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Infection dynamics of hepatitis E virus in naturally infected pigs in a Chinese farrow-to-finish farm
Infection, Genetics and Evolution 11 (2011) 1727–1731
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Infection dynamics of hepatitis E virus in naturally infected pigs in a Chinese farrow-to-finish farm Ruofei Feng a,1, Chenyan Zhao b,1, Mingsheng Li a, Tim J. Harrison c, Zilin Qiao a, Yuping Feng a, Zhongren Ma a, Youchun Wang b,⇑ a b c
Animal Cell Engineering & Technology Research Center of Gansu, Northwest University for Nationalities, No. 1 Xibeixincun, Lanzhou 730030, China Department of Cell Biology, National Institute for the Control of Pharmaceutical and Biological Products, No. 2 Tiantanxili, Beijing 100050, China Division of Medicine, University College London Medical School, Windeyer Building, 46 Cleveland Street, London W1T 4JF, UK
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Article history: Received 26 February 2011 Received in revised form 5 July 2011 Accepted 10 July 2011 Available online 23 July 2011 Keywords: Hepatitis E virus Genotype 4 Swine Infection Dynamics
a b s t r a c t To analyze the changes that occur in pigs during hepatitis E virus (HEV) infection, 256 serial serum samples were obtained from 32 pigs from one pig farm at ages 0 (cord blood), 15, 30, 60, 75, 90, 120, and 150 days. All HEV markers were assayed in these samples and showed that total anti-HEV antibodies and IgG formed two peaks. The first peak occurred at 0–60 days and the second after 75 days. No markers of infection, such as HEV RNA, antigen and anti-HEV IgM, were detectable during the first peak. Most newborn piglets (<24 h of age) were negative for total anti-HEV and IgG. However, colostrum from all of the sows had evidence of these antibodies. Thus, the anti-HEV in the first peak was assumed to be acquired from maternal milk. Some infectious markers were positive at the beginning of second peak. PCR products were cloned and sequenced and the results indicated those sequences belonged to HEV genotype 4. The antibody present during the second peak may be induced by natural infection with HEV. In conclusion, pigs are susceptible to HEV infection and may remain infectious after the first peak of anti-HEV antibody. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction Hepatitis E virus (HEV) is a small non-enveloped virus belonging to the genus Hepevirus (Emerson and Purcell, 2003) in the proposed Hepeviridae family of viruses. HEV is the most common cause of acute hepatitis in adults throughout Asia and the second most common cause in the Middle East, northern Africa and more industrialized countries (Emerson and Purcell, 2003). HEV is transmitted primarily via the fecal–oral route and contaminated drinking water is the most common vehicle of transmission. Recently, several reports have shown that various species of animals, including rodents, swine, wild boar, cows, horses, chicken, and rabbits, may have a high prevalence of anti-HEV antibodies, suggesting that zoonotic transmission of HEV is likely (Arankalle et al., 2002; Adlhoch et al., 2009; Hirano et al., 2003; Ma et al., 2010; Matsuura et al., 2007; Saad et al., 2007; Wang et al., 2002; Xue et al., 2007; Zhao et al., 2009). In 1997, swine HEV was first isolated in pigs from the Midwestern United States. Swine from other countries, such as Australia, China, Vietnam, Korea, Canada, Spain, New Zealand, Japan, Germany and Italy, were also found to be infected ⇑ Corresponding author. Tel.: +86 10 67095415; fax: +86 10 65113538. 1
E-mail addresses: [email protected], [email protected] (Y. Wang). These authors contributed equally to this work.
1567-1348/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2011.07.009
with HEV (Choi et al., 2003; Adlhoch et al., 2009; Garkavenko et al., 2001; Martelli et al., 2010; Okamoto et al., 2001; Pina et al., 2000; Wang et al., 2000; Yoo et al., 2001). Phylogenetic analysis of the swine isolates from China, Japan, and East Asia indicated that they are closely related to the human HEV strains from the same countries (Okamoto et al., 2001; Yu et al., 2009). Experimental transmission studies have shown that human isolates of HEV genotypes 1, 3, and 4 can infect monkeys, and genotypes 3 and 4 isolated from swine are similar to those isolated from humans (Aggarwal et al., 2001). Similarly, genotypes 3 and 4 HEV from pigs can infect rhesus monkeys (Ji et al., 2008). These results strongly suggest that HEV presents a significant zoonotic risk. A high seroprevalence of HEV infection has been shown in pigs (Kunio and Hiroshi, 2007; Drobeniuc et al., 2001; Meng et al., 2002; Yu et al., 2009) and these potentially are reservoirs of human disease (Kunio and Hiroshi, 2007; Pavio et al., 2010). More importantly, the prevalence of anti-HEV in pigs varies with age (Pavio et al., 2010; Wu et al., 2002). By studying the dynamic changes of HEV antigen, and HEV IgG and IgM in pigs of different ages, important information on the dynamics of natural HEV infection of pigs may be gained. The course of HEV genotype 3 infection in pigs had been described in several previous studies (Leblanc et al., 2007; De Deus et al., 2008; Bouwknegt et al., 2009; Kanai et al., 2010; Casas et al., 2011). This study focuses on HEV genotype
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4. This is likely to have important implications for limiting potential zoonotic infections of humans and controlling the spread of HEV in commercial herds of pigs. 2. Materials and methods
(Yu et al., 2009; Zhang et al., 2006) developed by Wantai Biopharmaceutical (Beijing, China). All assay procedures were carried out according to the manufacturers’ instructions. Anti-HEV IgM and IgG were detected by the assays described above. All colostrum samples were tested for anti-HEV IgM, IgG, and total anti-HEV antibody using the same methods.
