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Evaluation of recombinant Chinese avian hepatitis E virus (CaHEV) ORF2 and ORF3 proteins for protection of chickens against CaHEV infection
Vaccine xxx (2017) xxx–xxx
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Evaluation of recombinant Chinese avian hepatitis E virus (CaHEV) ORF2 and ORF3 proteins for protection of chickens against CaHEV infection Shahid Faraz Syed a,b,c,1, Yani Sun a,b,1, Taofeng Du a,b,1, Yiyang Chen a,b, Baoyuan Liu a,b, Xinjie Wang a,b, Huixia Li a,b, Yuchen Nan a,b, En-Min Zhou a,b,⇑, Qin Zhao a,b,⇑ a b c
Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, PR China Scientific Observing and Experimental Station of Veterinary Pharmacology and Diagnosis, China Ministry of Agriculture, Yangling 712100, Shaanxi, PR China Faculty of Veterinary and Animal Sciences, Lasbella University of Agriculture, Water and Marine Sciences, Uthal, Baluchistan, Pakistan
a r t i c l e
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Article history: Received 12 March 2017 Received in revised form 2 May 2017 Accepted 6 May 2017 Available online xxxx Keywords: Hepatitis E virus Avian HEV ORF2 protein ORF3 protein Protection Vaccine
a b s t r a c t Avian hepatitis E virus (HEV) is the etiologic agent of big liver and spleen disease in chickens. In 2010, the Chinese avian HEV (CaHEV) strain was isolated from chickens and demonstrated to cause the decreased egg production in layer hens. No avian HEV commercial vaccine has yet been developed to prevent virus infection in China. In this study, recombinant CaHEV truncated ORF2 and complete ORF3 proteins were evaluated separately for immunoprotection of chickens against CaHEV infection. First, truncated ORF2 and complete ORF3 proteins were expressed in Escherichia coli. Next, 48 specific-pathogen-free chickens were randomly divided into three groups. One group was immunized with truncated ORF2 protein, the second group was immunized with recombinant ORF3 protein, while the third group (control) was mock-immunized with PBS. After booster immunization, chickens in all three groups were challenged intravenously with CaHEV infectious stock and assessed for viremia, fecal virus shedding, seroconversion, and gross hepatic lesions. In the ORF2 protein-immunized group, no chickens showed evidence of avian HEV infection. In the ORF3 protein-immunized group, nine chickens exhibited viremia and seven had fecal virus shedding. In the control group, all 16 chickens showed viremia and fecal virus shedding. However, the durations in chickens from the ORF3 protein group (2–4 weeks) were shorter than the ones from the control group (4–8 weeks). Moreover, no gross liver lesions emerged in the ORF2 protein group, while lesions observed in the ORF3 protein group were milder than in controls. Therefore, the ORF2 protein can confer complete immunoprotection against chicken CaHEV infection, while the ORF3 protein only confers partial immunoprotection. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Avian hepatitis E virus (HEV) is the causative agent of big liver and spleen disease (hepatitis-splenomegaly syndrome) in chickens [1–3]. Avian HEV infection usually leads to 1–4% increased mortality, 20–40% decreased egg production, and occasionally enlarged livers and spleens in both broiler breeder and laying hens [3–5]. Moreover, anti-avian HEV antibodies and RNA have been frequently detected in apparently healthy chicken flocks [1,6]. Avian HEV was characterized in chickens with hepatitis-splenomegaly syndrome in 2001 in the US and shown to be a variant strain of ⇑ Corresponding authors at: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, PR China. E-mail addresses: [email protected] (E.-M. Zhou), qinzhao_2004@nwsuaf. edu.cn (Q. Zhao). 1 These authors contributed equally to this work.
big liver and spleen disease virus isolated in Australia [1]. To date, the virus has been detected from many countries [4,5,7–9]. Avian HEV is a non-enveloped, single-stranded, positive sense RNA virus in the Hepeviridae family [10]. Various avian HEV strains belong to four major genotypes comprising a single serotype [7,9,11–13]. The avian HEV genome, approximately 6.6 kb in size, is only 48% identical to that of human and swine HEVs and contains three open-reading frames (ORFs) and two noncoding regions [14,15]. The largest ORF, ORF1, encodes non-structural proteins, ORF2 encodes viral capsid protein, and ORF3 encodes a small multifunctional phosphoprotein [15]. Because the lack of a highly efficient in vitro cell culture system has hampered traditional HEV vaccine development, genetic engineering of a subunit vaccine is currently being pursued. Previously, a truncated ORF2 protein from a US viral isolate was expressed in Escherichia coli [16,17], which contains immunodominant antigenic domains, neutralizing epitopes and can induce efficient neu-
http://dx.doi.org/10.1016/j.vaccine.2017.05.030 0264-410X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Syed SF et al. Evaluation of recombinant Chinese avian hepatitis E virus (CaHEV) ORF2 and ORF3 proteins for protection of chickens against CaHEV infection. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.030
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tralizing antibodies to prevent avian HEV infection in chickens [18–21]. Because ORF3 protein can also induce strong immune responses in avian HEV-infected chickens [22,23], it was speculated that the protein may be also a vaccine candidate. But there was no report about evaluation of the ORF3 protein for protection against avian HEV infection in chickens. The Chinese avian HEV strain (CaHEV) was isolated from a chicken with hepatitis-splenomegaly syndrome in 2010 and the virus infection can cause an approximately 10–30% decreased egg production in layer hens [24]. Because there was no commercial vaccine for protection against CaHEV infection in chickens in China, evaluation of CaHEV ORF2 and ORF3 proteins for their ability to confer immunoprotection against CaHEV infection would be of value. Therefore, in this study recombinant CaHEV truncated ORF2 (ORF2-C) and complete ORF3 proteins were expressed in Escherichia coli and immunoprotective effects of purified proteins were compared for their ability to prevent CaHEV infection in chickens. 2. Materials & methods 2.1. Virus An avian HEV infectious stock was produced by intravenously inoculating four 8-week-old specific-pathogen-free (SPF) chickens with 200 ml of a clinical bile sample containing CaHEV (GenBank accession no. GU954430) [9]. At 21 days post-inoculation, viruspositive fecal samples were collected and 10% fecal suspensions were prepared in phosphate-buffered saline (PBS, pH 7.4) containing penicillin-streptomycin then pooled. This CaHEV stock contained 104 genomic equivalents (GE)/ml or 500 CID50/ml of the virus [23].
and after challenge, chicken serum and fecal samples were collected weekly from 0 to 8 week post-challenge (wpc) for nine collections. Serum samples were tested for alanine aminotransferase (ALT) and anti-aHEV IgG antibodies (specific for ORF2-C or ORF3 protein) by indirect enzyme-linked immunosorbent assay (ELISA). Serum and fecal samples were also tested qualitatively and quantitatively for avian HEV RNA by reverse transcription-nested PCR (RT-nPCR) and quantitative RT-PCR (qRT-PCR), respectively. Fecal virus shedding and viremia were monitored for 8 weeks. Two chickens/group were necropsied at 2, 4, 6, and 8 wpc and bile and liver samples were collected. The bile and a portion of the liver sample from each chicken was homogenized in 10% (w/v) sterile PBS, clarified by centrifugation (1000g, 15 min, 4 °C), and tested for avian HEV RNA by RT-nPCR. Gross liver lesions were evaluated and recorded using digital photography. 2.5. Indirect ELISA Before and after immunization and virus challenge, levels of anti-avian HEV antibodies specific for ORF2-C or ORF3 proteins were measured in serum samples from immunized chickens using indirect ELISAs with ORF2-C protein, ORF3 protein, or peptide 10 coating antigens as previously described [19,21,22]. 2.6. Qualitative and quantitative detection of avian HEV RNA using RTnPCR and qRT-PCR Avian HEV RNA was detected in fecal, serum, bile, and liver samples using RT-nPCR followed by sequencing as described previously [19]. In addition, the avian HEV RNA in fecal and serum samples was quantified by qRT-PCR as described by Troxler et al. [25]. 2.7. Determination of serum alanine aminotransferase (ALT) levels
2.2. Animals Forty-eight 6-week-old SPF chickens (Beijing Merial Vital Laboratory Animal Technology Co., Ltd.) tested negative for anti-avian HEV antibodies and RNA. Chickens were divided into three groups (16 chickens/group) that were housed separately in three rooms (2 chickens/cage). Animal experiments were approved by the Animal Care and Use Committee of Northwest A&F University (NWSUAF, Permit Number AE319121) and NWSUAF guidelines were followed. 2.3. Immunization of chickens with recombinant CaHEV ORF2-C and ORF3 proteins CaHEV ORF2-C (containing C-terminal 268 amino acids) and ORF3 recombinant proteins were expressed separately in Escherichia coli RosettaTM (DE3) pLysS host cells and purified using a NiNTA resin column as described previously [21,22]. After one week of acclimation (at age of 7 weeks), Group 1 chickens (Nos. 1–16) were each immunized intramuscularly in both sides of the breast with ORF2-C protein (200 mg/chicken) emulsified in Freund’s complete adjuvant (Sigma-Aldrich, St. Louis, MO, USA) then boosted with 200 mg ORF2-C protein in Freund’s incomplete adjuvant after two weeks. Group 2 chickens (Nos. 17–32) were immunized with purified ORF3 protein using the same procedures and dosages as for group 1. Group 3 chickens (Nos. 33–48) were immunized as above, but with PBS only (negative control). 2.4. Virus challenge and sample collection Two weeks after booster immunizations, all chickens were inoculated intravenously with 800 ml of CaHEV infectious stock. Before
ALT concentrations of serum samples collected weekly were measured (Hitachi 912; Roche, Indianapolis, IN, USA) following manufacturer’s instructions. Biochemical evidence of hepatitis was recorded when serum ALT concentrations exceeded the baseline ALT level by more than twofold, as defined by the peak ALT value that was equal to or greater than twice the geometric mean of the three immediate pre-challenge values. This criterion was based on a previous study correlating ALT level increase with liver pathology in another animal model [21,26]. 2.8. Statistical analysis T-tests (Microsoft Excel 2003) were performed to compare differences in ELISA absorbance values between pre-vaccinated and post-vaccinated chicken sera for ORF2-C and ORF3 antigens and to assess immunoprotective efficacy among ORF2-C protein, ORF3 protein, and PBS immunized groups. ALT values among groups were also analyzed using the same method [21]. P 0.05 was considered significant. 3. Results 3.1. Immune responses in chickens immunized with recombinant ORF2-C or ORF3 proteins In the CaHEV ORF2-C protein-immunized group, chickens showed high anti-ORF2-C protein IgG antibody titers in sera, defined as the highest serum dilution producing an OD450nm value > 0.368 [17]. After the second immunization (but before CaHEV challenge), all chickens immunized with ORF2-C protein had the seroconversion (Fig. 1A) and titers of approximately
Please cite this article in press as: Syed SF et al. Evaluation of recombinant Chinese avian hepatitis E virus (CaHEV) ORF2 and ORF3 proteins for protection of chickens against CaHEV infection. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.030
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Fig. 1. Seroconversion (A) and titers of antibodies (B) against CaHEV ORF2-C protein in chickens immunized with the CaHEV ORF2-C and against CaHEV ORF3 protein in chickens immunized with CaHEV ORF3 protein. Serum samples were collected from chickens at pre-immunization (pre-im) and 2 weeks after first and second immunization (1st-im and 2nd-im). ELISA plates were coated with 200 ng/well of CaHEV ORF2-C protein or the recombinant CaHEV ORF3 protein and each point represents a mean value of OD450nm obtained from the 16 chickens in the same group. The titers of anti-ORF2-C protein IgG antibody in sera were approximately 1:10,000, which was defined as the highest serum dilution producing an OD450nm value >0.368 (Fig. 1A). The titers of anti-ORF3 protein IgG antibody in sera collected 2 weeks after the second immunization were also approximately 1:10,000 defined as the highest serum dilution generating an OD450nm value >0.4.
