BacHEVΔ111ORF2 expressed large amounts of the recombinant rΔ111ORF2 protein cloned from a genotype 3 HEV strain, as confirmed by SDS-PAGE (Figure 1A) and Western blotting (Figure 1B). The rΔ111ORF2 protein was recognized by an anti-HEV hyperimmune swine serum (kindly provided by Nicole Pavio, ANSES-ENVA-INRA, Maisons-Alfort, France) , but not by an SPF swine serum (not shown). The rΔ111ORF2 protein purified from Sf9 lysates showed a size of 55 kDa, corresponding to the capsid protein lacking the first 111 amino acids.Electron microscopic examination of ultracentrifuged infected cell lysates (not shown) revealed that the rΔ111ORF2 protein did not self-assemble into virus-like particles (VLP). For this reason, rΔ111ORF2 was concentrated and purified by ion-exchange chromatography (see Methods) (Figure 1C). Despite the lack of VLP formation, concentrated rΔ111ORF2 elicited a specific immune response in Balb/c mice, which showed serum antibody titers of approximately 1:1000 by both ELISA-ORF2 and Western blotting (not shown). These mouse sera were used as additional positive controls throughout the study.
Seroprevalence of swine HEV antibodies
One hundred and four out of 111 (93.7%) swine sera were positive by the ELISA-ORF2 test, whereas 102 sera were found to be positive by the commercial ELISA (91.9%) (Table 1). Forty of the 46 pig sera taken at slaughterhouse had been found positive previously  by the commercial ELISA kit, and were again confirmed positive by the ELISA-ORF2 in this study despite long storage at −20°C.
All swine sera were also analyzed by Western blotting using the rΔ111ORF2 protein. Eighty-one sera (73%) reacted with the rΔ111ORF2 protein specifically (part of the 81 sera are shown in Figure 2), as did the swine positive control serum. Seventy-five Western blotting positive sera were also positive by both ELISA tests, whereas four sera tested positive only at ELISA-ORF2, one at ELISA-kit, and one resulted negative at both assays (Table 1). Overall, slaughtering animals showed a seroprevalence of 86.9% (40 seropositive/46 tested), whereas all 65 farmed swine were seropositive (seroprevalence 100%).
The mean S/CO ratios between the OD values obtained for swine serum samples and the Cut-Off for each of the ELISA tests were also calculated grouping samples according to the positivity by one or both ELISA tests and WB (Table 1). Although it was overall higher for ELISA- and WB-positive samples, a statistically significant difference (p < 0.01) of the S/CO ratio was shown for the ELISA-ORF2 test among samples that were positive by all three assays.
Validity of the ELISA-ORF2 diagnostic test
The expected and observed frequencies of HEV-positive swine sera by diagnostic test, and the probability of belonging to latent class HEV infection predicted by this model are reported in Table 1. The estimated mean sensitivities for ELISA-ORF2 and ELISA-kit tests were 0.961 (95% CI: 0.911-0.992) and 0.936 (95% CI: 0.880-0.976), respectively. The estimated mean specificities of ELISA-ORF2 and ELISA-kit tests were 0.599 (95% CI: 0.173-0.997) and 0.475 (95% CI: 0.112-0.918), respectively. The estimated sensitivity of Western blotting was 0.775 (95%CI: 0.676-0.892), and the specificity was 0.944 (95% CI: 0.886-0.979). The mean estimate of the posterior probability distribution of the unbiased proportion of seropositive swine was 0.921 (95% CI: 0.797-0.985). Credibility intervals (CI) were relatively large because of the greater uncertainty related to non-informative priors. However, the sensitivity was similar for both ELISA tests.
Highly sensitive molecular techniques for viral genome identification have helped increase the detection rate of HEV in swine, but the short duration of virus shedding in feces represents a major limitation when investigating the prevalence of HEV infection in farms. Serological diagnosis by immunoassays can represent a valid screening method, because HEV-specific antibodies remain detectable much longer than viral RNA. Although several HEV genotypes have been described, only one viral serotype is acknowledged to date. Tests developed for screening human serum antibodies have consequently been adapted to investigate also the swine by replacing the secondary antibodies with an anti-pig Ig serum for successful detection of specific swine antibody [28, 29]. Despite the cross-reaction between genotypes, immunoassays based on swine HEV antigens have been recently suggested to provide higher sensitivity for swine serum antibody detection than those developed on human HEV strains [25, 28].