2.1. Serum samples and colostrum 2.5. Detection of HEV RNA and cloning positive amplicons In this study, 9 L with a total of 45 newborn pigs were first selected within 2 days of birth from one farm in Gansu province, China in 2009. All the selected newborn pigs were labeled on the ears and farmed with their mothers and other pigs together so that the natural infection of HEV could be monitored. Finally, 32 newborn pigs were followed up because others had lost their ear labels or died during the follow-up. Serial serum samples were collected from the 32 pigs at 0, 15, 30, 60, 75, 90, 120 and 150 days of age and stored at 20 °C. Additionally, from the same farm, we collected the colostrum from 25 sows. Animal experimentation was approved by the Local Committee of Laboratory Animal Welfare and Ethics. The national regulations for laboratory animal welfare and ethics were followed. 2.2. Development of an enzyme immunoassay (EIA) to detect anti-HEV IgG HEV recombinant protein NE2 (supplied by Wantai Biotechnology Company, Beijing, China) with an ORF2 immunodominant epitope was used as the antigen to detect anti-HEV IgG. A horseradish peroxidase-conjugated goat anti-swine IgG (KPL, Maryland, USA) was used as the secondary antibody. The cutoff value was determined on the basis of the optical density (OD) values obtained from 100 swine sera (36 total antibody-negative sera and 64 total antibody-positive sera) collected from Beijing Entry–Exit Inspection and Quarantine. The cutoff value was set at 0.12 + NC (mean value of negative control), which was from approximately two standard deviations above the mean OD value of 36 normal sera to ensure that all normal sera were negative and all positive sera from 100 swine positive for IgG antibodies tested positive. All serum samples from swine were tested in duplicate at a dilution of 1:100 in 0.05% Tween–phosphate-buffered saline (PBST) blocking buffer containing 5% non-fat dried milk. 2.3. Development of a capture EIA to detect anti-HEV IgM The IgM capture EIA method was based on the capture of IgM antibodies in sera Plates (Nalge, Nunc International, USA) were coated at 4 °C overnight with anti-swine IgM. After washing and blocking, serum samples were added to the well at 100 lL per well. The plate was incubated at 37 °C for 1 h and then washed five times with 250 lL of PBST. The horseradish peroxidase-labeled recombinant HEV proteins (E2) at a pre-determined concentration (1 lg/mL) were added to each well. After incubation and stopping the reaction, the absorbance of each well was determined at 450 nm using a microplate reader. Cutoff values were determined using HEV-negative sera and defined as the mean OD450 + 3 standard deviations (3 SD = 0.12). Therefore, when the OD450 of a serum sample is greater than cutoff values, the sample is considered positive for IgM. 2.4. Detection of total anti-HEV antibody, anti-HEV IgM, IgG and HEV antigen by EIA All serum samples were tested for total anti-HEV antibody, antiHEV IgM, IgG and HEV antigen using EIA. The total anti-HEV antibody and HEV antigen were detected using direct sandwich EIAs
All serum samples were tested for HEV RNA by nested RT-PCR using degenerate oligonucleotide primers corresponding to a region of ORF2, as described previously (Wang et al., 2002). The expected molecular size of the product of nested RT-PCR was 345 bp. All positive amplicons from the nested RT-PCR were purified from agarose gel with a DNA Product Purification kit (Tiangen, Beijing, China) and ligated to the pMD-18T vector (Takara, China). After transformation of bacteria with the ligation mixture, several recombinant plasmids containing the amplicons were obtained for each positive sample. 2.6. Sequencing of HEV strains and phylogenetic analysis One to three clones were selected and sequenced using vector sequencing primers and an ABI sequencing ready reaction kit (Perkin-Elmer, USA) and the products analyzed on an ABI model 373 automated DNA sequence analyzer. The HEV reference sequences used in the phylogenetic and sequence analyses were as follows: genotype 1: AKL-90 (GenBank accession No. AF124407), Chi (AF141652), and Moro (AF065061); genotype 2: M1 (M74506); genotype 3: 01–9913 (AF466676), HE-JA5 (AB082561) and NLSW20 (AF336290); subtype 4a: Ch87 (AJ344171), HF-030 (AF134916), Ch266 (AJ344193), and Ch-T11 (AF151962); subtype 4b:Ch181 (AJ344188), Ch210 (AJ344192), Ch254 (AJ344186), and LZ105 (AF103940); subtype 4c: HE-JA1 (AB097812), HE-JK4 (AB099347), HE-JI4 (AB080575), JYW-Sap02 (AB161719) and JSMSap95(AB161717); subtype 4d: swCH25 (AY594199), Ch108 (AJ344181), Ch202(AJ344184), and T1(AJ272108); subtype 4e: IND-SW1(AF324501) and IND-SW2 (AF324502); subtype 4f: HEJA2 (AB082558) and subtype 4g: CCC220(AB108537). Sequence alignments were performed using ClustalW (Thompson et al., 1994) to calculate the evolutionary distances between sequences and to generate neighbor-joining phylogenetic tree with 1000 bootstrap replicates. The final output was visualized using the TreeView program (Page, 1996). The nucleotide identity between sequences was calculated using the GeneDoc program (version 3.2). 3. Results 3.1. Prevalence of total anti-HEV antibody, anti-HEV IgM, IgG, HEV antigen and HEV RNA in pigs In this study, 256 serum samples from 32 pigs of various ages (0–150 days) were tested for the presence of total anti-HEV antibody, HEV antigen and viral RNA (Table 1, Fig. 1). At day 0, only 12.5% of the piglets were positive for anti-HEV antibody, but the prevalence increased, reaching the first peak with 100% positive at 15 days. At 60 days, the seroprevalence decreased to 59.4%. After this time, the prevalence increased again from 68.8% at 75 days to 100% at days 120 and 96.9% at days 150. All samples collected at 0, 15, 30, and 60 days were negative for HEV antigen. However, some samples were positive for HEV antigen at 75, 90, 120, and 150 days, with 9.4%, 15.6%, 3.1%, and 3.1% positive, respectively. This indicated the incidence of HEV antigen reached a peak at 90 days, with 15.6% positivity.