1:10,000 (Fig. 1B), while the other two groups did not develop specific antibodies against ORF2-C protein (the average OD450 nm value was 0.201). For the CaHEV ORF3 protein-immunized group, anti-avian HEV ORF3 protein antibodies in serum samples collected after booster immunization (prior to CaHEV challenge) were detected via indirect ELISA (using ORF3 protein as coating antigen). Anti-ORF3 protein IgG antibody titers in sera were defined as the highest serum dilution generating an OD450nm value >0.4 [22]. After the second immunization, all the ORF3 immunized chickens exhibited seroconversion for the anti-ORF3 protein antibodies (Fig. 1A) and serum titers of approximately 1:10,000 (Fig. 1B). At a 1:1000 serum dilution, all 16 immunized chickens exhibited positive titers for anti-ORF3 protein IgG antibodies with OD450nm values >1.0. In the ORF2-C protein-immunized and negative control groups, no chickens developed specific antibodies against CaHEV ORF3 protein (the average OD450nm value was 0.247).
3.2. Detection of avian HEV RNA in serum, feces, bile, and liver samples Serum and fecal samples from all chickens were negative for avian HEV RNA at 0 wpc. Post-challenge, numbers of chickens in the three groups testing positive for viremia and fecal virus shedding at different wpc are shown in Table 1. In the CaHEV ORF2-C protein-immunized group, all chickens were negative for viral RNA from 1 to 8 wpc. In the CaHEV ORF3 protein-immunized group, the 7 chickens exhibited fecal virus shedding and 9 ones for viremia, with a lower number observed than in the negative control group. Moreover, the difference in numbers of chickens showing fecal virus shedding between ORF3 protein and PBS immunized groups was significant (P < 0.05). In the negative control group, four chickens exhibited fecal virus shedding throughout the entire experiment. However, in the ORF3 protein-immunized group, no chickens shed virus from 4 to 8 wpc. As for fecal virus shedding, serum viral RNA was first detected at 1 wpc in 5 chickens in the negative control group, but no chickens were positive in the ORF3 protein-immunized group. In addition, avian HEV RNA in feces and sera was detected approximately one week later in the
immunized ORF3 protein group than the negative control group (Table 1). For each non-necropsied chicken in the ORF2-C proteinimmunized group, all chickens were negative for viremia and virus shedding throughout the experiment (data not shown). In the ORF3 protein-immunized group, the duration of viremia and fecal virus shedding of the two chickens (Nos. 19 and 29) were 4 and 2 weeks, respectively (Table 2), with shorter durations of these parameters observed for six other chickens (Nos. 17, 18, 21, 24, 26, and 31). However, in the negative control group, the duration of fecal virus shedding in three chickens (Nos. 33, 35, and 44) was 7 weeks and durations of viremia and fecal virus shedding for the majority of chickens were above 3 weeks. The differences in duration of fecal virus shedding and viremia between ORF3 protein and PBS immunized groups were significant (P < 0.05). For necropsied chickens in the ORF2-C protein-immunized group, all samples were negative for avian HEV RNA in serum, feces, bile, and liver. In the ORF3 protein-immunized group, only one of two necropsied chickens tested positive for avian HEV RNA in serum, feces, bile, and liver at 2 and 4 wpc (Table 3). However, in the negative control group, serum, feces, bile, and liver samples from both necropsied chickens were positive for viral RNA at 2 wpc and one chicken remained positive for viral RNA at 6 and 8 wpc. Viral sequences recovered from infected chickens matched the 536-bp portion of the CaHEV ORF1 coding gene, thus confirming that virus in infected chickens originated from the inoculum. 3.3. Anti-avian HEV ORF3 immune responses after viral challenge Serum samples collected from the 30 chickens not necropsied before 8 wpc in the three groups were tested to evaluate the kinetics of the anti-avian HEV ORF3 protein antibody response. Chickens in the ORF2-C protein-immunized group showed no anti-avian HEV ORF3 protein IgG antibody response throughout the experiment. However, in the ORF3 protein-immunized group, antiORF3 protein antibody levels in the 10 chickens reached high levels after the second immunization, but declined slightly at 1 wpc. At 2–4 wpc, the titers of antibodies against ORF3 protein exhibited
Please cite this article in press as: Syed SF et al. Evaluation of recombinant Chinese avian hepatitis E virus (CaHEV) ORF2 and ORF3 proteins for protection of chickens against CaHEV infection. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.030
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Table 1 Detection of avian HEV RNA in weekly sera and fecal swabs from all chickens challenged with CaHEV during the course of the study. Chicken group
Group 1a Group 2b Group 3c a b c
No. of positive sera (no. of positive fecal swabs)/total no. tested at indicated wpc 0
1
2
3
4
5
6
7
8
0(0)/16 0(0)/16 0(0)/16
0(0)/16 0(0)/16 7(5)/16
0(0)/16 9(7)/16 16(16)/16
0(0)/14 7(3)/14 10(14)/14
0(0)/14 4(0)/14 7(13)/14
0(0)/12 2(0)/12 4(7)/12
0(0)/12 0(0)/12 0(4)/12
0(0)/10 0(0)/10 0(4)/10
0(0)/10 0(0)/10 0(4)/10
Chickens were immunized with recombinant ORF2-C protein. Chickens were immunized with recombinant ORF3 protein. Chickens were the negative control group immunized with PBS.
Table 2 Viremia and fecal virus shedding in chickens not necropsied from CaHEV ORF3 protein and PBS immunized groups.a Group and Chicken No.
Detection of viral RNA in serum samples/fecal swabs at the different wpc 0
a
1
2
3
4
5
6
7
8
Immunized with CaHEV ORF3 protein group 17 –/– 18 –/– 19 –/– 21 –/– 22 –/– 24 –/– 26 –/– 28 –/– 29 –/– 31 –/–
–/– –/– –/– –/– –/– –/– –/– –/– –/– –/–
+/+ +/– +/+ +/+ –/– +/+ +/+ –/– +/+ +/–
–/– +/– +/+ +/– –/– +/– +/– –/– +/+ –/–
–/– –/– +/– –/– –/– +/– –/– –/– +/– –/–
–/– –/– +/– –/– –/– –/– –/– –/– +/– –/–
–/– –/– –/– –/– –/– –/– –/– –/– –/– –/–
–/– –/– –/– –/– –/– –/– –/– –/– –/– –/–
–/– –/– –/– –/– –/– –/– –/– –/– –/– –/–
Negative group mock immunized with PBS 33 –/– 35 –/– 36 –/– 39 –/– 40 –/– 41 –/– 42 –/– 44 –/– 45 –/– 47 –/–
–/– –/– +/+ +/+ +/+ –/– +/– –/– +/+ +/–
+/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+
+/+ –/+ +/+ –/+ +/+ +/+ +/+ +/+ +/+ +/+
+/+ –/+ +/– –/+ +/+ –/+ +/+ +/+ +/+ –/+
+/+ –/+ –/– –/+ –/– –/– +/+ +/+ –/– –/+
–/+ –/+ –/– –/– –/– –/– –/– –/+ –/– –/–
–/+ –/+ –/– –/– –/– –/– –/– –/+ –/– –/–
–/+ –/+ –/– –/– –/– –/– –/– –/+ –/– –/–
Chickens from CaHEV ORF2-C protein immunized group were all negative.
Table 3 Detection of avian HEV RNA in fecal, serum, bile, and liver samples from the chickens necropsied at different wpc. Group
Sample type
No. of positive samples/total no. tested at the different wpc 2
4
6
8
Immunized with CaHEV ORF2-C protein
Feces Serum Bile Liver
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
Immunized with CaHEV ORF3 protein
Feces Serum Bile Liver
1/2 1/2 1/2 1/2
0/2 1/2 1/2 1/2
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
PBS
Feces Serum Bile Liver
2/2 2/2 2/2 2/2
2/2 1/2 2/2 2/2
1/2 0/2 1/2 1/2
1/2 0/2 1/2 1/2
a transient increase in the 10 chickens. The chickens in the negative control group were negative for anti-ORF3 protein antibodies before challenge and 8 chickens become positive for antibodies at 3 wpc. At 5 wpc, antibodies levels against ORF3 protein reach the peak and all the 10 chickens seroconverted and antibody levels declined rapidly (Fig. 2A). At 8 wpc, based on the cut-off values of the indirect ELISA, all the 10 chickens were negative for anti-ORF3 protein antibodies (Fig. 2A).