The expression of a N-terminal truncated form of the ORF2 capsid protein lacking the first 111 amino acids was previously shown to favor its self-assembly in VLPs for both a genotype 1 HEV strain  and a rat HEV , suggesting that post translational processing is required for proper protein refolding . Other authors  have reported VLP production also from swine genotype 3 ORF2, using a similar approach. Conversely, the truncated form of the swine genotype 3 capsid protein expressed in this study did not self-assemble into virus-like particles, despite being expressed in large amount. The reasons for the failure in generating VLPs in this study are not clear, and might involve different assembly or post-translational modifications of the capsid protein between different genotypes or strains of human and/or swine HEV. In fact, the capsid proteins of human genotype 1  and swine genotype 3 (this study) HEV shared 94.5% amino acid identity.
Nonetheless, the unassembled swine HEV protein was recognized both efficiently and specifically by a reference swine serum raised against the naïve virus , and was highly immunogenic in mice, suggesting some extent of folding and conservation of its antigenic structure. In a previous study, a recombinant genotype 1 HEV peptide (HEV 239; Hecolin; Xiamen Innovax Biotech, Xiamen, China) was found to occur in solution as large aggregates rather than true VLPs , but it was highly immunogenic. In this form, HEV 239 has been used as an efficacious recombinant hepatitis E vaccine for human use .
The suitability of the rΔ111ORF2 protein for testing HEV antibody in swine sera using ELISA was confirmed by comparing by LCA the in-house method with a commercial ELISA-kit test, produced for human use and present in the market since several years. Sensitivity of the commercial assay is expected to be high, since antigens of different species and strains are known to cross-react largely, in line with the knowledge of a single serotype among all HEV genotypes [23, 34, 35].
By the LC model adopted, ELISA-ORF2 and ELISA-kit presented sensitivities of 0.961 and 0.936, and specificities of 0.599 and 0.475, respectively. The low specificity value for both ELISAs is due to the assumption that the immune-stained protein band in the Western blotting assay is 100% specific, but does not consider that Western blotting can yield false-negative results. Therefore, the ORF2 band absence in Western blotting using sera that reacted in ELISA does not imply a corresponding false-positive result of ELISA. The ELISA-kit flyers indicate that specificity varies between 92.7 and 100% with human sera [36–38], even if some tests can cross-react with Epstein-Barr virus (EBV) and cytomegalovirus (CMV) antibodies, determining false positive results . Therefore, it is hard to believe that the ELISA-kit would perform very poorly in terms of specificity only when adapted to detection of antibodies from a different species, such as the swine.
In addition, the normalized ratios between the OD values determined for test sera and the Cut-Off ODs for the ELISA tests used (S/CO value) were higher for swine sera that also proved positive at WB analysis. Although a statistically different S/CO was confirmed only in the case of ELISA-ORF2 for the more numerous group of samples that were positive at all three tests, these results overall suggest that positivity at WB is correlated with higher ELISA OD values, which may imply a higher serum antibody titer.
In this study, we have determined a mean anti-HEV seroprevalence of up to 93.7% by ELISA. No differences were observed depending on the geographical origin of farmed swine, whereas a lower seroprevalence of anti-HEV IgG in pigs from slaughterhouse was observed. This might be due to a decline of infection rate and/or immunity among the older animals in this stage of pork production . In fact, most of domestic pigs get infected at 2–3 months of age , and even if animals are in contact with the virus throughout their life span they could be seronegative at slaughtering age . Furthermore, 25 of 40 (62.5%) HEV-seropositive pigs collected at slaughterhouse were found to be HEV infected by HEV genome testing in bile, liver and/or feces . This result confirms that swine remains susceptible to HEV infection at any age, even at slaughter, that in Italy normally involves animals of 9 months of age and more.