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R. Feng et al. / Infection, Genetics and Evolution 11 (2011) 1727–1731 Table 1 Seroprevalence of HEV antigen, anti-HEV IgM, and IgG in 32 pigs at various days after birth. Days
No.
No. positive for HEV Ag (%)
No. positive for anti-IgM (%)
No. positive for anti-IgG (%)
No. positive for total anti-HEV antibody (%)
No. positive for HEV RNA (%)
0 15 30 60 75 90 120 150
32 32 32 32 32 32 32 32
0 0 0 0 3 5 1 1
0 0 0 2 5 4 2 3
0 32 (100) 29 (90.6) 3 (9.4) 7 (21.9) 18 (56.3) 32 (100) 32 (100)
4 (12.5) 32 (100) 32 (100) 19 (59.4) 22 (68.8) 26 (81.3) 32 (100) 31 (96.9)
0 0 0 7 (21.9) 11 (34.4) 5 (15.6) 1 (3.1) 0
(9.4) (15.6) (3.1) (3.1)
(6.2) (15.6) (12.5) (6.3) (9.4)
25
100 90
20
70 60 50
Titer(S/CO)
Positive rate(%)
80
40 30 20 10
15
10
5
0 0
15
30
45
60
75
90
105 120 135 150 165
Days Fig. 1. The change in HEV markers in pigs after birth. (.) Total anti-HEV antibody; (N) anti-HEV IgG; () HEV RNA; (d) anti-HEV IgM; (j) HEV Ag.
All sera were tested for anti-HEV IgM and IgG to determine the type of antibody. Anti-HEV IgG varied from 0% to 100% from birth to 15 days of age, and then decreased to 9.4% at 60 days. After that time, the prevalence of anti-HEV IgG began to increase and reached 100% at 120 and 150 days. Thus, anti-HEV IgG was detected in two peaks. During the first peak of anti-HEV IgG, all the samples were negative for anti-HEV IgM. However, some samples became positive for anti-HEV IgM after the first peak. The incidences of antiHEV IgM were 6.2%, 15.6%, 12.5%, 6.3%, and 9.4% at 60, 75, 90, 120, and 150 days, respectively. Similarly, all samples at the first peak of anti-HEV antibody were negative for HEV RNA. After the first peak, however, some samples at 60, 75, 90, and 120 days were positive for HEV RNA, with positivity of 21.9%, 34.4%, 15.6%, and 3.1%, respectively. At 75 days, the incidence of HEV RNA increased to 34.4% and then decreased to the minimum at 150 days. 3.2. Titers of anti-HEV IgG and total anti-HEV antibody in pigs Two peaks of total anti-HEV and IgG antibody were detected. The titers of the positive samples also were analyzed. The results indicated that the antibody titer (S/CO) in the first peak was lower than that in the second peak (Fig. 2). 3.3. Detection of anti-HEV IgM, IgG and total anti-HEV antibodies in colostrum The colostrum from 25 sows from the same farm was collected and tested for anti-HEV IgM, IgG, and total anti-HEV antibody by ELISA. All colostrum samples were positive for anti-HEV IgG and total anti-HEV antibody, with mean values (S/CO) of 9.15 ± 5.14 and 16.89 ± 4.81, respectively, however, they were negative for
0 0
15
30
45
60
75
90 10 5 120 135 150 165
Days Fig. 2. Dynamic change of titers of total anti-HEV antibody, IgG and IgM. (N) Total antibody; (d) anti-HEV IgG; (j) anti-HEV IgM.