3.4. Anti-avian HEV ORF2-C and peptide 10 antibody responses after viral challenge Avian HEV ORF2 protein contains the major epitopes of HEV viral particles and is currently universally used as antigen to diagnose avian HEV infection in chickens. In this study, an indirect ELISA using CaHEV ORF2-C protein as coating antigen was used to detect anti-avian HEV ORF2 protein antibodies [17]. For the ORF2-C protein-immunized group, all the 10 chickens were positive for antibodies against the ORF2-C protein at 0 wpc and at 3
Please cite this article in press as: Syed SF et al. Evaluation of recombinant Chinese avian hepatitis E virus (CaHEV) ORF2 and ORF3 proteins for protection of chickens against CaHEV infection. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.030
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The peptide 10 is the epitope located in the N-terminal of entire avian HEV ORF2 protein, which is lacking in the ORF2-C protein, and can induce the strong immune response in the avian HEV challenged chickens [21]. Therefore, to differentiate between antibodies produced in response to immunization with ORF2-C protein from those produced during avian HEV infection, another indirect ELISA using peptide 10 as coating antigen was performed as described previously [21]. Anti-peptide 10 antibodies were not detected in chickens immunized with the ORF2-C protein from 0 to 8 wpc (Fig. 2C). Moreover, in the ORF3 protein-immunized and negative groups, the changing trends in serum samples for antipeptide 10 antibodies were similar as the ones for anti-ORF2-C protein antibodies from 0 to 8 wpc (Fig. 2C), but the titres were lower. 3.5. Quantitation of avian HEV RNA in fecal and serum samples Besides the qualitative detection of avian HEV RNA in fecal and serum samples via RT-nPCR, viral concentrations were also determined by qRT-PCR as described previously [25]. In the ORF2-C protein-immunized group, the avian HEV RNA concentrations in all fecal and serum samples were <100 copies/mL from 0 to 8 wpc (Fig. 3A). In the ORF3 protein-immunized group, the avian HEV RNA concentrations in serum and fecal samples were ranged from 0 to 4.5 105 copies/mL from 0 to 8 wpc and peaked at 2 wpc, but lower than that from the negative group (Fig. 3A). 3.6. Serum ALT In the ORF2-C protein-immunized group, no ALT level changes were observed in serum samples from the 10 chickens at any wpc (Fig. 3B). In the ORF3 protein-immunized group, 6 chickens were protected against significant ALT elevation following challenge, but the other 4 chickens were only evaluated at 1 wpc (Fig. 3B). However, in the negative control group, the 6 chickens not necropsied before 8 wpc showed significantly increased ALT levels at 1 wpc. In addition, the increasing serum ALT levels form the chickens of the negative group were higher than the ones of the CaHEV ORF3 protein group (Fig. 3B). Thus, serum ALT levels differed significantly at 1 wpc among the three groups, but not at other wpc. 3.7. Gross hepatic lesions
Fig. 2. Variation of anti-avian HEV ORF3 (A), ORF2 (B) proteins and peptide 10 (C) antibody responses in the immunized chickens not necropised before 8 wpi after challenge with CaHEV among the three groups. Sera from 30 chickens were diluted 1:100 for the detection of antibodies against CaHEV ORF3, ORF2 proteins and peptide 10. Each point represents the mean value of OD450nm obtained from 10 chickens of each group ± range.
In the ORF2-C protein-immunized group, no liver hemorrhages were found (Fig. 4). Severe sub-capsular hemorrhages were observed in the livers of necropsied chickens in the negative control and ORF3 protein-immunized groups at 4, 6, and 8 wpc. However, in the ORF3 protein-immunized group, liver hemorrhages in necropsied chickens were less severe than in negative controls. 4. Discussion
wpc, the titers of anti-ORF2 protein antibodies declined slightly. However, in the negative control and ORF3 protein-immunized groups, at 0 wpc (two weeks after booster immunization), there was no detectable antibody against ORF2-C protein. At 3 wpc, the 8 chickens from the negative control group and the 5 chickens from the ORF3 protein-immunized group seroconverted to antiORF2-C protein antibodies (Fig. 2B). At 4 wpc, in the negative group all the 10 chickens seroconverted, but in the ORF3 protein immunized group, the 2 chickens were still negative. In addition, the antibody titers in the negative control group were higher than that in the ORF3 protein-immunized group (Fig. 2B).
Avian HEV infection causes hepatitis-splenomegaly syndrome, resulting in serious economic losses for the poultry industry worldwide [2,4,27,28]. However, no commercial vaccine is yet available for preventing disease in chickens. Previously, it was shown that a truncated ORF2 protein from a US avian HEV strain expressed in Escherichia coli could induce immunoprotection against US avian HEV infection in chickens [21]. However, it is not yet known if the CaHEV ORF2 protein can prevent CaHEV infection in chickens, which can cause decreased egg production in layer hens in China [24]. In addition, the immunoprotective efficacy of avian HEV ORF3 protein is not known, even though it contains known immunodominant epitopes and induces strong immune responses
Please cite this article in press as: Syed SF et al. Evaluation of recombinant Chinese avian hepatitis E virus (CaHEV) ORF2 and ORF3 proteins for protection of chickens against CaHEV infection. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.030
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Fig. 3. Dynamics of viral load in fecal and serum samples (A) and serum levels of ALT in sera (B) from all chickens after the challenge with CaHEV. (A) Y axis represents log10 [copies/ml] of avian HEV RNA in serum and feces. Each point represents the mean value of viral copy numbers obtained from 10 chickens of each group ± range. (B) The mean ALT values at each week post-challenge were generated from the 10 chickens of each group ± range. Statistical significance (*P 0.05).
Fig. 4. Gross hepatic lesions in chickens challenged with CaHEV. None (top row), moderate (middle row), and severe (bottom row) subcapsular liver hemorrhages (arrows) were found in the chickens after virus challenge as indicated. Livers in the top row of images were from CaHEV ORF2-C protein immunized chickens necropsied at 2 (A), 4 (B), 6 (C), and 8 (D) wpc. Middle row images were from chickens in the CaHEV ORF3 protein immunized group necropsied at 2 (E), 4 (F), 6 (G), and 8 (H) wpc. Bottom row images were from chickens in the negative control group necropsied at 2 (I), 4 (J), 6 (K), and 8 (L) wpc.
in virus-infected chickens [22]. In the present study, immunoprotection using CaHEV ORF2-C and ORF3 proteins expressed in Escherichia coli were separately evaluated and suggest that ORF2C protein can provide complete protection against CaHEV infection in chickens, while ORF3 protein only generates partial immunoprotection. Avian HEV strains can be divided into four major distinct genotypes which share approximately 82% identity at the nucleotide level [9,12,13,29,30]. However, these viral strains collectively belong to only a single serotype; therefore, it may be possible to produce a broadly protective vaccine based on the ORF2 viral capsid protein. In this study, immunization of chickens with CaHEV ORF2-C protein induced immunoprotection against CaHEV infec-
tion. These results align with results of a previous study showing that the truncated ORF2 protein from a US avian HEV strain could also provide protection against US avian HEV infection [21]. Therefore, we can only speculate that either CaHEV or US avian HEV ORF2 proteins may induce protection against different avian HEV isolates in chickens, but animal experiments are needed to confirm this speculation. Alternatively, ORF3 protein may also serve as a candidate for a broadly protective vaccine, since one human HEV ORF3 genotype has been documented by several researchers to protect animals against infection with other human HEV genotype strains [31]. Additional animal experiments are still needed to evaluate use of ORF3 protein as a broadly protective vaccine.
Please cite this article in press as: Syed SF et al. Evaluation of recombinant Chinese avian hepatitis E virus (CaHEV) ORF2 and ORF3 proteins for protection of chickens against CaHEV infection. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.030
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Notably, a previous study reported that anti-HEV ORF3 protein antibody titer decreases much more quickly after natural infection than does anti-avian HEV ORF2 protein antibody titer [32,33]. However, how long anti-avian HEV ORF3 protein antibody persists and whether or not duration of persistence depends on original titer are not known. More experiments will be needed to characterize the question. In this study, all ORF3 protein-immunized chickens produced a higher anti-avian HEV ORF3 protein antibody titer and exhibited fewer liver lesions, shortened durations of fecal virus shedding and viremia than controls. But the anti-ORF3 protein antibodies alone did not entirely eliminate viral infection. Previously, three C-terminal antigenic epitopes of the CaHEV ORF3 protein were identified, but their neutralizing characteristics were not studied [22]. Therefore, further studies are needed to determine if these are neutralizing epitopes and to determine which epitope(s) confer protection. In contrast, neutralizing epitopes identified in the avian HEV ORF2 protein could induce immunoprotective responses in chickens [34] and were confirmed here using chickens immunized with CaHEV ORF2-C protein. Unexpectedly, immunization with the CaHEV ORF3 protein only induced partial protection against CaHEV infection in chickens. Possible reasons for lack of protection may be attributed to the various roles that ORF3 protein plays at various times during virus infection. ORF3 encodes a small multifunctional phosphoprotein associated with virus release from host cells in vitro [35–37] and is essential for establishing viral infection in vivo [38,39]. Interestingly, anti-HEV ORF3 protein antibodies have only been observed to capture HEV from serum, not from fecal samples [35]. This observation suggests that the physical location of ORF3 protein within viral particles or its accessibility to antibody binding may differ between ORF3 proteins within serum versus fecal virus particles. Therefore, more experiments are needed to characterize the function and role played by the viral ORF3 protein in the anti-HEV protein immune response. Moreover, the mechanism of immunoprotection achieved by ORF3 protein-specific neutralizing antibodies also remains to be elucidated. This is the first report demonstrating that recombinant CaHEV ORF3 protein expressed in a bacterial system partially protects chickens against CaHEV infection. However, ORF2-C protein provides complete protection that surpasses protection using ORF3 protein. These results should aid understanding of the kinetics of infection and the immune response to virus in its natural host to facilitate vaccine development and prevent avian HEV infection.
Conflict of interest The authors have declared no competing interests exist. Acknowledgments The study is mainly funded by grants from National Natural Science Foundation of China (No. 31402233 and No. 31672583) to QZ and (No. 31372464) to EMZ, Project of Zhongying Distinguished Young Scholars to QZ, Technology Research and innovation of Shaanxi province (2015NY170) to YNS, the Fundamental Research Funds for the Central Universities of China (2452015035) to YNS, (2014YB013) to TFD and (2452016048) to QZ. References [1] Haqshenas G, Shivaprasad HL, Woolcock PR, Read DH, Meng XJ. Genetic identification and characterization of a novel virus related to human hepatitis E virus from chickens with hepatitis-splenomegaly syndrome in the United States. J Gen Virol 2001;82:2449–62.