Overall the results presented in this paper confirm that the swine HEV rΔ111ORF2 may be suitably applied to large seroprevalence studies in pig herds using an ELISA format. We found the in house assay to be at least as sensitive as the commercial ELISA kit including a human genotype viral antigen. These data support further that the human and swine HEV strains belonging to different genotypes are highly cross-reactive, if not even identical, in their antigenic determinants. Finally, the use of over one-hundred swine sera for diagnostic assays comparisons in this study permitted to determine a mean anti-HEV seroprevalence of up to 93.7% among pigs which are part of the Italian food chain, confirming the high HEV circulation among Italian farmed pigs previously reported in Italy and other countries [16, 18, 24, 25, 28].
Generation of recombinant baculovirus and expression of HEV capsid protein
Total RNA was extracted from the HEV-positive pig bile sample SwHEV/BO85/06 collected in Northern Italy, using the Qiamp Viral RNA Extraction kit (Qiagen). The cDNA corresponding to HEV ORF2 was obtained using oligo(dT)20 primer and SuperScript™III reverse transcriptase (First-Strand SuperScript™III Synthesis System, Life Technologies), following the manufacturer’s instructions. Amplification of an ORF2 fragment lacking the first 333 nucleotides was performed by PCR with primers designed on an Italian swine HEV ORF2 available in [GenBank NCBI: GU117636], denominated F∆111HEV (5′-GCTCTAGAGCATGGCCGTATCACCGGCTCCCGATACA GCC-3′), flanked by XbaI restriction site (italics), and RHEV (5′-GACTCGAGA TTAAGACTC CCGGGTTTTACC-3′), flanked by XhoI site (italics) and annealing in the ORF2 3′-terminal portion (stop codon indicated in bold). A methionine codon (in bold in F∆111HEV primer) was introduced into the forward primer F∆111HEV between XbaI cleavage site and the alanine codon at position 112. The 1652 bp DNA fragment obtained, flanked by XbaI (italics) and XhoI sites, was ligated into the pFastBac™1 (Life Technologies) donor vector. The construct obtained, which was named pFast::HEVΔ111ORF2, was transformed in the E. coli DH10Bac™ host strain containing a Baculovirus shuttle vector (bacmid) and a helper plasmid. The recombinant baculovirus DNA BacHEVΔ111ORF2 was generated by transposition in the E. coli DH10Bac host, and the resulting bacmid was purified and transfected into Sf9 cells using Cellfectin-II (Life Technologies) to produce infectious recombinant baculovirus. Based on comparison of ORF2 sequence with HEV reference strains available on GenBank, the identity of the HEV swine strain as genotype 3 subtype e, and its correct reading frame was confirmed [GenBank NCBI: GU556929].
Antigen preparation and purification
Sf9 cell monolayers grown in Sf900 medium (Life Technologies) in ten T75 cm2 flasks were infected with BacHEVΔ111ORF2 baculovirus. When a diffuse cytopathic effect was observed, cell cultures were lysed by three cycles of freezing and thawing. The recombinant protein rΔ111ORF2 was partially purified from the supernatant by anion exchange chromatography using a Macro-Prep Hydroxyapatite column (Bio-Rad), following the manufacturer’s instructions. A 55 kDa protein corresponding to the 111-aa deletion fragment of the capsid protein was produced, and analyzed by SDS-PAGE.
Immunization of Balb/c mice with recombinant HEV capsid protein
Animal work was conducted according to the Italian legislation enacting the EU directives (D.L. 116/92), following approval of the specific experimental protocols (mouse immunization and serum withdrawal, granted to Franco Maria Ruggeri) by the Biological and Animal Experimental Managing Service of the Istituto Superiore di Sanità and the legal authorization by the Italian Ministry of Health (Decree no. 97/2011 – B, 24th May 2011).
Three adult Balb/c mice (12 week-old) were immunized three times intraperitoneally at 3-week intervals with 15 μg of purified HEV capsid protein, in the presence of complete Freund’s adjuvant for the first inoculation, and incomplete adjuvant for the following immunizations.
