Expression and Immunoreactivities of Hepatitis E Virus Genotype 3 Open Reading Frame-2 (ORF-2) Recombinant Proteins Expressed in Insect Cells

  • Nereida Jiménez de Oya
  • Inmaculada Galindo
  • Estela Escribano-Romero
  • Ana-Belén Blázquez
  • Julio Alonso-Padilla
  • Nabil Halaihel
  • José M. Escribano
  • Juan-Carlos Saiz
Original Paper


Hepatitis E virus (HEV) is a fecal-orally transmitted virus that is endemic in many geographical areas with poor sanitary conditions and inadequate water supplies. In Europe, a low-endemic area, an increased number of autochthonous sporadic human cases of patients infected with HEV strains of genotype 3, have been reported lately. The relatively high prevalence of HEV genotype 3 infections in European pigs has raised concerns about a potential zoonotic transmission to humans. Determination of HEV seroprevalence in pigs would help to clarify its incidence and possible zoonotic implications. To this purpose, we have expressed and partially characterized swine genotype 3 HEV open reading frame-2 proteins upon infection of Sf21 insect cells with recombinant baculoviruses. The use of the expressed proteins as diagnostic antigens for the detection of antibodies to HEV has been further assayed with human and swine sera.


Zoonosis Emerging virus Immunogenicity ELISA 


Hepatitis E is an infection with clinical and epidemiological features of acute hepatitis. The causative agent, the hepatitis E virus (HEV), is a spherical, non-enveloped viral particle of around 32–34 nm in diameter, the genome of which is a single-stranded RNA molecule of positive polarity of approximately 7.2 kb, containing three overlapping open reading frames (ORF) and a 3′poly (A) tail (Pintó and Saiz 2007; Worm et al. 2002).

HEV is a fecal-orally transmitted pathogen that causes frequent epidemics in areas with inadequate water supplies and poor sanitary conditions (Pintó and Saiz 2007). However, an increased number of indigenous HEV cases have recently been reported in several European countries (Borgen et al. 2008; Buti et al. 2004; Dalton et al. 2008; Herremans et al. 2007a; Mansuy et al. 2004; Preiss et al. 2006; Widdowson et al. 2003). In contrast to what is seen in endemic areas, where the infecting viruses usually belong to genotype 1 or 2, these indigenous infections are mainly caused by genotype 3 strains (Pintó and Saiz 2007; Worm et al. 2002).

The detection of HEV infection in pigs that shed high quantities of virus in feces and the genetic similarity between human and swine strains from the same geographical region raised concerns about a possible zoonotic potential for HEV (Meng 2003), which was further confirmed in people who ate uncooked deer meat or liver from pork or wild boar (Tei et al. 2003; Yazaki et al. 2003; Matsuda et al. 2003). Latter on, HEV-RNA and infectious virus have been detected in commercial pig livers sold in local grocery stores (Bouwknegt et al. 2007b; Feagins et al. 2007; Yazaki et al. 2003).

In addition, several studies have found a higher prevalence of HEV antibodies in veterinarians and swine farmers than in control groups, suggesting that people in close contact with pigs may present a higher risk of infection (Bouwknegt et al. 2008, Drobeniuc et al. 2001; Galiana et al. 2008; Meng et al. 2002; Mizuo et al. 2005; Withers et al. 2002), although other reports did not find such relationship (Olsen et al. 2006). Therefore, assessment of the incidence of HEV infection in swine herds would help to clarify its prevalence and possible zoonotic implications; however, the lack of an efficient and validated serological test for HEV detection in swine has hampered the assessment of the seroprevalence of HEV in Europe, where only a limited number of reports have been conducted in swine herds (Banks et al. 2004; Casas et al. 2008; De Deus et al. 2008; Seminati et al. 2008).

To date, there are only a few commercially available kits for use with human sera that include short fragments of ORF-2 and ORF-3 of genotypes 1 and 2, but not of genotype 3 HEV, the most prevalent in Europe in swine and humans (Pintó and Saiz 2007), and, in some instances, these assays have failed to detect specific antibodies in sera from patients with proven HEV genotype 3 infections (Bouwknegt et al. 2007a; Daniel et al. 2004; Herremans et al. 2007b; Zhang et al. 2002). So far, different synthetic peptides and recombinant polypeptides, mainly corresponding to viral capsid protein ORF-2, expressed in various heterologous systems have been assayed for specific antibody detection, but, to the best of our knowledge, none of them have been derived from genotype 3 strains (Pintó and Saiz 2007; Worm et al. 2002).