anti-HEV IgM. Because almost all adult pigs were positive for anti-HEV antibody in serum, bovine colostrum was used to validate the EIA and determine whether the higher protein and fat content in colostrum would induce the false positive results. All the colostrum samples from cattle were negative (data not provided) and the higher protein and fat content in colostrum did not influence the EIA results. IgG was present in sow colostrum and these antibodies likely afforded the piglets acquired immunity (Klobasa and Butler, 1987; Takeshi et al., 1980). Therefore, the positive rate and titer curves for anti-HEV IgG and total antibody were higher and appeared during the first antibody peak at 0–30 days, but the sera were negative for anti-HEV IgM. 3.4. Analysis of nucleotide sequences from serum samples of pigs In this study, 24 of the 256 (9.4%) serum samples were positive for HEV RNA using nested RT-PCR which produced a specific ampli con of 345 bp from ORF2. The 24 amplicons were cloned and se quenced, and these sequences were compared with the corre sponding regions of HEV genotypes 1, 2, 3, and 4 reported previously. The 24 sequences were 74–79%, 73–76%, 73–78%, and 80– 100% identical to HEV genotypes 1, 2, 3, and 4, respectively, and appear to belong to HEV genotype 4. All sequences shared 82–100% identity with each other at the nucleotide level. The 24 sequences were grouped into three distinct clades. One clade included 15 isolates (five samples at 60 days, seven sample at 75 days, two sample at 90 days and one sample at 120 days) sharing 86–88% identity with HEV subtype 4f and likely belong to subtype 4f. The second clade, comprising eight isolates (two
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Fig. 3. Phylogenetic tree of hepatitis E virus genotype 4 isolates, based on partial open reading frame 2 sequences (345 nt).
samples at 60 days, four samples at 75 days and two samples at 90 days) was 88–99% identical to the representative isolates of HEV subtype 4d and likely belonged to subtype 4d. One isolate (at 90 days) shared 91% identity with HEV subtype 4a. The neighbor-joining phylogenetic tree (Fig. 3) also showed that these sequences may represent 4f, 4d, and 4a subtypes. Among those sequences, 22 were isolated from individual pigs at one time point. Only two sequences which were isolated from the same pig at two time points, 90 days and 120 days, possessed 100% identity.
4. Discussion It is recognized that pigs are the animal host of HEV genotypes 3 and 4. The prevalence of anti-HEV antibody in swine is very high. According to previous reports, seropositivity for HEV antibody in varies with age. Pig less than 3 months of age have a significantly lower seroprevalence than pigs older than 3 months. To investigate the natural history of HEV infection and dynamic changes of HEV markers, we followed 32 piglets after birth and tested serial serum samples for HEV markers. The current study indicates that two peaks of anti-HEV antibody are typically observed during infection of pigs during the first few months of life. In the first peak, only anti-HEV IgG was present and infectious markers such as anti-HEV IgM, HEV antigen and RNA were not observed. Thus, the first peak of antibody detected likely is acquired from maternal colostrum. Pigs possess an
epithelial-chorial placenta that can prevent IgG from sows being transferred to piglets. Thus, newborn piglets are likely to have no antibody at birth but are able to acquire passive immunity through colostrum because the alimentary tract can absorb whole immunoglobulins during the 36 h after birth (Speer et al., 1957). In this study, total HEV antibody and anti-HEV IgG were detected in all samples of sow colostrum. The antibodies present in the first peak are likely acquired from the colostrum but are only transient. However, the absorbed anti-HEV antibody in piglets may provide temporary protection from HEV infection because no HEV antigens and RNA were detectable during the first peak. Few pigs showed evidence of HEV antigen and/or RNA after the first peak and before the second peak. Because the markers of infection last for only a short time, they were not detected in most pigs. The second peak was of anti-HEV IgG and IgM. However, IgG persists longer than IgM. The antibody detected in this peak is likely to be induced by HEV infection. As shown in Fig. 1, a peak of HEV RNA and antiHEV IgM occurred at 75 days and the peak HEV antigen occurred at 90 days. Thus, the HEV RNA and anti-HEV IgM markers have significant meaning for the early diagnosis of HEV, especially HEV RNA because it was detected before anti-HEV IgM. HEV RNA was detected at 60, 75, 90, and 120 days while HEV antigen was detected from day 75 to day 150. Higher correlations were observed for HEV antigen and HEV RNA. Meanwhile, we noticed that HEV RNA detected by RT-PCR was more sensitive than HEV antigen detected by EIA in this study, whereas the duration of HEV antigen was longer than that of HEV RNA. That viremia
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appeared transiently and the copy number of the RNA shed was low may be the reason. Therefore, the use of HEV antigen in combination with HEV RNA can be very useful for making clear the state of HEV infection. Genomic analysis of the HEV isolates indicates that HEV isolated from this study belonged to genotype 4, specifically the subtypes 4a, 4d and 4f. Multiple HEV strains were identified on the selected farm. This may be due to the introduction of infected sows making several strains prevalent in one farm. A similar situation was reported in another study (Nakai et al., 2006). These subtypes were markedly similar to the strains isolated from humans and other pigs in the same region (Feng et al., 2010; Ma et al., 2010). The close genetic relationship between human and swine HEV suggests zoonotic transmission of genotype 4 HEV in Gansu province. This study indicates that pigs become susceptible to HEV infection somewhere in the interval between 30 and 60 days of age. Therefore, during that period, improvement of feeding condition, facilities or type of farming and control of contaminated sources on the farm may be important to reduce the risk of HEV infection in pigs.