A mouse inoculated with sterile saline solution was used as negative control. Fourteen days after the final boosting, sera were tested for anti-HEV antibodies by Western blotting, as described below . Mice were euthanized following isoflurane anesthesia, and serum was collected from the intracardiac clot.
Sixty-five sera were aliquots from a larger sample of sera previously analyzed and stored within the regional monitoring plan for African swine fever (ASF) and Classical swine fever (CSF) enforced in Sardinia, in compliance with the European Community requirements. Sera had been collected from clinically healthy pigs by staff veterinarians of the Italian Public Health System (ASL), according to Decrees No. 9, 16.05.2007, and No. 1567/decA/23, 14.07.2009, of the “Assessorato dell’Igiene e Sanità e dell’Assistenza Sociale, Regione Autonoma della Sardegna”, at five farms located in different areas in Sardinia . Additional 46 sera had been collected post-mortem from the intracardiac clot of slaughtered pigs at pork slaughterhouses in Northern Italy . Sera were stored at −20°C until use.
Purified rΔ111ORF2 was separated by SDS-PAGE, and either stained with Coomassie Brilliant Blue R-250 (Bio-Rad), or transferred to nitrocellulose membrane (Trans-blot transfer medium, Bio-Rad). After blocking with 5% skim milk in phosphate-buffered saline (PBS), the membrane was incubated with test (diluted 1:160) or control (1:1000) sera in PBS containing 0.05% Tween-20 and 1% skim milk, for 4 hours. Three sera from Specific Pathogen Free (SPF) pigs were used as negative control, while an experimentally infected swine serum  and a hyperimmune anti-HEVORF2 mouse serum were used as positive controls. Membranes were then incubated with alkaline phosphatase-conjugated anti-pig IgG (1:12000; SIGMA) or anti-mouse IgG (1:3000; Bio-Rad). Bands were visualized with 1-step NBT/BCIP solution (Pierce).
Recombinant ORF2-based ELISA procedure (ELISA-ORF2)
Polystyrene 96-well microplates (Maxisorp, Nunc) were coated with purified rΔ111ORF2 (0.01 μg/well). A control plate was coated with an Sf9 lysate containing an irrelevant protein (bovine norovirus capsid) . After 18 hours at 4°C, wells were washed 3 times with PBS containing 0.05% Tween-20 (PBS-T), and blocked with 5% skim milk in PBS at 37°C for 2 hours. After washings, plates were incubated with test sera diluted 1:20 in PBS containing 0.05% Tween-20 and 2% skim milk (PBS-T-milk), at 37°C for 90 min, in triplicate. Swine positive and control sera were used in all tests.
After washing with PBS-T, anti-pig (1:12000) and anti-mouse (1:3000) conjugates were added. Following 1 hour incubation at 37°C and washings, the reaction was developed with p-nitrophenol phosphate (SIGMA) in 10 mM diethanolamine, pH 10, for 1 hour at 37°C. Optical densities were measured at a wavelength of 405 nm (OD405). The Cut-Off value (COV) for the ELISA-ORF2 was established from internal controls for each test, using the commercial ELISA test formula: [mean OD of negative controls + 0.1]. Throughout this study, this value was 0.18.
Commercial ELISA test (ELISA-kit)
Swine sera (diluted 1:20) were also tested by the BioChain kit (http://www.biochain.com, USA), commercially available for anti-HEV IgG detection in human sera, with the following modifications: the secondary antibody was replaced with a horseradish peroxidase-labeled anti-swine IgG antibody (SIGMA), diluted 1:10000. Test sera were considered as positive if OD450 ≥ COV, as indicated by the kit manufacturer. Positive and negative swine control sera (see above) were included in all assays.
All reporting adheres to the NC3Rs ARRIVE guidelines (Animal Research: Reporting In Vivo Experiments) (Additional file 1).
A Bayesian approach was used to obtain estimates for the test accuracies of the three tests. The data were processed with Bayes Latent Class Models software (http://www.medicine.mcgill.ca/epidemiology/Joseph/Bayesian-Software-Diagnostic-Testing.html). Parameter estimates were based on analytical summaries of 10,000 iterations of the Gibbs sampler with a burn-in phase of 1000 iterations.