Here, we describe the production and partial characterization of genotype 3 HEV-ORF2 recombinant proteins produced upon infection of insect cells with recombinant baculoviruses. The expressed proteins are recognized by human and swine sera and might be suitable diagnostic reagents for the detection of HEV antibodies.

Materials and Methods

Cloning of HEV Genotype 3 ORF-2 Proteins

RNA was extracted from 140 μl of a swine fecal extract (Fernández-Barredo et al. 2006) using QIAamp®Viral RNA mini kit (Qiagen, Valencia, CA, USA) according to manufacturer’s instructions. An aliquot of 10 μl of isolated RNA was reverse transcribed using a commercial Super-Script One-step RT-PCR® amplification kit (Invitrogen, Carlsbad, CA, USA) and primers HE-E-EcoF and HE-E-Bam-R (Table 1) to render a 2015-nt fragment. Cycling conditions for RT-PCR amplification were: 45°C for 1 h, 94°C for 2 min, followed by 40 cycles at 94°C for 30 s, 45°C for 1 min, and 68°C for 3 min, with a final elongation step at 68°C for 10 min. Amplified product was identified by electrophoresis in a 1% agarose gel stained with ethidium bromide, excised from it, purified using QIAquick® Gel Extraction Kit (Qiagen) and cloned into the TOPO TA Cloning vector® (Invitrogen) to render TOPO-TA-ORF-2 plasmid. Then, a PCR was performed to obtain a full-length ORF-2 insert of 2006 nt using 10 ng of TOPO-TA-ORF-2 as template and primers HE-Bam-Bac-F and HE-Kpn-Bac-R (Table 1) with a commercial Expand High Fidelity PCR® System (Roche Diagnostics, Mannheim, Germany). Reaction conditions were: 94°C for 1 min, followed by 5 cycles at 94°C for 1 min, 40°C for 1 min, and 72°C for 2 min and 35 additional cycles at 94°C for 1 min, 47°C for 1 min and 72°C for 2 min, with a final elongation step at 72°C for 10 min. The PCR product obtained was purified and cloned again into TOPO TA Cloning vector® to render the TOPO-TA-ORF2-Bac plasmid.
Table 1

Oligonucleotide primers used in this study






















The restriction sites introduced in the primers are shown underlined, and the ATG start codon and the ACT codon, complementary to the in-frame stop codon (TGA), are shown in bold face. Nucleotide positions are according to Burmese prototype strains B1 (Tam et al. 1991)

Similarly, in order to obtain a truncated form of the ORF-2, ∆-ORF-2, lacking the 111 first amino acids at the N-termini, a PCR was performed as described above using 10 ng of TOPO-TA-ORF2-Bac as template and primers T-HE-Bam-Bac and HE-Kpn-Bac-R (Table 1) to produce a 1675-nt fragment. Reaction conditions were: 94°C for 2 min, followed by 39 cycles at 94°C for 30 s, 50°C for 1 min, and 68°C for 3 min, with a final elongation step at 68°C for 10 min. The amplified fragment was cloned as above to render TOPO-TA-Δ-ORF2-Bac plasmid.

Generation of Genotype 3 ORF-2 Recombinant Baculoviruses

TOPO-TA-ORF2-Bac and TOPO-TA-Δ-ORF2-Bac plasmids were digested with Bam HI and Kpn I to yield Bac-ORF2 and Bac-∆-ORF-2. The purified fragments were then inserted into Bam HI and Kpn I digested pFastBacMel-B2 vector under the control of the polyhedrin promoter to obtain Bac-ORF2r-His, which includes the entire ORF-2 product, and Bac-∆-ORF2r-His, which include the 3′-end 1600 nt of ORF-2, and bidirectionally sequenced. pFastBacMel-B2 plasmid, recently developed by J.M. Escribano and coworkers, is a modification of the pFastBac-1 vector® (Invitrogen) that incorporates the sequences of the melitonine peptide signal at the 5′ end and of a 6-histidine tail at the 3′end. Bac-ORF2r-His and Bac-∆-ORF2r-His were used to obtain recombinant baculoviruses (Bac1-ORF2r and Bac1-∆-ORF2r) expressing the entire genotype 3 ORF-2 and a truncated form of it, respectively, using the Bac-to-Bac Baculovirus® Expression System (Invitrogen) upon co-transfection into Sf21 insect cells following manufacturer’s instructions. Selected recombinant baculoviruses were grown on Sf21 cells following manufacturer’s instructions (Invitrogen) to reach a concentration of 108 pfu/ml, keeping a working stock at 4°C and storing aliquots at −80°C.