Acknowledgments This study was supported by the Ministry of Science and Technology, China (‘‘863’’ project) (Grant No. 2006AA02Z453).
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Infection dynamics of hepatitis E virus in naturally infected pigs in a Chinese farrow-to-finish farm Ruofei Feng a,1, Chenyan Zhao b,1, Mingsheng Li a, Tim J. Harrison c, Zilin Qiao a, Yuping Feng a, Zhongren Ma a, Youchun Wang b,⇑ a b c
Animal Cell Engineering & Technology Research Center of Gansu, Northwest University for Nationalities, No. 1 Xibeixincun, Lanzhou 730030, China Department of Cell Biology, National Institute for the Control of Pharmaceutical and Biological Products, No. 2 Tiantanxili, Beijing 100050, China Division of Medicine, University College London Medical School, Windeyer Building, 46 Cleveland Street, London W1T 4JF, UK
a r t i c l e
i n f o
Article history: Received 26 February 2011 Received in revised form 5 July 2011 Accepted 10 July 2011 Available online 23 July 2011 Keywords: Hepatitis E virus Genotype 4 Swine Infection Dynamics
a b s t r a c t To analyze the changes that occur in pigs during hepatitis E virus (HEV) infection, 256 serial serum samples were obtained from 32 pigs from one pig farm at ages 0 (cord blood), 15, 30, 60, 75, 90, 120, and 150 days. All HEV markers were assayed in these samples and showed that total anti-HEV antibodies and IgG formed two peaks. The first peak occurred at 0–60 days and the second after 75 days. No markers of infection, such as HEV RNA, antigen and anti-HEV IgM, were detectable during the first peak. Most newborn piglets (<24 h of age) were negative for total anti-HEV and IgG. However, colostrum from all of the sows had evidence of these antibodies. Thus, the anti-HEV in the first peak was assumed to be acquired from maternal milk. Some infectious markers were positive at the beginning of second peak. PCR products were cloned and sequenced and the results indicated those sequences belonged to HEV genotype 4. The antibody present during the second peak may be induced by natural infection with HEV. In conclusion, pigs are susceptible to HEV infection and may remain infectious after the first peak of anti-HEV antibody. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction Hepatitis E virus (HEV) is a small non-enveloped virus belonging to the genus Hepevirus (Emerson and Purcell, 2003) in the proposed Hepeviridae family of viruses. HEV is the most common cause of acute hepatitis in adults throughout Asia and the second most common cause in the Middle East, northern Africa and more industrialized countries (Emerson and Purcell, 2003). HEV is transmitted primarily via the fecal–oral route and contaminated drinking water is the most common vehicle of transmission. Recently, several reports have shown that various species of animals, including rodents, swine, wild boar, cows, horses, chicken, and rabbits, may have a high prevalence of anti-HEV antibodies, suggesting that zoonotic transmission of HEV is likely (Arankalle et al., 2002; Adlhoch et al., 2009; Hirano et al., 2003; Ma et al., 2010; Matsuura et al., 2007; Saad et al., 2007; Wang et al., 2002; Xue et al., 2007; Zhao et al., 2009). In 1997, swine HEV was first isolated in pigs from the Midwestern United States. Swine from other countries, such as Australia, China, Vietnam, Korea, Canada, Spain, New Zealand, Japan, Germany and Italy, were also found to be infected ⇑ Corresponding author. Tel.: +86 10 67095415; fax: +86 10 65113538. 1
E-mail addresses: [email protected], [email protected] (Y. Wang). These authors contributed equally to this work.
1567-1348/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2011.07.009
with HEV (Choi et al., 2003; Adlhoch et al., 2009; Garkavenko et al., 2001; Martelli et al., 2010; Okamoto et al., 2001; Pina et al., 2000; Wang et al., 2000; Yoo et al., 2001). Phylogenetic analysis of the swine isolates from China, Japan, and East Asia indicated that they are closely related to the human HEV strains from the same countries (Okamoto et al., 2001; Yu et al., 2009). Experimental transmission studies have shown that human isolates of HEV genotypes 1, 3, and 4 can infect monkeys, and genotypes 3 and 4 isolated from swine are similar to those isolated from humans (Aggarwal et al., 2001). Similarly, genotypes 3 and 4 HEV from pigs can infect rhesus monkeys (Ji et al., 2008). These results strongly suggest that HEV presents a significant zoonotic risk. A high seroprevalence of HEV infection has been shown in pigs (Kunio and Hiroshi, 2007; Drobeniuc et al., 2001; Meng et al., 2002; Yu et al., 2009) and these potentially are reservoirs of human disease (Kunio and Hiroshi, 2007; Pavio et al., 2010). More importantly, the prevalence of anti-HEV in pigs varies with age (Pavio et al., 2010; Wu et al., 2002). By studying the dynamic changes of HEV antigen, and HEV IgG and IgM in pigs of different ages, important information on the dynamics of natural HEV infection of pigs may be gained. The course of HEV genotype 3 infection in pigs had been described in several previous studies (Leblanc et al., 2007; De Deus et al., 2008; Bouwknegt et al., 2009; Kanai et al., 2010; Casas et al., 2011). This study focuses on HEV genotype
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4. This is likely to have important implications for limiting potential zoonotic infections of humans and controlling the spread of HEV in commercial herds of pigs. 2. Materials and methods
(Yu et al., 2009; Zhang et al., 2006) developed by Wantai Biopharmaceutical (Beijing, China). All assay procedures were carried out according to the manufacturers’ instructions. Anti-HEV IgM and IgG were detected by the assays described above. All colostrum samples were tested for anti-HEV IgM, IgG, and total anti-HEV antibody using the same methods.