Sensitivity and specificity were estimated by the classic validation method as well as by Latent-class analysis modeling. In Latent class analysis (LCA), we started by fitting the basic two latent class model.
Beta distributions Be (a, b) were used as priors for the parameters of interest (sensitivities, and specificities). The same Beta prior distributions  were used for the sensitivities of both ELISA tests: <0.6; mode = 0.9 corresponding to Be (8.30, 1.81) priors. For both ELISA tests a uniform prior distribution for specificity Be (0, 1) was assumed.
Swine sera testing by HEV WB were considered 100% specific. The Beta prior distribution for WB specificity had mode = 0.9 and a 5th percentile of 0.9. For WB tests we assumed a uniform prior distribution for sensitivity Be (0, 1).
Sensitivity and specificity of tests were reported with an approximate 95% confidence interval (CI), as estimated by the model.
Garbuglia AR, Scognamiglio P, Petrosillo N, Mastroianni CM, Sordillo P, Gentile D, La Scala P, Girardi E, Capobianchi MR: Hepatitis E virus genotype 4 outbreak, Italy, 2011. Emerg Infect Dis. 2013, 19 (1): 110-114. 10.3201/eid1901.120983.
Dalton HR, Stableforth W, Thurairajah P, Hazeldine S, Remnarace R, Usama W, Farrington L, Hamad N, Sieberhagen C, Ellis V, Mitchell J, Hussaini SH, Banks M, Ijaz S, Bendall RP: Autochthonous hepatitis E in Southwest England: natural history, complications and seasonal variation, and hepatitis E virus IgG seroprevalence in blood donors, the elderly and patients with chronic liver disease. Eur J Gastroenterol Hepatol. 2008, 20 (8): 784-790. 10.1097/MEG.0b013e3282f5195a.
Ijaz S, Vyse AJ, Morgan D, Pebody RG, Tedder RS, Brown D: Indigenous hepatitis E virus infection in England: more common than it seems. J Clin Virol. 2009, 44 (4): 272-276. 10.1016/j.jcv.2009.01.005.
Colson P, Romanet P, Moal V, Borentain P, Purgus R, Benezech A, Motte A, Gerolami R: Autochthonous infections with hepatitis E virus genotype 4, France. Emerg Health Serv Rev. 2012, 18 (8): 1361-1364.
Emerson SU AA, Arankalle XJ, Meng XJ, Purdy M, Schlauder GG, Tsarev SA: Hepevirus. Virus Taxonomy, VIIth Report of the ICTV. Edited by: CM Fauquet MM, Maniloff J, Desselberg U, Ball LA. 2004, London: Elsevier/Academic Press, 851-855.
Yamada K, Takahashi M, Hoshino Y, Takahashi H, Ichiyama K, Nagashima S, Tanaka T, Okamoto H: ORF3 protein of hepatitis E virus is essential for virion release from infected cells. J Gen Virol. 2009, 90 (Pt 8): 1880-1891.
Meng XJ: Zoonotic and foodborne transmission of hepatitis E virus. Semin Liver Dis. 2013, 33 (1): 41-49.
Johne R, Heckel G, Plenge-Bonig A, Kindler E, Maresch C, Reetz J, Schielke A, Ulrich RG: Novel hepatitis E virus genotype in Norway rats, Germany. Emerg Infect Dis. 2010, 16 (9): 1452-1455. 10.3201/eid1609.100444.
Raj VS, Smits SL, Pas SD, Provacia LB, Moorman-Roest H, Osterhaus AD, Haagmans BL: Novel hepatitis E virus in ferrets, the Netherlands. Emerg Infect Dis. 2012, 18 (8): 1369-1370. 10.3201/eid1808.111659.
Bodewes R, van der Giessen J, Haagmans BL, Osterhaus AD, Smits SL: Identification of multiple novel viruses, including a parvovirus and a hepevirus, in feces of red foxes. J Virol. 2013, 87 (13): 7758-7764. 10.1128/JVI.00568-13.