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[2] Payne CJ, Ellis TM, Plant SL, Gregory AR, Wilcox GE. Sequence data suggests big liver and spleen disease virus (BLSV) is genetically related to hepatitis E virus. Vet Microbiol 1999;68:119–25. [3] Troxler S, Pac K, Prokofieva I, Liebhart D, Chodakowska B, Furmanek D, et al. Subclinical circulation of avian hepatitis E virus within a multiple-age rearing and broiler breeder farm indicates persistence and vertical transmission of the virus. Avian Pathol 2014;43:310–8. [4] Agunos AC, Yoo D, Youssef SA, Ran D, Binnington B, Hunter DB. Avian hepatitis E virus in an outbreak of hepatitis–splenomegaly syndrome and fatty liver haemorrhage syndrome in two flaxseed-fed layer flocks in Ontario. Avian Pathol 2006;35:404–12. [5] Morrow CJ, Samu G, Matrai E, Klausz A, Wood AM, Richter S, et al. Avian hepatitis E virus infection and possible associated clinical disease in broiler breeder flocks in Hungary. Avian Pathol 2008;37:527–35. [6] Sun ZF, Larsen CT, Dunlop A, Huang FF, Pierson FW, Toth TE, et al. Genetic identification of avian hepatitis E virus (HEV) from healthy chicken flocks and characterization of the capsid gene of 14 avian HEV isolates from chickens with hepatitis-splenomegaly syndrome in different geographical regions of the United States. J Gen Virol 2004;85:693–700. [7] Bilic I, Jaskulska B, Basic A, Morrow CJ, Hess M. Sequence analysis and comparison of avian hepatitis E viruses from Australia and Europe indicate the existence of different genotypes. J Gen Virol 2009;90:863–73. [8] Moon HW, Lee BW, Sung HW, Yoon BI, Kwon HM. Identification and characterization of avian hepatitis E virus genotype 2 from chickens with hepatitis-splenomegaly syndrome in Korea. Virus Genes 2016. [9] Zhao Q, Zhou EM, Dong SW, Qiu HK, Zhang L, Hu SB, et al. Analysis of avian hepatitis E virus from chickens. China. Emerg Infect Dis 2010;16:1469–72. [10] Smith DB, Simmonds P, International Committee on Taxonomy of Viruses Hepeviridae Study G, Jameel S, Emerson SU, Harrison TJ, et al. Consensus proposals for classification of the family Hepeviridae. J Gen Virol 2014;95:2223–32. [11] Gerber PF, Trampel DW, Opriessnig T. Identification and characterization of avian hepatitis E virus in 2013 outbreaks of hepatitis-splenomegaly syndrome in two US layer operations. Avian Pathol 2014;43:357–63. [12] Kwon HM, Sung HW, Meng XJ. Serological prevalence, genetic identification, and characterization of the first strains of avian hepatitis E virus from chickens in Korea. Virus Genes 2012;45:237–45. [13] Banyai K, Toth AG, Ivanics E, Glavits R, Szentpali-Gavaller K, Dan A. Putative novel genotype of avian hepatitis E virus, Hungary, 2010. Emerg Infect Dis 2012;18:1365–8. [14] Huang FF, Sun ZF, Emerson SU, Purcell RH, Shivaprasad HL, Pierson FW, et al. Determination and analysis of the complete genomic sequence of avian hepatitis E virus (avian HEV) and attempts to infect rhesus monkeys with avian HEV. J Gen Virol 2004;85:1609–18. [15] Tam AW, Smith MM, Guerra ME, Huang CC, Bradley DW, Fry KE, et al. Hepatitis E virus (HEV): molecular cloning and sequencing of the full-length viral genome. Virology 1991;185:120–31. [16] Haqshenas G, Huang FF, Fenaux M, Guenette DK, Pierson FW, Larsen CT, et al. The putative capsid protein of the newly identified avian hepatitis E virus shares antigenic epitopes with that of swine and human hepatitis E viruses and chicken big liver and spleen disease virus. J Gen Virol 2002;83:2201–9. [17] Zhao Q, Sun Y, Zhao J, Hu S, Zhao F, Chen F, et al. Development and application of an indirect ELISA for detection of antibodies against avian hepatitis E virus. J Virol Methods 2013;187:32–6. [18] Wang L, Sun Y, Du T, Wang C, Xiao S, Mu Y, et al. Identification of an antigenic domain in the N-terminal region of avian hepatitis E virus (HEV) capsid protein that is not common to swine and human HEVs. J Gen Virol 2014;95:2710–5. [19] Dong S, Zhao Q, Lu M, Sun P, Qiu H, Zhang L, et al. Analysis of epitopes in the capsid protein of avian hepatitis E virus by using monoclonal antibodies. J Virol Methods 2011;171:374–80. [20] Guo H, Zhou EM, Sun ZF, Meng XJ. Immunodominant epitopes mapped by synthetic peptides on the capsid protein of avian hepatitis E virus are nonprotective. Viral Immunol 2008;21:61–7. [21] Guo H, Zhou EM, Sun ZF, Meng XJ. Protection of chickens against avian hepatitis E virus (avian HEV) infection by immunization with recombinant avian HEV capsid protein. Vaccine 2007;25:2892–9. [22] Zhao Q, Sun YN, Hu SB, Wang XJ, Xiao YH, Hsu WH, et al. Characterization of antigenic domains and epitopes in the ORF3 protein of a Chinese isolate of avian hepatitis E virus. Vet Microbiol 2013;167:242–9. [23] Wang X, Zhao Q, Dang L, Sun Y, Gao J, Liu B, et al. Characterization of Two Novel Linear B-Cell Epitopes in the Capsid Protein of Avian Hepatitis E Virus (HEV) That Are Common to Avian, Swine, and Human HEVs. J Virol 2015;89:5491–501. [24] Zhao Q, Liu B, Sun Y, Du T, Chen Y, Wang X, et al. Decreased egg production in laying hens associated with infection with genotype 3 avian hepatitis E virus strain from China. Vet Microbiol 2017;203:174–80. [25] Troxler S, Marek A, Prokofieva I, Bilic I, Hess M. TaqMan real-time reverse transcription-PCR assay for universal detection and quantification of avian hepatitis E virus from clinical samples in the presence of a heterologous internal control RNA. J Clin Microbiol 2011;49:1339–46. [26] Ma H, Zheng L, Liu Y, Zhao C, Harrison TJ, Ma Y, et al. Experimental infection of rabbits with rabbit and genotypes 1 and 4 hepatitis E viruses. PLoS One 2010;5:e9160. [27] Clarke JK, Allan GM, Bryson DG, Williams W, Todd D, Mackie DP, et al. Big liver and spleen disease of broiler breeders. Avian Pathol 1990;19:41–50.
Please cite this article in press as: Syed SF et al. Evaluation of recombinant Chinese avian hepatitis E virus (CaHEV) ORF2 and ORF3 proteins for protection of chickens against CaHEV infection. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.030
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S.F. Syed et al. / Vaccine xxx (2017) xxx–xxx
[28] Handlinger JH, Williams W. An egg drop associated with splenomegaly in broiler breeders. Avian Dis 1988;32:773–8. [29] Hsu IW, Tsai HJ. Avian hepatitis E virus in chickens, Taiwan, 2013. Emerg Infect Dis 2014;20:149–51. [30] Billam P, Sun ZF, Meng XJ. Analysis of the complete genomic sequence of an apparently avirulent strain of avian hepatitis E virus (avian HEV) identified major genetic differences compared with the prototype pathogenic strain of avian HEV. J Gen Virol 2007;88:1538–44. [31] Ma H, Song X, Harrison TJ, Li R, Huang G, Zhang H, et al. Immunogenicity and efficacy of a bacterially expressed HEV ORF3 peptide, assessed by experimental infection of primates. Arch Virol 2009;154:1641–8. [32] Li F, Zhuang H, Kolivas S, Locarnini SA, Anderson DA. Persistent and transient antibody responses to hepatitis E virus detected by western immunoblot using open reading frame 2 and 3 and glutathione S-transferase fusion proteins. J Clin Microbiol 1994;32:2060–6. [33] Ruan B, Zhuang H, Ma Y. Dynamics of anti-HEV ORF2, ORF3, IgM and IgG in serial sera of patients with hepatitis E and their clinical significance. Zhonghua Yi Xue Za Zhi 1998;78:498–500.
[34] Guo H, Zhou EM, Sun ZF, Meng XJ, Halbur PG. Identification of B-cell epitopes in the capsid protein of avian hepatitis E virus (avian HEV) that are common to human and swine HEVs or unique to avian HEV. J Gen Virol 2006;87:217–23. [35] Takahashi M, Yamada K, Hoshino Y, Takahashi H, Ichiyama K, Tanaka T, et al. Monoclonal antibodies raised against the ORF3 protein of hepatitis E virus (HEV) can capture HEV particles in culture supernatant and serum but not those in feces. Arch Virol 2008;153:1703–13. [36] Kenney SP, Pudupakam RS, Huang YW, Pierson FW, LeRoith T, Meng XJ. The PSAP motif within the ORF3 protein of an avian strain of the hepatitis E virus is not critical for viral infectivity in vivo but plays a role in virus release. J Virol 2012;86:5637–46. [37] Nagashima S, Takahashi M, Jirintai Tanaka T, Yamada K, Nishizawa T, et al. A PSAP motif in the ORF3 protein of hepatitis E virus is necessary for virion release from infected cells. J Gen Virol 2011;92:269–78. [38] Graff J, Nguyen H, Kasorndorkbua C, Halbur PG, St Claire M, Purcell RH, et al. In vitro and in vivo mutational analysis of the 3’-terminal regions of hepatitis e virus genomes and replicons. J Virol 2005;79:1017–26. [39] Huang YW, Opriessnig T, Halbur PG, Meng XJ. Initiation at the third in-frame AUG codon of open reading frame 3 of the hepatitis E virus is essential for viral infectivity in vivo. J Virol 2007;81:3018–26.