2.1. Serum samples and colostrum 2.5. Detection of HEV RNA and cloning positive amplicons In this study, 9 L with a total of 45 newborn pigs were first selected within 2 days of birth from one farm in Gansu province, China in 2009. All the selected newborn pigs were labeled on the ears and farmed with their mothers and other pigs together so that the natural infection of HEV could be monitored. Finally, 32 newborn pigs were followed up because others had lost their ear labels or died during the follow-up. Serial serum samples were collected from the 32 pigs at 0, 15, 30, 60, 75, 90, 120 and 150 days of age and stored at 20 °C. Additionally, from the same farm, we collected the colostrum from 25 sows. Animal experimentation was approved by the Local Committee of Laboratory Animal Welfare and Ethics. The national regulations for laboratory animal welfare and ethics were followed. 2.2. Development of an enzyme immunoassay (EIA) to detect anti-HEV IgG HEV recombinant protein NE2 (supplied by Wantai Biotechnology Company, Beijing, China) with an ORF2 immunodominant epitope was used as the antigen to detect anti-HEV IgG. A horseradish peroxidase-conjugated goat anti-swine IgG (KPL, Maryland, USA) was used as the secondary antibody. The cutoff value was determined on the basis of the optical density (OD) values obtained from 100 swine sera (36 total antibody-negative sera and 64 total antibody-positive sera) collected from Beijing Entry–Exit Inspection and Quarantine. The cutoff value was set at 0.12 + NC (mean value of negative control), which was from approximately two standard deviations above the mean OD value of 36 normal sera to ensure that all normal sera were negative and all positive sera from 100 swine positive for IgG antibodies tested positive. All serum samples from swine were tested in duplicate at a dilution of 1:100 in 0.05% Tween–phosphate-buffered saline (PBST) blocking buffer containing 5% non-fat dried milk. 2.3. Development of a capture EIA to detect anti-HEV IgM The IgM capture EIA method was based on the capture of IgM antibodies in sera Plates (Nalge, Nunc International, USA) were coated at 4 °C overnight with anti-swine IgM. After washing and blocking, serum samples were added to the well at 100 lL per well. The plate was incubated at 37 °C for 1 h and then washed five times with 250 lL of PBST. The horseradish peroxidase-labeled recombinant HEV proteins (E2) at a pre-determined concentration (1 lg/mL) were added to each well. After incubation and stopping the reaction, the absorbance of each well was determined at 450 nm using a microplate reader. Cutoff values were determined using HEV-negative sera and defined as the mean OD450 + 3 standard deviations (3 SD = 0.12). Therefore, when the OD450 of a serum sample is greater than cutoff values, the sample is considered positive for IgM. 2.4. Detection of total anti-HEV antibody, anti-HEV IgM, IgG and HEV antigen by EIA All serum samples were tested for total anti-HEV antibody, antiHEV IgM, IgG and HEV antigen using EIA. The total anti-HEV antibody and HEV antigen were detected using direct sandwich EIAs
All serum samples were tested for HEV RNA by nested RT-PCR using degenerate oligonucleotide primers corresponding to a region of ORF2, as described previously (Wang et al., 2002). The expected molecular size of the product of nested RT-PCR was 345 bp. All positive amplicons from the nested RT-PCR were purified from agarose gel with a DNA Product Purification kit (Tiangen, Beijing, China) and ligated to the pMD-18T vector (Takara, China). After transformation of bacteria with the ligation mixture, several recombinant plasmids containing the amplicons were obtained for each positive sample. 2.6. Sequencing of HEV strains and phylogenetic analysis One to three clones were selected and sequenced using vector sequencing primers and an ABI sequencing ready reaction kit (Perkin-Elmer, USA) and the products analyzed on an ABI model 373 automated DNA sequence analyzer. The HEV reference sequences used in the phylogenetic and sequence analyses were as follows: genotype 1: AKL-90 (GenBank accession No. AF124407), Chi (AF141652), and Moro (AF065061); genotype 2: M1 (M74506); genotype 3: 01–9913 (AF466676), HE-JA5 (AB082561) and NLSW20 (AF336290); subtype 4a: Ch87 (AJ344171), HF-030 (AF134916), Ch266 (AJ344193), and Ch-T11 (AF151962); subtype 4b:Ch181 (AJ344188), Ch210 (AJ344192), Ch254 (AJ344186), and LZ105 (AF103940); subtype 4c: HE-JA1 (AB097812), HE-JK4 (AB099347), HE-JI4 (AB080575), JYW-Sap02 (AB161719) and JSMSap95(AB161717); subtype 4d: swCH25 (AY594199), Ch108 (AJ344181), Ch202(AJ344184), and T1(AJ272108); subtype 4e: IND-SW1(AF324501) and IND-SW2 (AF324502); subtype 4f: HEJA2 (AB082558) and subtype 4g: CCC220(AB108537). Sequence alignments were performed using ClustalW (Thompson et al., 1994) to calculate the evolutionary distances between sequences and to generate neighbor-joining phylogenetic tree with 1000 bootstrap replicates. The final output was visualized using the TreeView program (Page, 1996). The nucleotide identity between sequences was calculated using the GeneDoc program (version 3.2). 3. Results 3.1. Prevalence of total anti-HEV antibody, anti-HEV IgM, IgG, HEV antigen and HEV RNA in pigs In this study, 256 serum samples from 32 pigs of various ages (0–150 days) were tested for the presence of total anti-HEV antibody, HEV antigen and viral RNA (Table 1, Fig. 1). At day 0, only 12.5% of the piglets were positive for anti-HEV antibody, but the prevalence increased, reaching the first peak with 100% positive at 15 days. At 60 days, the seroprevalence decreased to 59.4%. After this time, the prevalence increased again from 68.8% at 75 days to 100% at days 120 and 96.9% at days 150. All samples collected at 0, 15, 30, and 60 days were negative for HEV antigen. However, some samples were positive for HEV antigen at 75, 90, 120, and 150 days, with 9.4%, 15.6%, 3.1%, and 3.1% positive, respectively. This indicated the incidence of HEV antigen reached a peak at 90 days, with 15.6% positivity.