Protein Expression in Insect Cells and Immunoblot Analysis

In order to optimize protein expression, Sf21 cells were infected with recombinant baculoviruses and incubated at 27°C. Cellular suspensions, harvested at different days of post-infection, were centrifuged at 1200 rpm for 5 min at 4°C and the pellets were suspended in RIPA buffer (150 mM NaCl, 5 mM β-Mercaptoethanol, 1% NP40, 0.1% SDS, and 50 mM Tris–HCl pH 8) with 1× protease inhibitors, kept on ice for 30 min, and centrifuged again at 12000 rpm for 20 min at 4°C. The supernatants were recovered and stored at −20°C for further analyses of protein expression. Cellular extracts obtained upon inoculation of Sf21 cells with a baculovirus expressing an irrelevant histidine-tagged protein (Bac-Irr-His), or from mock-infected cells, and similarly processed, were used as control.

For Western blot analysis, processed protein extracts were resolved in 10% SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Richmond, CA, USA), which were then blocked for 1 h with PBS-0.05% Tween-20 (PBST) containing 5% skim milk. The membranes were subsequently incubated at 4°C overnight with a 1/7500 dilution of a commercial mouse anti-histidine monoclonal antibody (Clontech Laboratories, Inc., Mountain View, CA, USA) and then with a 1/4000 dilution of a goat anti-mouse horse-radish-peroxidase (HRP) conjugated secondary antibody (Sigma-Aldrich, St. Louis, MO, USA). Protein bands were then detected by the addition of 2.5 mM luminol–0.4 mM p-cumaric acid–100 mM Tris–HCl pH 8.5–0.018% H2O2.

In the evaluation phase, membranes were incubated with previously characterized sera from pigs (Seminati et al. 2008), or human who were unlinked to the identity of their donors (Buti et al. 2006), which tested positive (n = 4) or negative (n = 4) for anti-HEV antibodies. Next, goat anti-pig, or anti-human, immunoglobulin IgG-specific HRP conjugated (MorphoSys AG, Martinsried-Plangg, Germany) were added as secondary antibodies, and proteins were detected as described above.


Crude cellular suspensions from Sf21 cells (5 × 106) infected with Bac1-ORF2r or Bac1-∆-ORF2r recombinant baculoviruses were harvested, centrifuged at 1200 rpm for 5 min at 4°C and, then, the pellets were suspended in 250 μl of RIPA buffer with 1× protease inhibitors and tested as ELISA antigens. Microplates (Polysorp, Nunc, Roskilde, Denmark) were coated with 50 μl/well of serial dilutions of crude baculoviruses infected Sf21 cellular extract in 50 mM carbonate/bicarbonate buffer, pH 9.6, and incubated overnight at 4°C. All the following incubations were carried out for 1 h at 37°C under constant shaking. After washing thrice with PBST, plates were incubated with 100 μl/well of blocking solution (PBST—3% skim milk); then, serum diluted in blocking buffer was added in duplicate wells. To minimize unspecific reactivity, similar concentrations of cellular extracts obtained from Sf21 cells infected with an irrelevant baculovirus (Bac-Irr), and similarly processed, were included in the buffer. Plates were washed again with PBST before addition of 50 μl/well of goat HRP conjugated anti-swine, or anti-human, IgG secondary antibody diluted 1:30.000 and 1:10.000, respectively (MorphoSys). After three additional washes, 50 μl/well of substrate solution (OPD—0.056% H2O2) was added. Plates were incubated (dark, room temperature, 10 min) and then the reaction was stopped by adding 50 μl/well of 3 N H2SO4 and read at 495 nm in an ELISA microplate reader (Tecan Genios Ag, Vienna, VA, USA). Four previously characterized negative and four positive swine (Seminati et al. 2008) and human (Buti et al. 2006) sera were included in the assay as controls.