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Evaluation of recombinant Chinese avian hepatitis E virus (CaHEV) ORF2 and ORF3 proteins for protection of chickens against CaHEV infection Shahid Faraz Syed a,b,c,1, Yani Sun a,b,1, Taofeng Du a,b,1, Yiyang Chen a,b, Baoyuan Liu a,b, Xinjie Wang a,b, Huixia Li a,b, Yuchen Nan a,b, En-Min Zhou a,b,⇑, Qin Zhao a,b,⇑ a b c
Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, PR China Scientific Observing and Experimental Station of Veterinary Pharmacology and Diagnosis, China Ministry of Agriculture, Yangling 712100, Shaanxi, PR China Faculty of Veterinary and Animal Sciences, Lasbella University of Agriculture, Water and Marine Sciences, Uthal, Baluchistan, Pakistan
a r t i c l e
i n f o
Article history: Received 12 March 2017 Received in revised form 2 May 2017 Accepted 6 May 2017 Available online xxxx Keywords: Hepatitis E virus Avian HEV ORF2 protein ORF3 protein Protection Vaccine
a b s t r a c t Avian hepatitis E virus (HEV) is the etiologic agent of big liver and spleen disease in chickens. In 2010, the Chinese avian HEV (CaHEV) strain was isolated from chickens and demonstrated to cause the decreased egg production in layer hens. No avian HEV commercial vaccine has yet been developed to prevent virus infection in China. In this study, recombinant CaHEV truncated ORF2 and complete ORF3 proteins were evaluated separately for immunoprotection of chickens against CaHEV infection. First, truncated ORF2 and complete ORF3 proteins were expressed in Escherichia coli. Next, 48 specific-pathogen-free chickens were randomly divided into three groups. One group was immunized with truncated ORF2 protein, the second group was immunized with recombinant ORF3 protein, while the third group (control) was mock-immunized with PBS. After booster immunization, chickens in all three groups were challenged intravenously with CaHEV infectious stock and assessed for viremia, fecal virus shedding, seroconversion, and gross hepatic lesions. In the ORF2 protein-immunized group, no chickens showed evidence of avian HEV infection. In the ORF3 protein-immunized group, nine chickens exhibited viremia and seven had fecal virus shedding. In the control group, all 16 chickens showed viremia and fecal virus shedding. However, the durations in chickens from the ORF3 protein group (2–4 weeks) were shorter than the ones from the control group (4–8 weeks). Moreover, no gross liver lesions emerged in the ORF2 protein group, while lesions observed in the ORF3 protein group were milder than in controls. Therefore, the ORF2 protein can confer complete immunoprotection against chicken CaHEV infection, while the ORF3 protein only confers partial immunoprotection. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Avian hepatitis E virus (HEV) is the causative agent of big liver and spleen disease (hepatitis-splenomegaly syndrome) in chickens [1–3]. Avian HEV infection usually leads to 1–4% increased mortality, 20–40% decreased egg production, and occasionally enlarged livers and spleens in both broiler breeder and laying hens [3–5]. Moreover, anti-avian HEV antibodies and RNA have been frequently detected in apparently healthy chicken flocks [1,6]. Avian HEV was characterized in chickens with hepatitis-splenomegaly syndrome in 2001 in the US and shown to be a variant strain of ⇑ Corresponding authors at: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, PR China. E-mail addresses: [email protected] (E.-M. Zhou), qinzhao_2004@nwsuaf. edu.cn (Q. Zhao). 1 These authors contributed equally to this work.
big liver and spleen disease virus isolated in Australia [1]. To date, the virus has been detected from many countries [4,5,7–9]. Avian HEV is a non-enveloped, single-stranded, positive sense RNA virus in the Hepeviridae family [10]. Various avian HEV strains belong to four major genotypes comprising a single serotype [7,9,11–13]. The avian HEV genome, approximately 6.6 kb in size, is only 48% identical to that of human and swine HEVs and contains three open-reading frames (ORFs) and two noncoding regions [14,15]. The largest ORF, ORF1, encodes non-structural proteins, ORF2 encodes viral capsid protein, and ORF3 encodes a small multifunctional phosphoprotein [15]. Because the lack of a highly efficient in vitro cell culture system has hampered traditional HEV vaccine development, genetic engineering of a subunit vaccine is currently being pursued. Previously, a truncated ORF2 protein from a US viral isolate was expressed in Escherichia coli [16,17], which contains immunodominant antigenic domains, neutralizing epitopes and can induce efficient neu-
http://dx.doi.org/10.1016/j.vaccine.2017.05.030 0264-410X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Syed SF et al. Evaluation of recombinant Chinese avian hepatitis E virus (CaHEV) ORF2 and ORF3 proteins for protection of chickens against CaHEV infection. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.030
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tralizing antibodies to prevent avian HEV infection in chickens [18–21]. Because ORF3 protein can also induce strong immune responses in avian HEV-infected chickens [22,23], it was speculated that the protein may be also a vaccine candidate. But there was no report about evaluation of the ORF3 protein for protection against avian HEV infection in chickens. The Chinese avian HEV strain (CaHEV) was isolated from a chicken with hepatitis-splenomegaly syndrome in 2010 and the virus infection can cause an approximately 10–30% decreased egg production in layer hens [24]. Because there was no commercial vaccine for protection against CaHEV infection in chickens in China, evaluation of CaHEV ORF2 and ORF3 proteins for their ability to confer immunoprotection against CaHEV infection would be of value. Therefore, in this study recombinant CaHEV truncated ORF2 (ORF2-C) and complete ORF3 proteins were expressed in Escherichia coli and immunoprotective effects of purified proteins were compared for their ability to prevent CaHEV infection in chickens. 2. Materials & methods 2.1. Virus An avian HEV infectious stock was produced by intravenously inoculating four 8-week-old specific-pathogen-free (SPF) chickens with 200 ml of a clinical bile sample containing CaHEV (GenBank accession no. GU954430) [9]. At 21 days post-inoculation, viruspositive fecal samples were collected and 10% fecal suspensions were prepared in phosphate-buffered saline (PBS, pH 7.4) containing penicillin-streptomycin then pooled. This CaHEV stock contained 104 genomic equivalents (GE)/ml or 500 CID50/ml of the virus [23].
and after challenge, chicken serum and fecal samples were collected weekly from 0 to 8 week post-challenge (wpc) for nine collections. Serum samples were tested for alanine aminotransferase (ALT) and anti-aHEV IgG antibodies (specific for ORF2-C or ORF3 protein) by indirect enzyme-linked immunosorbent assay (ELISA). Serum and fecal samples were also tested qualitatively and quantitatively for avian HEV RNA by reverse transcription-nested PCR (RT-nPCR) and quantitative RT-PCR (qRT-PCR), respectively. Fecal virus shedding and viremia were monitored for 8 weeks. Two chickens/group were necropsied at 2, 4, 6, and 8 wpc and bile and liver samples were collected. The bile and a portion of the liver sample from each chicken was homogenized in 10% (w/v) sterile PBS, clarified by centrifugation (1000g, 15 min, 4 °C), and tested for avian HEV RNA by RT-nPCR. Gross liver lesions were evaluated and recorded using digital photography. 2.5. Indirect ELISA Before and after immunization and virus challenge, levels of anti-avian HEV antibodies specific for ORF2-C or ORF3 proteins were measured in serum samples from immunized chickens using indirect ELISAs with ORF2-C protein, ORF3 protein, or peptide 10 coating antigens as previously described [19,21,22]. 2.6. Qualitative and quantitative detection of avian HEV RNA using RTnPCR and qRT-PCR Avian HEV RNA was detected in fecal, serum, bile, and liver samples using RT-nPCR followed by sequencing as described previously [19]. In addition, the avian HEV RNA in fecal and serum samples was quantified by qRT-PCR as described by Troxler et al. [25]. 2.7. Determination of serum alanine aminotransferase (ALT) levels
2.2. Animals Forty-eight 6-week-old SPF chickens (Beijing Merial Vital Laboratory Animal Technology Co., Ltd.) tested negative for anti-avian HEV antibodies and RNA. Chickens were divided into three groups (16 chickens/group) that were housed separately in three rooms (2 chickens/cage). Animal experiments were approved by the Animal Care and Use Committee of Northwest A&F University (NWSUAF, Permit Number AE319121) and NWSUAF guidelines were followed. 2.3. Immunization of chickens with recombinant CaHEV ORF2-C and ORF3 proteins CaHEV ORF2-C (containing C-terminal 268 amino acids) and ORF3 recombinant proteins were expressed separately in Escherichia coli RosettaTM (DE3) pLysS host cells and purified using a NiNTA resin column as described previously [21,22]. After one week of acclimation (at age of 7 weeks), Group 1 chickens (Nos. 1–16) were each immunized intramuscularly in both sides of the breast with ORF2-C protein (200 mg/chicken) emulsified in Freund’s complete adjuvant (Sigma-Aldrich, St. Louis, MO, USA) then boosted with 200 mg ORF2-C protein in Freund’s incomplete adjuvant after two weeks. Group 2 chickens (Nos. 17–32) were immunized with purified ORF3 protein using the same procedures and dosages as for group 1. Group 3 chickens (Nos. 33–48) were immunized as above, but with PBS only (negative control). 2.4. Virus challenge and sample collection Two weeks after booster immunizations, all chickens were inoculated intravenously with 800 ml of CaHEV infectious stock. Before
ALT concentrations of serum samples collected weekly were measured (Hitachi 912; Roche, Indianapolis, IN, USA) following manufacturer’s instructions. Biochemical evidence of hepatitis was recorded when serum ALT concentrations exceeded the baseline ALT level by more than twofold, as defined by the peak ALT value that was equal to or greater than twice the geometric mean of the three immediate pre-challenge values. This criterion was based on a previous study correlating ALT level increase with liver pathology in another animal model [21,26]. 2.8. Statistical analysis T-tests (Microsoft Excel 2003) were performed to compare differences in ELISA absorbance values between pre-vaccinated and post-vaccinated chicken sera for ORF2-C and ORF3 antigens and to assess immunoprotective efficacy among ORF2-C protein, ORF3 protein, and PBS immunized groups. ALT values among groups were also analyzed using the same method [21]. P 0.05 was considered significant. 3. Results 3.1. Immune responses in chickens immunized with recombinant ORF2-C or ORF3 proteins In the CaHEV ORF2-C protein-immunized group, chickens showed high anti-ORF2-C protein IgG antibody titers in sera, defined as the highest serum dilution producing an OD450nm value > 0.368 [17]. After the second immunization (but before CaHEV challenge), all chickens immunized with ORF2-C protein had the seroconversion (Fig. 1A) and titers of approximately
Please cite this article in press as: Syed SF et al. Evaluation of recombinant Chinese avian hepatitis E virus (CaHEV) ORF2 and ORF3 proteins for protection of chickens against CaHEV infection. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.030
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Fig. 1. Seroconversion (A) and titers of antibodies (B) against CaHEV ORF2-C protein in chickens immunized with the CaHEV ORF2-C and against CaHEV ORF3 protein in chickens immunized with CaHEV ORF3 protein. Serum samples were collected from chickens at pre-immunization (pre-im) and 2 weeks after first and second immunization (1st-im and 2nd-im). ELISA plates were coated with 200 ng/well of CaHEV ORF2-C protein or the recombinant CaHEV ORF3 protein and each point represents a mean value of OD450nm obtained from the 16 chickens in the same group. The titers of anti-ORF2-C protein IgG antibody in sera were approximately 1:10,000, which was defined as the highest serum dilution producing an OD450nm value >0.368 (Fig. 1A). The titers of anti-ORF3 protein IgG antibody in sera collected 2 weeks after the second immunization were also approximately 1:10,000 defined as the highest serum dilution generating an OD450nm value >0.4.