Drexler JF, Seelen A, Corman VM, Fumie Tateno A, Cottontail V, Melim Zerbinati R, Gloza-Rausch F, Klose SM, Adu-Sarkodie Y, Oppong SK, Kalko EK, Osterman A, Rasche A, Adam A, Müller MA, Ulrich RG, Leroy EM, Lukashev AN, Drosten C: Bats worldwide carry hepatitis E virus-related viruses that form a putative novel genus within the family Hepeviridae. J Virol. 2012, 86 (17): 9134-9147. 10.1128/JVI.00800-12.
Meng XJ, Purcell RH, Halbur PG, Lehman JR, Webb DM, Tsareva TS, Haynes JS, Thacker BJ, Emerson SU: A novel virus in swine is closely related to the human hepatitis E virus. Proc Natl Acad Sci U S A. 1997, 94 (18): 9860-9865. 10.1073/pnas.94.18.9860.
Schlauder GG, Dawson GJ, Erker JC, Kwo PY, Knigge MF, Smalley DL, Rosenblatt JE, Desai SM, Mushahwar IK: The sequence and phylogenetic analysis of a novel hepatitis E virus isolated from a patient with acute hepatitis reported in the United States. J Gen Virol. 1998, 79 (Pt 3): 447-456.
Huang FF, Haqshenas G, Guenette DK, Halbur PG, Schommer SK, Pierson FW, Toth TE, Meng XJ: Detection by reverse transcription-PCR and genetic characterization of field isolates of swine hepatitis E virus from pigs in different geographic regions of the United States. J Clin Microbiol. 2002, 40 (4): 1326-1332. 10.1128/JCM.40.4.1326-1332.2002.
Mansuy JM, Peron JM, Abravanel F, Poirson H, Dubois M, Miedouge M, Vischi F, Alric L, Vinel JP, Izopet J: Hepatitis E in the south west of France in individuals who have never visited an endemic area. J Med Virol. 2004, 74 (3): 419-424. 10.1002/jmv.20206.
Di Bartolo I, Ponterio E, Castellini L, Ostanello F, Ruggeri FM: Viral and antibody HEV prevalence in swine at slaughterhouse in Italy. Vet Microbiol. 2011, 149 (3–4): 330-338.
Colson P, Borentain P, Queyriaux B, Kaba M, Moal V, Gallian P, Heyries L, Raoult D, Gerolami R: Pig liver sausage as a source of hepatitis E virus transmission to humans. J Infect Dis. 2010, 202 (6): 825-834. 10.1086/655898.
Di Bartolo I, Diez-Valcarce M, Vasickova P, Kralik P, Hernandez M, Angeloni G, Ostanello F, Bouwknegt M, Rodriguez-Lazaro D, Pavlik I, Ruggeri FM: Hepatitis E virus in pork production chain in Czech Republic, Italy, and Spain, 2010. Emerg Infect Dis. 2012, 18 (8): 1282-1289.
Feagins AR, Opriessnig T, Guenette DK, Halbur PG, Meng XJ: Detection and characterization of infectious Hepatitis E virus from commercial pig livers sold in local grocery stores in the USA. J Gen Virol. 2007, 88 (Pt 3): 912-917.
Choi JY, Lee JM, Jo YW, Min HJ, Kim HJ, Jung WT, Lee OJ, Yun H, Yoon YS: Genotype-4 hepatitis E in a human after ingesting roe deer meat in South Korea. Clin Mol Hepatol. 2013, 19 (3): 309-314. 10.3350/cmh.2013.19.3.309.
Kanai Y, Tsujikawa M, Yunoki M, Nishiyama S, Ikuta K, Hagiwara K: Long-term shedding of hepatitis E virus in the feces of pigs infected naturally, born to sows with and without maternal antibodies. J Med Virol. 2010, 82 (1): 69-76. 10.1002/jmv.21647.
Panda SK, Thakral D, Rehman S: Hepatitis E virus. Rev Med Virol. 2007, 17 (3): 151-180. 10.1002/rmv.522.
Satou K, Nishiura H: Transmission dynamics of hepatitis E among swine: potential impact upon human infection. BMC Vet Res. 2007, 3: 9-10.1186/1746-6148-3-9.