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R. Feng et al. / Infection, Genetics and Evolution 11 (2011) 1727–1731 Table 1 Seroprevalence of HEV antigen, anti-HEV IgM, and IgG in 32 pigs at various days after birth. Days
No.
No. positive for HEV Ag (%)
No. positive for anti-IgM (%)
No. positive for anti-IgG (%)
No. positive for total anti-HEV antibody (%)
No. positive for HEV RNA (%)
0 15 30 60 75 90 120 150
32 32 32 32 32 32 32 32
0 0 0 0 3 5 1 1
0 0 0 2 5 4 2 3
0 32 (100) 29 (90.6) 3 (9.4) 7 (21.9) 18 (56.3) 32 (100) 32 (100)
4 (12.5) 32 (100) 32 (100) 19 (59.4) 22 (68.8) 26 (81.3) 32 (100) 31 (96.9)
0 0 0 7 (21.9) 11 (34.4) 5 (15.6) 1 (3.1) 0
(9.4) (15.6) (3.1) (3.1)
(6.2) (15.6) (12.5) (6.3) (9.4)
25
100 90
20
70 60 50
Titer(S/CO)
Positive rate(%)
80
40 30 20 10
15
10
5
0 0
15
30
45
60
75
90
105 120 135 150 165
Days Fig. 1. The change in HEV markers in pigs after birth. (.) Total anti-HEV antibody; (N) anti-HEV IgG; () HEV RNA; (d) anti-HEV IgM; (j) HEV Ag.
All sera were tested for anti-HEV IgM and IgG to determine the type of antibody. Anti-HEV IgG varied from 0% to 100% from birth to 15 days of age, and then decreased to 9.4% at 60 days. After that time, the prevalence of anti-HEV IgG began to increase and reached 100% at 120 and 150 days. Thus, anti-HEV IgG was detected in two peaks. During the first peak of anti-HEV IgG, all the samples were negative for anti-HEV IgM. However, some samples became positive for anti-HEV IgM after the first peak. The incidences of antiHEV IgM were 6.2%, 15.6%, 12.5%, 6.3%, and 9.4% at 60, 75, 90, 120, and 150 days, respectively. Similarly, all samples at the first peak of anti-HEV antibody were negative for HEV RNA. After the first peak, however, some samples at 60, 75, 90, and 120 days were positive for HEV RNA, with positivity of 21.9%, 34.4%, 15.6%, and 3.1%, respectively. At 75 days, the incidence of HEV RNA increased to 34.4% and then decreased to the minimum at 150 days. 3.2. Titers of anti-HEV IgG and total anti-HEV antibody in pigs Two peaks of total anti-HEV and IgG antibody were detected. The titers of the positive samples also were analyzed. The results indicated that the antibody titer (S/CO) in the first peak was lower than that in the second peak (Fig. 2). 3.3. Detection of anti-HEV IgM, IgG and total anti-HEV antibodies in colostrum The colostrum from 25 sows from the same farm was collected and tested for anti-HEV IgM, IgG, and total anti-HEV antibody by ELISA. All colostrum samples were positive for anti-HEV IgG and total anti-HEV antibody, with mean values (S/CO) of 9.15 ± 5.14 and 16.89 ± 4.81, respectively, however, they were negative for
0 0
15
30
45
60
75
90 10 5 120 135 150 165
Days Fig. 2. Dynamic change of titers of total anti-HEV antibody, IgG and IgM. (N) Total antibody; (d) anti-HEV IgG; (j) anti-HEV IgM.