1:10,000 (Fig. 1B), while the other two groups did not develop specific antibodies against ORF2-C protein (the average OD450 nm value was 0.201). For the CaHEV ORF3 protein-immunized group, anti-avian HEV ORF3 protein antibodies in serum samples collected after booster immunization (prior to CaHEV challenge) were detected via indirect ELISA (using ORF3 protein as coating antigen). Anti-ORF3 protein IgG antibody titers in sera were defined as the highest serum dilution generating an OD450nm value >0.4 [22]. After the second immunization, all the ORF3 immunized chickens exhibited seroconversion for the anti-ORF3 protein antibodies (Fig. 1A) and serum titers of approximately 1:10,000 (Fig. 1B). At a 1:1000 serum dilution, all 16 immunized chickens exhibited positive titers for anti-ORF3 protein IgG antibodies with OD450nm values >1.0. In the ORF2-C protein-immunized and negative control groups, no chickens developed specific antibodies against CaHEV ORF3 protein (the average OD450nm value was 0.247).
3.2. Detection of avian HEV RNA in serum, feces, bile, and liver samples Serum and fecal samples from all chickens were negative for avian HEV RNA at 0 wpc. Post-challenge, numbers of chickens in the three groups testing positive for viremia and fecal virus shedding at different wpc are shown in Table 1. In the CaHEV ORF2-C protein-immunized group, all chickens were negative for viral RNA from 1 to 8 wpc. In the CaHEV ORF3 protein-immunized group, the 7 chickens exhibited fecal virus shedding and 9 ones for viremia, with a lower number observed than in the negative control group. Moreover, the difference in numbers of chickens showing fecal virus shedding between ORF3 protein and PBS immunized groups was significant (P < 0.05). In the negative control group, four chickens exhibited fecal virus shedding throughout the entire experiment. However, in the ORF3 protein-immunized group, no chickens shed virus from 4 to 8 wpc. As for fecal virus shedding, serum viral RNA was first detected at 1 wpc in 5 chickens in the negative control group, but no chickens were positive in the ORF3 protein-immunized group. In addition, avian HEV RNA in feces and sera was detected approximately one week later in the
immunized ORF3 protein group than the negative control group (Table 1). For each non-necropsied chicken in the ORF2-C proteinimmunized group, all chickens were negative for viremia and virus shedding throughout the experiment (data not shown). In the ORF3 protein-immunized group, the duration of viremia and fecal virus shedding of the two chickens (Nos. 19 and 29) were 4 and 2 weeks, respectively (Table 2), with shorter durations of these parameters observed for six other chickens (Nos. 17, 18, 21, 24, 26, and 31). However, in the negative control group, the duration of fecal virus shedding in three chickens (Nos. 33, 35, and 44) was 7 weeks and durations of viremia and fecal virus shedding for the majority of chickens were above 3 weeks. The differences in duration of fecal virus shedding and viremia between ORF3 protein and PBS immunized groups were significant (P < 0.05). For necropsied chickens in the ORF2-C protein-immunized group, all samples were negative for avian HEV RNA in serum, feces, bile, and liver. In the ORF3 protein-immunized group, only one of two necropsied chickens tested positive for avian HEV RNA in serum, feces, bile, and liver at 2 and 4 wpc (Table 3). However, in the negative control group, serum, feces, bile, and liver samples from both necropsied chickens were positive for viral RNA at 2 wpc and one chicken remained positive for viral RNA at 6 and 8 wpc. Viral sequences recovered from infected chickens matched the 536-bp portion of the CaHEV ORF1 coding gene, thus confirming that virus in infected chickens originated from the inoculum. 3.3. Anti-avian HEV ORF3 immune responses after viral challenge Serum samples collected from the 30 chickens not necropsied before 8 wpc in the three groups were tested to evaluate the kinetics of the anti-avian HEV ORF3 protein antibody response. Chickens in the ORF2-C protein-immunized group showed no anti-avian HEV ORF3 protein IgG antibody response throughout the experiment. However, in the ORF3 protein-immunized group, antiORF3 protein antibody levels in the 10 chickens reached high levels after the second immunization, but declined slightly at 1 wpc. At 2–4 wpc, the titers of antibodies against ORF3 protein exhibited
Please cite this article in press as: Syed SF et al. Evaluation of recombinant Chinese avian hepatitis E virus (CaHEV) ORF2 and ORF3 proteins for protection of chickens against CaHEV infection. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.030
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Table 1 Detection of avian HEV RNA in weekly sera and fecal swabs from all chickens challenged with CaHEV during the course of the study. Chicken group
Group 1a Group 2b Group 3c a b c
No. of positive sera (no. of positive fecal swabs)/total no. tested at indicated wpc 0
1
2
3
4
5
6
7
8
0(0)/16 0(0)/16 0(0)/16
0(0)/16 0(0)/16 7(5)/16
0(0)/16 9(7)/16 16(16)/16
0(0)/14 7(3)/14 10(14)/14
0(0)/14 4(0)/14 7(13)/14
0(0)/12 2(0)/12 4(7)/12
0(0)/12 0(0)/12 0(4)/12
0(0)/10 0(0)/10 0(4)/10
0(0)/10 0(0)/10 0(4)/10
Chickens were immunized with recombinant ORF2-C protein. Chickens were immunized with recombinant ORF3 protein. Chickens were the negative control group immunized with PBS.
Table 2 Viremia and fecal virus shedding in chickens not necropsied from CaHEV ORF3 protein and PBS immunized groups.a Group and Chicken No.
Detection of viral RNA in serum samples/fecal swabs at the different wpc 0
a
1
2
3
4
5
6
7
8
Immunized with CaHEV ORF3 protein group 17 –/– 18 –/– 19 –/– 21 –/– 22 –/– 24 –/– 26 –/– 28 –/– 29 –/– 31 –/–
–/– –/– –/– –/– –/– –/– –/– –/– –/– –/–
+/+ +/– +/+ +/+ –/– +/+ +/+ –/– +/+ +/–
–/– +/– +/+ +/– –/– +/– +/– –/– +/+ –/–
–/– –/– +/– –/– –/– +/– –/– –/– +/– –/–
–/– –/– +/– –/– –/– –/– –/– –/– +/– –/–
–/– –/– –/– –/– –/– –/– –/– –/– –/– –/–
–/– –/– –/– –/– –/– –/– –/– –/– –/– –/–
–/– –/– –/– –/– –/– –/– –/– –/– –/– –/–
Negative group mock immunized with PBS 33 –/– 35 –/– 36 –/– 39 –/– 40 –/– 41 –/– 42 –/– 44 –/– 45 –/– 47 –/–
–/– –/– +/+ +/+ +/+ –/– +/– –/– +/+ +/–
+/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+
+/+ –/+ +/+ –/+ +/+ +/+ +/+ +/+ +/+ +/+
+/+ –/+ +/– –/+ +/+ –/+ +/+ +/+ +/+ –/+
+/+ –/+ –/– –/+ –/– –/– +/+ +/+ –/– –/+
–/+ –/+ –/– –/– –/– –/– –/– –/+ –/– –/–
–/+ –/+ –/– –/– –/– –/– –/– –/+ –/– –/–
–/+ –/+ –/– –/– –/– –/– –/– –/+ –/– –/–
Chickens from CaHEV ORF2-C protein immunized group were all negative.
Table 3 Detection of avian HEV RNA in fecal, serum, bile, and liver samples from the chickens necropsied at different wpc. Group
Sample type
No. of positive samples/total no. tested at the different wpc 2
4
6
8
Immunized with CaHEV ORF2-C protein
Feces Serum Bile Liver
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
Immunized with CaHEV ORF3 protein
Feces Serum Bile Liver
1/2 1/2 1/2 1/2
0/2 1/2 1/2 1/2
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
PBS
Feces Serum Bile Liver
2/2 2/2 2/2 2/2
2/2 1/2 2/2 2/2
1/2 0/2 1/2 1/2
1/2 0/2 1/2 1/2
a transient increase in the 10 chickens. The chickens in the negative control group were negative for anti-ORF3 protein antibodies before challenge and 8 chickens become positive for antibodies at 3 wpc. At 5 wpc, antibodies levels against ORF3 protein reach the peak and all the 10 chickens seroconverted and antibody levels declined rapidly (Fig. 2A). At 8 wpc, based on the cut-off values of the indirect ELISA, all the 10 chickens were negative for anti-ORF3 protein antibodies (Fig. 2A).