Peralta B, Casas M, de Deus N, Martin M, Ortuno A, Perez-Martin E, Pina S, Mateu E: Anti-HEV antibodies in domestic animal species and rodents from Spain using a genotype 3-based ELISA. Vet Microbiol. 2009, 137 (1–2): 66-73.
Rose N, Boutrouille A, Fablet C, Madec F, Eloit M, Pavio N: The use of Bayesian methods for evaluating the performance of a virus-like particles-based ELISA for serology of hepatitis E virus infection in swine. J Virol Methods. 2010, 163 (2): 329-335. 10.1016/j.jviromet.2009.10.019.
Casas M, Pujols J, Rosell R, de Deus N, Peralta B, Pina S, Casal J, Martin M: Retrospective serological study on hepatitis E infection in pigs from 1985 to 1997 in Spain. Vet Microbiol. 2009, 135 (3–4): 248-252.
Hadgu A, Qu Y: A biomedical appication of latent class models with random effects. Appl Stat. 1998, 47: 603-616.
Jimenez de Oya N, Galindo I, Girones O, Duizer E, Escribano JM, Saiz JC: Serological immunoassay for detection of hepatitis E virus on the basis of genotype 3 open reading frame 2 recombinant proteins produced in Trichoplusia ni larvae. J Clin Microbiol. 2009, 47 (10): 3276-3282. 10.1128/JCM.00750-09.
Engle RE, Yu C, Emerson SU, Meng XJ, Purcell RH: Hepatitis E virus (HEV) capsid antigens derived from viruses of human and swine origin are equally efficient for detecting anti-HEV by enzyme immunoassay. J Clin Microbiol. 2002, 40 (12): 4576-4580. 10.1128/JCM.40.12.4576-4580.2002.
Li TC, Yamakawa Y, Suzuki K, Tatsumi M, Razak MA, Uchida T, Takeda N, Miyamura T: Expression and self-assembly of empty virus-like particles of hepatitis E virus. J Virol. 1997, 71 (10): 7207-7213.
Li TC, Yoshimatsu K, Yasuda SP, Arikawa J, Koma T, Kataoka M, Ami Y, Suzaki Y, le Mai TQ, Hoa NT, Yamashiro T, Hasebe F, Takeda N, Wakita T: Characterization of self-assembled virus-like particles of rat hepatitis E virus generated by recombinant baculoviruses. J Gen Virol. 2011, 92 (Pt 12): 2830-2837.
Li SW, Zhang J, Li YM, Ou SH, Huang GY, He ZQ, Ge SX, Xian YL, Pang SQ, Ng MH, Xia NS: A bacterially expressed particulate hepatitis E vaccine: antigenicity, immunogenicity and protectivity on primates. Vaccine. 2005, 23 (22): 2893-2901. 10.1016/j.vaccine.2004.11.064.
Zhu FC, Zhang J, Zhang XF, Zhou C, Wang ZZ, Huang SJ, Wang H, Yang CL, Jiang HM, Cai JP, Wang YJ, Ai X, Hu YM, Tang Q, Yao X, Yan Q, Xian YL, Wu T, Li YM, Miao J, Ng MH, Shih JW, Xia NS: Efficacy and safety of a recombinant hepatitis E vaccine in healthy adults: a large-scale, randomised, double-blind placebo-controlled, phase 3 trial. Lancet. 2010, 376 (9744): 895-902. 10.1016/S0140-6736(10)61030-6.
Meng J, Dai X, Chang JC, Lopareva E, Pillot J, Fields HA, Khudyakov YE: Identification and characterization of the neutralization epitope(s) of the hepatitis E virus. Virology. 2001, 288 (2): 203-211. 10.1006/viro.2001.1093.
Lee WJ, Shin MK, Cha SB, Yoo HS: Development of a novel enzyme-linked immunosorbent assay to detect Anti-IgG against swine hepatitis E virus. J Vet Sci. 2013, 14 (4): 467-472. 10.4142/jvs.2013.14.4.467.