Expression of HEV Genotype 3 ORF-2r Proteins in Insect Cells

Western blot analysis with an anti-His monoclonal antibody of Sf21 cells infected with Bac1-ORF2r recombinant baculovirus that expresses the full-length HEV genotype 3 ORF-2 showed a major protein with an apparent molecular mass of around 78 kDa (Fig. 1a), although some minor species with a lower molecular mass of around 65–68 kDa could also be observed. When similar analyses were carried out with anti-HEV IgG positive swine or human sera, the same major specific species were observed together with some minor cells associated immunoreactive proteins (Fig. 1b, c). The specific proteins were not presented in uninfected Sf21 cell extracts (mock) or in extracts obtained from Sf21 cells infected with a baculovirus expressing an irrelevant protein fused to a His tail (Bac-Irr-His). Blot staining using anti-HEV IgG negative swine or human sera did not detect any specific band of the expected mass (data not shown). As shown in Fig. 1, Sf21 cells infected with Bac1-∆-ORF2r yield a major protein species of around 65–68 kDa. HEV-specific proteins obtained after infection with both recombinant baculoviruses were difficult to dissolve, but the yield of soluble protein was higher when the truncated form was expressed.
Fig. 1

Western blot analysis of HEV ORF-2r and ∆-ORF-2r proteins. Western blots were stained with: a an anti-His Mab; b swine serum; and c human serum. Lanes 1, 4, and 7 mock-infected Sf21 cellular extracts; lanes 2, 5, and 8 Bac1-ORF2r-infected Sf21 cellular extracts; lanes 3, 6, and 9 Bac1-∆-ORF-2r-infected Sf21 cellular extracts. Molecular weight markers are shown. Arrows mark the position of the HEV ORF-2 protein

In order to optimize protein expression, Sf21 cells were infected with 1 pfu/cell of each HEV recombinant baculoviruses and cellular extracts were harvested and analyzed at days 1–8 post-infection. Protein expression was observed from day 2 post-infection and stayed at similar levels until day 5, as shown for Bac1-∆-ORF2r in Fig. 2.
Fig. 2

Time course of the expression of ∆-ORF-2r in Sf21 cells infected with Bac1-∆-ORF-2r recombinant baculovirus. Infection was carried out as described in section “Materials and methods.” Western blots were stained with swine serum. M mock infected Sf21 cells. Lanes 1–8, Sf21 cellular extracts harvested 1–8 days post-infection, respectively. Molecular weight markers are shown

ELISA Based on the Expressed Proteins

In order to optimize the ELISA, different dilutions of crude cellular extracts from Sf21 cells infected with Bac1-ORF2r, or Bac1-∆-ORF2r recombinant baculoviruses, were tested with a battery (n = 8) of previously characterized positive and negative swine and human sera as described in section “Materials and methods.” Optimal conditions of antigen dilution were established at a ratio of 1/800 dilution (Fig. 3). Similarly, a 1/100 serum dilution ratio was established as the optimal working dilution (Fig. 4). The same sera were tested against crude extracts obtained from Sf21 cells infected with an irrelevant recombinant baculovirus. HEV positive sera produced A495 values significantly higher in Ag positive than in Ag negative-coated wells, whereas negative sera showed similar A495 values in both cases and these values were significantly lower than that of positive serum samples.
Fig. 3

Optimization of antigen concentration. Previously characterized positive (solid circles) and negative (open squares), swine (upper panel) and human (lower panel) sera were tested by ELISA against twofold serial dilutions (1/50 to 1/6400) of crude extracts from Sf21 cells infected with Bac1-ORF2r (a), Bac1-∆-ORF2r (b), or an irrelevant baculovirus, Bac-Irr, (c) that were processed as described in section “Materials and methods.” Data in each panel are shown as the average A495 of four negative and four positive sera. Standard deviations for each point are shown

Fig. 4

Optimization of sera dilution. Different twofold serial dilutions of sera (1/25 to 1/800) were tested by ELISA against crude extracts from Bac1-ORF2r (a), Bac1-∆-ORF2r (b), and mock (c) Sf21-infected cells, processed as described in section “Materials and methods,” using previously tested positive (solid lines) and negative (dotted lines) swine (upper panel) and human (lower panel) sera. Data in each panel are shown as the average A495 of four negative and four positive control sera. Standard deviations for each point are shown