3.4. Anti-avian HEV ORF2-C and peptide 10 antibody responses after viral challenge Avian HEV ORF2 protein contains the major epitopes of HEV viral particles and is currently universally used as antigen to diagnose avian HEV infection in chickens. In this study, an indirect ELISA using CaHEV ORF2-C protein as coating antigen was used to detect anti-avian HEV ORF2 protein antibodies [17]. For the ORF2-C protein-immunized group, all the 10 chickens were positive for antibodies against the ORF2-C protein at 0 wpc and at 3
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The peptide 10 is the epitope located in the N-terminal of entire avian HEV ORF2 protein, which is lacking in the ORF2-C protein, and can induce the strong immune response in the avian HEV challenged chickens [21]. Therefore, to differentiate between antibodies produced in response to immunization with ORF2-C protein from those produced during avian HEV infection, another indirect ELISA using peptide 10 as coating antigen was performed as described previously [21]. Anti-peptide 10 antibodies were not detected in chickens immunized with the ORF2-C protein from 0 to 8 wpc (Fig. 2C). Moreover, in the ORF3 protein-immunized and negative groups, the changing trends in serum samples for antipeptide 10 antibodies were similar as the ones for anti-ORF2-C protein antibodies from 0 to 8 wpc (Fig. 2C), but the titres were lower. 3.5. Quantitation of avian HEV RNA in fecal and serum samples Besides the qualitative detection of avian HEV RNA in fecal and serum samples via RT-nPCR, viral concentrations were also determined by qRT-PCR as described previously [25]. In the ORF2-C protein-immunized group, the avian HEV RNA concentrations in all fecal and serum samples were <100 copies/mL from 0 to 8 wpc (Fig. 3A). In the ORF3 protein-immunized group, the avian HEV RNA concentrations in serum and fecal samples were ranged from 0 to 4.5 105 copies/mL from 0 to 8 wpc and peaked at 2 wpc, but lower than that from the negative group (Fig. 3A). 3.6. Serum ALT In the ORF2-C protein-immunized group, no ALT level changes were observed in serum samples from the 10 chickens at any wpc (Fig. 3B). In the ORF3 protein-immunized group, 6 chickens were protected against significant ALT elevation following challenge, but the other 4 chickens were only evaluated at 1 wpc (Fig. 3B). However, in the negative control group, the 6 chickens not necropsied before 8 wpc showed significantly increased ALT levels at 1 wpc. In addition, the increasing serum ALT levels form the chickens of the negative group were higher than the ones of the CaHEV ORF3 protein group (Fig. 3B). Thus, serum ALT levels differed significantly at 1 wpc among the three groups, but not at other wpc. 3.7. Gross hepatic lesions
Fig. 2. Variation of anti-avian HEV ORF3 (A), ORF2 (B) proteins and peptide 10 (C) antibody responses in the immunized chickens not necropised before 8 wpi after challenge with CaHEV among the three groups. Sera from 30 chickens were diluted 1:100 for the detection of antibodies against CaHEV ORF3, ORF2 proteins and peptide 10. Each point represents the mean value of OD450nm obtained from 10 chickens of each group ± range.
In the ORF2-C protein-immunized group, no liver hemorrhages were found (Fig. 4). Severe sub-capsular hemorrhages were observed in the livers of necropsied chickens in the negative control and ORF3 protein-immunized groups at 4, 6, and 8 wpc. However, in the ORF3 protein-immunized group, liver hemorrhages in necropsied chickens were less severe than in negative controls. 4. Discussion
wpc, the titers of anti-ORF2 protein antibodies declined slightly. However, in the negative control and ORF3 protein-immunized groups, at 0 wpc (two weeks after booster immunization), there was no detectable antibody against ORF2-C protein. At 3 wpc, the 8 chickens from the negative control group and the 5 chickens from the ORF3 protein-immunized group seroconverted to antiORF2-C protein antibodies (Fig. 2B). At 4 wpc, in the negative group all the 10 chickens seroconverted, but in the ORF3 protein immunized group, the 2 chickens were still negative. In addition, the antibody titers in the negative control group were higher than that in the ORF3 protein-immunized group (Fig. 2B).
Avian HEV infection causes hepatitis-splenomegaly syndrome, resulting in serious economic losses for the poultry industry worldwide [2,4,27,28]. However, no commercial vaccine is yet available for preventing disease in chickens. Previously, it was shown that a truncated ORF2 protein from a US avian HEV strain expressed in Escherichia coli could induce immunoprotection against US avian HEV infection in chickens [21]. However, it is not yet known if the CaHEV ORF2 protein can prevent CaHEV infection in chickens, which can cause decreased egg production in layer hens in China [24]. In addition, the immunoprotective efficacy of avian HEV ORF3 protein is not known, even though it contains known immunodominant epitopes and induces strong immune responses
Please cite this article in press as: Syed SF et al. Evaluation of recombinant Chinese avian hepatitis E virus (CaHEV) ORF2 and ORF3 proteins for protection of chickens against CaHEV infection. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.030
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Fig. 3. Dynamics of viral load in fecal and serum samples (A) and serum levels of ALT in sera (B) from all chickens after the challenge with CaHEV. (A) Y axis represents log10 [copies/ml] of avian HEV RNA in serum and feces. Each point represents the mean value of viral copy numbers obtained from 10 chickens of each group ± range. (B) The mean ALT values at each week post-challenge were generated from the 10 chickens of each group ± range. Statistical significance (*P 0.05).
Fig. 4. Gross hepatic lesions in chickens challenged with CaHEV. None (top row), moderate (middle row), and severe (bottom row) subcapsular liver hemorrhages (arrows) were found in the chickens after virus challenge as indicated. Livers in the top row of images were from CaHEV ORF2-C protein immunized chickens necropsied at 2 (A), 4 (B), 6 (C), and 8 (D) wpc. Middle row images were from chickens in the CaHEV ORF3 protein immunized group necropsied at 2 (E), 4 (F), 6 (G), and 8 (H) wpc. Bottom row images were from chickens in the negative control group necropsied at 2 (I), 4 (J), 6 (K), and 8 (L) wpc.
in virus-infected chickens [22]. In the present study, immunoprotection using CaHEV ORF2-C and ORF3 proteins expressed in Escherichia coli were separately evaluated and suggest that ORF2C protein can provide complete protection against CaHEV infection in chickens, while ORF3 protein only generates partial immunoprotection. Avian HEV strains can be divided into four major distinct genotypes which share approximately 82% identity at the nucleotide level [9,12,13,29,30]. However, these viral strains collectively belong to only a single serotype; therefore, it may be possible to produce a broadly protective vaccine based on the ORF2 viral capsid protein. In this study, immunization of chickens with CaHEV ORF2-C protein induced immunoprotection against CaHEV infec-
tion. These results align with results of a previous study showing that the truncated ORF2 protein from a US avian HEV strain could also provide protection against US avian HEV infection [21]. Therefore, we can only speculate that either CaHEV or US avian HEV ORF2 proteins may induce protection against different avian HEV isolates in chickens, but animal experiments are needed to confirm this speculation. Alternatively, ORF3 protein may also serve as a candidate for a broadly protective vaccine, since one human HEV ORF3 genotype has been documented by several researchers to protect animals against infection with other human HEV genotype strains [31]. Additional animal experiments are still needed to evaluate use of ORF3 protein as a broadly protective vaccine.
Please cite this article in press as: Syed SF et al. Evaluation of recombinant Chinese avian hepatitis E virus (CaHEV) ORF2 and ORF3 proteins for protection of chickens against CaHEV infection. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.030
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Notably, a previous study reported that anti-HEV ORF3 protein antibody titer decreases much more quickly after natural infection than does anti-avian HEV ORF2 protein antibody titer [32,33]. However, how long anti-avian HEV ORF3 protein antibody persists and whether or not duration of persistence depends on original titer are not known. More experiments will be needed to characterize the question. In this study, all ORF3 protein-immunized chickens produced a higher anti-avian HEV ORF3 protein antibody titer and exhibited fewer liver lesions, shortened durations of fecal virus shedding and viremia than controls. But the anti-ORF3 protein antibodies alone did not entirely eliminate viral infection. Previously, three C-terminal antigenic epitopes of the CaHEV ORF3 protein were identified, but their neutralizing characteristics were not studied [22]. Therefore, further studies are needed to determine if these are neutralizing epitopes and to determine which epitope(s) confer protection. In contrast, neutralizing epitopes identified in the avian HEV ORF2 protein could induce immunoprotective responses in chickens [34] and were confirmed here using chickens immunized with CaHEV ORF2-C protein. Unexpectedly, immunization with the CaHEV ORF3 protein only induced partial protection against CaHEV infection in chickens. Possible reasons for lack of protection may be attributed to the various roles that ORF3 protein plays at various times during virus infection. ORF3 encodes a small multifunctional phosphoprotein associated with virus release from host cells in vitro [35–37] and is essential for establishing viral infection in vivo [38,39]. Interestingly, anti-HEV ORF3 protein antibodies have only been observed to capture HEV from serum, not from fecal samples [35]. This observation suggests that the physical location of ORF3 protein within viral particles or its accessibility to antibody binding may differ between ORF3 proteins within serum versus fecal virus particles. Therefore, more experiments are needed to characterize the function and role played by the viral ORF3 protein in the anti-HEV protein immune response. Moreover, the mechanism of immunoprotection achieved by ORF3 protein-specific neutralizing antibodies also remains to be elucidated. This is the first report demonstrating that recombinant CaHEV ORF3 protein expressed in a bacterial system partially protects chickens against CaHEV infection. However, ORF2-C protein provides complete protection that surpasses protection using ORF3 protein. These results should aid understanding of the kinetics of infection and the immune response to virus in its natural host to facilitate vaccine development and prevent avian HEV infection.
Conflict of interest The authors have declared no competing interests exist. Acknowledgments The study is mainly funded by grants from National Natural Science Foundation of China (No. 31402233 and No. 31672583) to QZ and (No. 31372464) to EMZ, Project of Zhongying Distinguished Young Scholars to QZ, Technology Research and innovation of Shaanxi province (2015NY170) to YNS, the Fundamental Research Funds for the Central Universities of China (2452015035) to YNS, (2014YB013) to TFD and (2452016048) to QZ. References [1] Haqshenas G, Shivaprasad HL, Woolcock PR, Read DH, Meng XJ. Genetic identification and characterization of a novel virus related to human hepatitis E virus from chickens with hepatitis-splenomegaly syndrome in the United States. J Gen Virol 2001;82:2449–62.