Chaussade H, Rigaud E, Allix A, Carpentier A, Touze A, Delzescaux D, Choutet P, Garcia-Bonnet N, Coursaget P: Hepatitis E virus seroprevalence and risk factors for individuals in working contact with animals. J Clin Virol. 2013, 58 (3): 504-508. 10.1016/j.jcv.2013.08.030.
Shenyang G, Dandan L, Chen F, Shuliang W, Tiezhong Z: Design and evaluation of a recombinant multi-epitope-based ELISA for the serological surveillance of HEV infection in northern China. Arch Virol. 2011, 156 (9): 1621-1626. 10.1007/s00705-011-1007-y.
Herremans M, Bakker J, Duizer E, Vennema H, Koopmans MP: Use of serological assays for diagnosis of hepatitis E virus genotype 1 and 3 infections in a setting of low endemicity. Clin Vaccine Immunol. 2007, 14 (5): 562-568. 10.1128/CVI.00231-06.
Fogeda M, de Ory F, Avellon A, Echevarria JM: Differential diagnosis of hepatitis E virus, cytomegalovirus and Epstein-Barr virus infection in patients with suspected hepatitis E. J Clin Virol. 2009, 45 (3): 259-261. 10.1016/j.jcv.2009.05.022.
Meng XJ: Hepatitis E virus: animal reservoirs and zoonotic risk. Vet Microbiol. 2010, 140 (3–4): 256-265.
Casas M, Cortes R, Pina S, Peralta B, Allepuz A, Cortey M, Casal J, Martin M: Longitudinal study of hepatitis E virus infection in Spanish farrow-to-finish swine herds. Vet Microbiol. 2011, 148 (1): 27-34. 10.1016/j.vetmic.2010.08.010.
Masia G, Orru G, Liciardi M, Desogus G, Coppola RC, Murru V, Argiolas M, Orru G: Evidence of hepatitis E virus (HEV) infection in human and pigs in Sardinia, Italy. J Prev Med Hyg. 2009, 50 (4): 227-231.
Zakhour M, Maalouf H, Di Bartolo I, Haugarreau L, Le Guyader FS, Ruvoen-Clouet N, Le Saux JC, Ruggeri FM, Pommepuy M, Le Pendu J: Bovine norovirus: carbohydrate ligand, environmental contamination, and potential cross-species transmission via oysters. Appl Environ Microbiol. 2010, 76 (19): 6404-6411. 10.1128/AEM.00671-10.
This research was supported by grants: European Commission Framework Program 7 projects “Integrated monitoring and control of foodborne viruses in European food supply chains (VITAL)” (Grant No. KBBE 213178), led by the coordination team of Nigel Cook (FERA, UK), Martin D’Agostino (FERA, UK), and Franco Maria Ruggeri (ISS, Italy), and Preparedness, Prediction and Prevention of Emerging Zoonotic Viruses with Pandemic Potential using Multidisciplinary Approaches (PREDEMICS)” (Grant No. 278433).
All authors read and approved the final manuscript, and agreed with the conclusions of the work. The authors declare that they have no competing interests.
EP participated to cloning and to production of protein, conducted serological testing, and contributed to data analysis and drafted the first version of the manuscript. IDB participated to cloning, supervised laboratory testing, and contributed to data analysis and participated to final draft preparation. GO and ML organized and performed animal sampling and sera collection. FO performed statistical analysis, and participated to final draft preparation. FMR supervised the activities, and reviewed the final manuscript. All authors read and approved the final manuscript.
Electronic supplementary material
Authors’ original submitted files for images
Below are the links to the authors’ original submitted files for images.
Rights and permissions
Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( https://creativecommons.org/publicdomain/zero/1.0/ ) applies to the data made available in this article, unless otherwise stated.
About this article
Cite this article
Ponterio, E., Di Bartolo, I., Orrù, G. et al. Detection of serum antibodies to hepatitis E virus in domestic pigs in Italy using a recombinant swine HEV capsid protein. BMC Vet Res 10, 133 (2014). https://doi.org/10.1186/1746-6148-10-133
- Hepatitis E virus
- Western blotting