Criteria for Determining Positive Reactivity in Anti-HEV ELISA

In order to establish the cut-off of the assay, a battery of eight HEV-RNA negative swine sera (N. Jiménez de Oya et al., unpublished results) that did not react by Western blot was used. These serum samples showed A495 values below 0.15 (between 0.08 and 0.14) when tested with crude extracts from Sf21 cells infected with HEV recombinant baculoviruses, values similar to those obtained with extracts from Sf21 cells infected with an irrelevant baculovirus (0.08–0.12). These negative sera were pooled and used as controls. Similarly, four IgG-negative previously characterized human sera (Buti et al. 2006) that gave A495 values below 0.10 (between 0.06 and 0.09) in the in-house test and did not react in Western blot were pooled and used as controls. Then, the cut-off value was established as 2.5 times the A495 value of the negative serum pool that was included in each plate. Samples above the cut-off value were considered positive.

Next the ELISA immunoreactivity of both recombinant proteins was tested against a battery of 32 swine and 32 human sera at the above established conditions, and a complete concordance was found between both antigens (Fig. 5).
Fig. 5

Comparison of ELISA immunoreactivity of ORF-2r and ∆-ORF2r recombinant proteins. ELISA results of human (a) and swine (b) sera using ORF-2r and ∆-ORF2r recombinant proteins as antigens. ELISA was performed as described in “Materials and methods.” Data are expressed as P/N ratio, which was calculated by dividing the mean A495 value of the test sera by the mean A495 value of the negative control serum pool. Vertical and horizontal dotted lines represent the cut-off value of the assays (P/N ≥ 2.5)


HEV is a waterborne virus that is endemic in many geographical areas with poor sanitary conditions and inadequate water supplies (Pintó and Saiz 2007). In these geographical regions, HEV strains of genotypes 1 and 2 are a major cause of outbreaks and sporadic cases. In Western countries most sporadic cases reported correspond to people that had usually travelled to endemic regions and were infected with these genotypes. Nevertheless, an increasing number of autochthonous sporadic cases of patients infected with HEV strains of genotype 3 have been recently reported in Europe.

The origin of these locally acquired infections is unknown, but concerns have arisen over a potential zoonotic transmission of HEV to humans from infected pigs. Although currently definite evidence has not been obtained, several investigations suggest that such zoonotic transmission is possible. For instance, in Europe viral strains found in pigs and humans are genetically closely related (Peréz-Gracia et al. 2004; Preiss et al. 2006; Van der Poel et al. 2001; Widdowson et al. 2003). The virus has been found in pork livers sold in markets (Bouwknegt et al. 2007b; Feagins et al. 2007; Yazaki et al. 2003), food-borne transmission has been proven (Matsuda et al. 2003; Tei et al. 2003; Yazaki et al. 2003), and people in close contact with pigs seem to present a higher prevalence of HEV antibodies (Bouwknegt et al. 2008; Drobeniuc et al. 2001; Galiana et al. 2008; Meng et al. 2002; Mizuo et al. 2005; Withers et al. 2002). All these data have stressed the need for a proper assessment of the incidence of HEV in the European swine population, but these studies have been hampered by the lack of an appropriate serological test for use in pigs.

The few serological diagnostic studies conducted in Europe (Banks et al. 2004; Casas et al. 2008; De Deus et al. 2008; Seminati et al. 2008) have applied in house tests based on recombinant proteins of genotypes other than 3, the most prevalent in Europe. In addition, available commercial kits are designed for use in humans, and have not been validated for use in swine samples, and may be inaccurate when detecting sera from patients infected with genotypes other than 1 (Daniel et al. 2004; Herremans et al. 2007b; Wang et al. 2001; Zhang et al. 2002). Therefore, availability of diagnostic methods based on HEV strains circulating in non-endemic regions could help to clarify the incidence of HEV infection in these regions.

For this purpose, we have obtained and partially characterized ORF-2 recombinant proteins from genotype 3 HEV upon infection of Sf21 insect cells with recombinant baculoviruses. Next, extracts from infected insect cells were tested for their usefulness as diagnostic antigens. ORF-2 proteins of genotype other than 3 have been produced in several eukaryotic heterologous systems (Pintó and Saiz 2007; Worm et al. 2002), but, to the best of our knowledge, this is the first time that ORF-2 proteins of genotype 3 have been expressed, partially characterized, and tested as specific ELISA antigens.