7
[2] Payne CJ, Ellis TM, Plant SL, Gregory AR, Wilcox GE. Sequence data suggests big liver and spleen disease virus (BLSV) is genetically related to hepatitis E virus. Vet Microbiol 1999;68:119–25. [3] Troxler S, Pac K, Prokofieva I, Liebhart D, Chodakowska B, Furmanek D, et al. Subclinical circulation of avian hepatitis E virus within a multiple-age rearing and broiler breeder farm indicates persistence and vertical transmission of the virus. Avian Pathol 2014;43:310–8. [4] Agunos AC, Yoo D, Youssef SA, Ran D, Binnington B, Hunter DB. Avian hepatitis E virus in an outbreak of hepatitis–splenomegaly syndrome and fatty liver haemorrhage syndrome in two flaxseed-fed layer flocks in Ontario. Avian Pathol 2006;35:404–12. [5] Morrow CJ, Samu G, Matrai E, Klausz A, Wood AM, Richter S, et al. Avian hepatitis E virus infection and possible associated clinical disease in broiler breeder flocks in Hungary. Avian Pathol 2008;37:527–35. [6] Sun ZF, Larsen CT, Dunlop A, Huang FF, Pierson FW, Toth TE, et al. Genetic identification of avian hepatitis E virus (HEV) from healthy chicken flocks and characterization of the capsid gene of 14 avian HEV isolates from chickens with hepatitis-splenomegaly syndrome in different geographical regions of the United States. J Gen Virol 2004;85:693–700. [7] Bilic I, Jaskulska B, Basic A, Morrow CJ, Hess M. Sequence analysis and comparison of avian hepatitis E viruses from Australia and Europe indicate the existence of different genotypes. J Gen Virol 2009;90:863–73. [8] Moon HW, Lee BW, Sung HW, Yoon BI, Kwon HM. Identification and characterization of avian hepatitis E virus genotype 2 from chickens with hepatitis-splenomegaly syndrome in Korea. Virus Genes 2016. [9] Zhao Q, Zhou EM, Dong SW, Qiu HK, Zhang L, Hu SB, et al. Analysis of avian hepatitis E virus from chickens. China. Emerg Infect Dis 2010;16:1469–72. [10] Smith DB, Simmonds P, International Committee on Taxonomy of Viruses Hepeviridae Study G, Jameel S, Emerson SU, Harrison TJ, et al. Consensus proposals for classification of the family Hepeviridae. J Gen Virol 2014;95:2223–32. [11] Gerber PF, Trampel DW, Opriessnig T. Identification and characterization of avian hepatitis E virus in 2013 outbreaks of hepatitis-splenomegaly syndrome in two US layer operations. Avian Pathol 2014;43:357–63. [12] Kwon HM, Sung HW, Meng XJ. Serological prevalence, genetic identification, and characterization of the first strains of avian hepatitis E virus from chickens in Korea. Virus Genes 2012;45:237–45. [13] Banyai K, Toth AG, Ivanics E, Glavits R, Szentpali-Gavaller K, Dan A. Putative novel genotype of avian hepatitis E virus, Hungary, 2010. Emerg Infect Dis 2012;18:1365–8. [14] Huang FF, Sun ZF, Emerson SU, Purcell RH, Shivaprasad HL, Pierson FW, et al. Determination and analysis of the complete genomic sequence of avian hepatitis E virus (avian HEV) and attempts to infect rhesus monkeys with avian HEV. J Gen Virol 2004;85:1609–18. [15] Tam AW, Smith MM, Guerra ME, Huang CC, Bradley DW, Fry KE, et al. Hepatitis E virus (HEV): molecular cloning and sequencing of the full-length viral genome. Virology 1991;185:120–31. [16] Haqshenas G, Huang FF, Fenaux M, Guenette DK, Pierson FW, Larsen CT, et al. The putative capsid protein of the newly identified avian hepatitis E virus shares antigenic epitopes with that of swine and human hepatitis E viruses and chicken big liver and spleen disease virus. J Gen Virol 2002;83:2201–9. [17] Zhao Q, Sun Y, Zhao J, Hu S, Zhao F, Chen F, et al. Development and application of an indirect ELISA for detection of antibodies against avian hepatitis E virus. J Virol Methods 2013;187:32–6. [18] Wang L, Sun Y, Du T, Wang C, Xiao S, Mu Y, et al. Identification of an antigenic domain in the N-terminal region of avian hepatitis E virus (HEV) capsid protein that is not common to swine and human HEVs. J Gen Virol 2014;95:2710–5. [19] Dong S, Zhao Q, Lu M, Sun P, Qiu H, Zhang L, et al. Analysis of epitopes in the capsid protein of avian hepatitis E virus by using monoclonal antibodies. J Virol Methods 2011;171:374–80. [20] Guo H, Zhou EM, Sun ZF, Meng XJ. Immunodominant epitopes mapped by synthetic peptides on the capsid protein of avian hepatitis E virus are nonprotective. Viral Immunol 2008;21:61–7. [21] Guo H, Zhou EM, Sun ZF, Meng XJ. Protection of chickens against avian hepatitis E virus (avian HEV) infection by immunization with recombinant avian HEV capsid protein. Vaccine 2007;25:2892–9. [22] Zhao Q, Sun YN, Hu SB, Wang XJ, Xiao YH, Hsu WH, et al. Characterization of antigenic domains and epitopes in the ORF3 protein of a Chinese isolate of avian hepatitis E virus. Vet Microbiol 2013;167:242–9. [23] Wang X, Zhao Q, Dang L, Sun Y, Gao J, Liu B, et al. Characterization of Two Novel Linear B-Cell Epitopes in the Capsid Protein of Avian Hepatitis E Virus (HEV) That Are Common to Avian, Swine, and Human HEVs. J Virol 2015;89:5491–501. [24] Zhao Q, Liu B, Sun Y, Du T, Chen Y, Wang X, et al. Decreased egg production in laying hens associated with infection with genotype 3 avian hepatitis E virus strain from China. Vet Microbiol 2017;203:174–80. [25] Troxler S, Marek A, Prokofieva I, Bilic I, Hess M. TaqMan real-time reverse transcription-PCR assay for universal detection and quantification of avian hepatitis E virus from clinical samples in the presence of a heterologous internal control RNA. J Clin Microbiol 2011;49:1339–46. [26] Ma H, Zheng L, Liu Y, Zhao C, Harrison TJ, Ma Y, et al. Experimental infection of rabbits with rabbit and genotypes 1 and 4 hepatitis E viruses. PLoS One 2010;5:e9160. [27] Clarke JK, Allan GM, Bryson DG, Williams W, Todd D, Mackie DP, et al. Big liver and spleen disease of broiler breeders. Avian Pathol 1990;19:41–50.
Please cite this article in press as: Syed SF et al. Evaluation of recombinant Chinese avian hepatitis E virus (CaHEV) ORF2 and ORF3 proteins for protection of chickens against CaHEV infection. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.030
8
S.F. Syed et al. / Vaccine xxx (2017) xxx–xxx
[28] Handlinger JH, Williams W. An egg drop associated with splenomegaly in broiler breeders. Avian Dis 1988;32:773–8. [29] Hsu IW, Tsai HJ. Avian hepatitis E virus in chickens, Taiwan, 2013. Emerg Infect Dis 2014;20:149–51. [30] Billam P, Sun ZF, Meng XJ. Analysis of the complete genomic sequence of an apparently avirulent strain of avian hepatitis E virus (avian HEV) identified major genetic differences compared with the prototype pathogenic strain of avian HEV. J Gen Virol 2007;88:1538–44. [31] Ma H, Song X, Harrison TJ, Li R, Huang G, Zhang H, et al. Immunogenicity and efficacy of a bacterially expressed HEV ORF3 peptide, assessed by experimental infection of primates. Arch Virol 2009;154:1641–8. [32] Li F, Zhuang H, Kolivas S, Locarnini SA, Anderson DA. Persistent and transient antibody responses to hepatitis E virus detected by western immunoblot using open reading frame 2 and 3 and glutathione S-transferase fusion proteins. J Clin Microbiol 1994;32:2060–6. [33] Ruan B, Zhuang H, Ma Y. Dynamics of anti-HEV ORF2, ORF3, IgM and IgG in serial sera of patients with hepatitis E and their clinical significance. Zhonghua Yi Xue Za Zhi 1998;78:498–500.
[34] Guo H, Zhou EM, Sun ZF, Meng XJ, Halbur PG. Identification of B-cell epitopes in the capsid protein of avian hepatitis E virus (avian HEV) that are common to human and swine HEVs or unique to avian HEV. J Gen Virol 2006;87:217–23. [35] Takahashi M, Yamada K, Hoshino Y, Takahashi H, Ichiyama K, Tanaka T, et al. Monoclonal antibodies raised against the ORF3 protein of hepatitis E virus (HEV) can capture HEV particles in culture supernatant and serum but not those in feces. Arch Virol 2008;153:1703–13. [36] Kenney SP, Pudupakam RS, Huang YW, Pierson FW, LeRoith T, Meng XJ. The PSAP motif within the ORF3 protein of an avian strain of the hepatitis E virus is not critical for viral infectivity in vivo but plays a role in virus release. J Virol 2012;86:5637–46. [37] Nagashima S, Takahashi M, Jirintai Tanaka T, Yamada K, Nishizawa T, et al. A PSAP motif in the ORF3 protein of hepatitis E virus is necessary for virion release from infected cells. J Gen Virol 2011;92:269–78. [38] Graff J, Nguyen H, Kasorndorkbua C, Halbur PG, St Claire M, Purcell RH, et al. In vitro and in vivo mutational analysis of the 3’-terminal regions of hepatitis e virus genomes and replicons. J Virol 2005;79:1017–26. [39] Huang YW, Opriessnig T, Halbur PG, Meng XJ. Initiation at the third in-frame AUG codon of open reading frame 3 of the hepatitis E virus is essential for viral infectivity in vivo. J Virol 2007;81:3018–26.
Please cite this article in press as: Syed SF et al. Evaluation of recombinant Chinese avian hepatitis E virus (CaHEV) ORF2 and ORF3 proteins for protection of chickens against CaHEV infection. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.030