anti-HEV IgM. Because almost all adult pigs were positive for anti-HEV antibody in serum, bovine colostrum was used to validate the EIA and determine whether the higher protein and fat content in colostrum would induce the false positive results. All the colostrum samples from cattle were negative (data not provided) and the higher protein and fat content in colostrum did not influence the EIA results. IgG was present in sow colostrum and these antibodies likely afforded the piglets acquired immunity (Klobasa and Butler, 1987; Takeshi et al., 1980). Therefore, the positive rate and titer curves for anti-HEV IgG and total antibody were higher and appeared during the first antibody peak at 0–30 days, but the sera were negative for anti-HEV IgM. 3.4. Analysis of nucleotide sequences from serum samples of pigs In this study, 24 of the 256 (9.4%) serum samples were positive for HEV RNA using nested RT-PCR which produced a specific ampli con of 345 bp from ORF2. The 24 amplicons were cloned and se quenced, and these sequences were compared with the corre sponding regions of HEV genotypes 1, 2, 3, and 4 reported previously. The 24 sequences were 74–79%, 73–76%, 73–78%, and 80– 100% identical to HEV genotypes 1, 2, 3, and 4, respectively, and appear to belong to HEV genotype 4. All sequences shared 82–100% identity with each other at the nucleotide level. The 24 sequences were grouped into three distinct clades. One clade included 15 isolates (five samples at 60 days, seven sample at 75 days, two sample at 90 days and one sample at 120 days) sharing 86–88% identity with HEV subtype 4f and likely belong to subtype 4f. The second clade, comprising eight isolates (two
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Fig. 3. Phylogenetic tree of hepatitis E virus genotype 4 isolates, based on partial open reading frame 2 sequences (345 nt).
samples at 60 days, four samples at 75 days and two samples at 90 days) was 88–99% identical to the representative isolates of HEV subtype 4d and likely belonged to subtype 4d. One isolate (at 90 days) shared 91% identity with HEV subtype 4a. The neighbor-joining phylogenetic tree (Fig. 3) also showed that these sequences may represent 4f, 4d, and 4a subtypes. Among those sequences, 22 were isolated from individual pigs at one time point. Only two sequences which were isolated from the same pig at two time points, 90 days and 120 days, possessed 100% identity.
4. Discussion It is recognized that pigs are the animal host of HEV genotypes 3 and 4. The prevalence of anti-HEV antibody in swine is very high. According to previous reports, seropositivity for HEV antibody in varies with age. Pig less than 3 months of age have a significantly lower seroprevalence than pigs older than 3 months. To investigate the natural history of HEV infection and dynamic changes of HEV markers, we followed 32 piglets after birth and tested serial serum samples for HEV markers. The current study indicates that two peaks of anti-HEV antibody are typically observed during infection of pigs during the first few months of life. In the first peak, only anti-HEV IgG was present and infectious markers such as anti-HEV IgM, HEV antigen and RNA were not observed. Thus, the first peak of antibody detected likely is acquired from maternal colostrum. Pigs possess an
epithelial-chorial placenta that can prevent IgG from sows being transferred to piglets. Thus, newborn piglets are likely to have no antibody at birth but are able to acquire passive immunity through colostrum because the alimentary tract can absorb whole immunoglobulins during the 36 h after birth (Speer et al., 1957). In this study, total HEV antibody and anti-HEV IgG were detected in all samples of sow colostrum. The antibodies present in the first peak are likely acquired from the colostrum but are only transient. However, the absorbed anti-HEV antibody in piglets may provide temporary protection from HEV infection because no HEV antigens and RNA were detectable during the first peak. Few pigs showed evidence of HEV antigen and/or RNA after the first peak and before the second peak. Because the markers of infection last for only a short time, they were not detected in most pigs. The second peak was of anti-HEV IgG and IgM. However, IgG persists longer than IgM. The antibody detected in this peak is likely to be induced by HEV infection. As shown in Fig. 1, a peak of HEV RNA and antiHEV IgM occurred at 75 days and the peak HEV antigen occurred at 90 days. Thus, the HEV RNA and anti-HEV IgM markers have significant meaning for the early diagnosis of HEV, especially HEV RNA because it was detected before anti-HEV IgM. HEV RNA was detected at 60, 75, 90, and 120 days while HEV antigen was detected from day 75 to day 150. Higher correlations were observed for HEV antigen and HEV RNA. Meanwhile, we noticed that HEV RNA detected by RT-PCR was more sensitive than HEV antigen detected by EIA in this study, whereas the duration of HEV antigen was longer than that of HEV RNA. That viremia
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appeared transiently and the copy number of the RNA shed was low may be the reason. Therefore, the use of HEV antigen in combination with HEV RNA can be very useful for making clear the state of HEV infection. Genomic analysis of the HEV isolates indicates that HEV isolated from this study belonged to genotype 4, specifically the subtypes 4a, 4d and 4f. Multiple HEV strains were identified on the selected farm. This may be due to the introduction of infected sows making several strains prevalent in one farm. A similar situation was reported in another study (Nakai et al., 2006). These subtypes were markedly similar to the strains isolated from humans and other pigs in the same region (Feng et al., 2010; Ma et al., 2010). The close genetic relationship between human and swine HEV suggests zoonotic transmission of genotype 4 HEV in Gansu province. This study indicates that pigs become susceptible to HEV infection somewhere in the interval between 30 and 60 days of age. Therefore, during that period, improvement of feeding condition, facilities or type of farming and control of contaminated sources on the farm may be important to reduce the risk of HEV infection in pigs.
Acknowledgments This study was supported by the Ministry of Science and Technology, China (‘‘863’’ project) (Grant No. 2006AA02Z453).
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