Full-length ORF-2 was expressed in Sf21 cells as a major single protein of around 78 kDa. This molecular mass is slightly larger than the predicted size (71 kDa). Presence of the melitonine peptide signal and the histidine tail added to the recombinant protein, together with the already described post-translational modifications of this protein (Pintó and Saiz 2007; Worm et al. 2002) and the inaccuracy of the estimation of molecular masses from SDS-PAGE, may have contributed to the difference observed. In any case, different molecular sizes have been previously described for ORF-2 proteins of genotypes 1, 2, and 4 expressed by recombinant baculoviruses in different cell lines (Arankalle et al. 2007; He et al. 1995; Li et al. 1997b; Tsarev et al. 1993; Zhang et al. 1997). Similarly, previous studies have reported the detection of multiple immunoreactive proteins with a wide size range in insect cells infected with recombinant ORF-2 baculovirus of genotypes other than 3 (Arankalle et al. 2007; Li et al. 1997b; Robinson et al. 1998; Tsarev et al. 1993). In our experiments, minor species with lower molecular mass could be also observed, including a doublet of around 65–68 kDa. This doublet was most probably due to the proteolytic processing of the complete ORF-2 protein that occurs by truncating N-terminus of ORF-2 (Li et al. 1997b; Robinson et al. 1998; Tsarev et al. 1993), as suggested by the similar pattern observed when the truncated form (∆-ORF-2) of the protein was expressed. The specificity of both recombinant proteins was confirmed by Western blot analysis using the well-characterized human and pig sera.

Both expressed recombinant proteins demonstrated to be good antigens for diagnostic purposes. As shown in Fig. 5, concordance between them was total when human and pig sera were tested. This concordance is in agreement with previous reports (Li et al. 1997a; Wang et al. 2001) describing that convalescent-reactive sera mainly react with epitopes located within the C-terminal region of the protein, which are likely presented in both recombinant species tested here. However, as described by others (Li et al. 1997b; He et al. 1993, 1995), the full-length protein seems to be more tightly associated cell than the truncated forms and therefore, its diagnostic utility may be hampered by its lower solubility and higher sensitivity to protease digestion.

In conclusion, we have successfully expressed and characterized, for the first time, ORF-2 HEV genotype 3 proteins upon inoculation of recombinant baculoviruses in Sf21 insect cells. Crude extracts containing the HEV recombinant proteins have been shown to be suitable as ELISA antigens for screening of IgG antibodies against HEV both in human and swine sera, and are now being applied to a large seroprevalence study in European swine herds.



The study was supported in part by grants (AGL2004-06071 and CSD2006-0007) from the Spanish Ministerio de Ciencia e Innovación (MICINN) to J.C.S. N.J.O. has been supported by a scholarship from Instituto Nacional de Investigación y Tecnología Agraraia y Alimentaria (INIA) and by a COST-929 short-term mission. J.A. was supported by a scholarship from the MICIIN. E.E.R. was supported by a contract ascribed to grant CSD2006-0007 from MICIIN. We are indebted to M.T. Pérez-Gracia (U. Cardenal Herrera-CEU, Valencia, Spain), M. Buti (Hospital Vall d′Hebron. Barcelona, Spain), and M. Martín (CReSA, UAB, Barcelona, Spain) for kindly providing us with swine fecal extract, human and swine sera, respectively.


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Copyright information

© Springer Science + Business Media, LLC 2009

Authors and Affiliations

  • Nereida Jiménez de Oya
    • 1
  • Inmaculada Galindo
    • 1
  • Estela Escribano-Romero
    • 1
  • Ana-Belén Blázquez
    • 1
  • Julio Alonso-Padilla
    • 1
  • Nabil Halaihel
    • 2
  • José M. Escribano
    • 1
  • Juan-Carlos Saiz
    • 1
  1. 1.Department of BiotechnologyInstituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)MadridSpain
  2. 2.Department of Infectious Pathology and Epidemiology, Faculty of VeterinariaUniversity of ZaragozaZaragozaSpain

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