Detection of Hepatitis E Virus in Raw Pork and Pig Viscera As Food in Hebei Province of China

Abstract

Hepatitis E virus (HEV) is an important human pathogen with pigs serving as the main natural animal reservoir. In China, pork is the most popular meat, while pig viscera are also widely consumed. The aim of this study was to evaluate the prevalence of HEV among pigs at slaughter, and assess the presence of HEV in raw pork and pig viscera as food. Samples of pig blood, raw pork, liver, kidney, and blood curd were collected from slaughterhouse or (and) retail market. Anti-HEV antibody in serum samples was detected using enzyme-linked immunosorbent assay based on an ORF2 antigen sandwich kit. HEV RNA was tested by reverse transcription nested polymerase chain reaction (RT-nested PCR) and the viral load was further assessed using quantitative real-time PCR. The final amplicons of RT-nested PCR were sequenced and undergone phylogenetic analysis. Prevalence of antibodies to HEV was 90.4% (104/115) in pigs at slaughtered level, and one serum sample was HEV RNA positive (0.9%, 1/115). HEV RNA was detected in liver, kidney, and blood samples with positivity of 6.1% (7/114), 3.1% (4/129), and 1.2% (2/170) respectively with viral loads ranged 10^2.4–10^4.4 (2.4Log–4.4Log) genome equivalents per gram, but not in pork. The HEV RNA prevalence in both liver and kidney were statistically higher than in pork. Phylogenetic analysis showed that all obtained sequences belonged to HEV genotype 4, which were divided into subtypes 4a, 4b, 4d, and 4i, highly identical to the known human and swine HEV sequences in China. The results indicate that raw pig viscera are more likely to harbor HEV than pork, suggesting a higher transmission risk related to consuming pig organs.

Introduction

Hepatitis E virus (HEV) is a small nonenveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family (Doceul et al., 2016). Within this family, there are four genotypes of HEV capable of infecting humans, which are classified into the Orthohepevirus A species of Orthohepevirus genus (Smith et al., 2014; Doceul et al., 2016). The four HEV genotypes differ in geographic distribution, host range, and pathogenicity: genotypes 1 and 2 HEV infect only humans and are associated with large waterborne epidemics in developing countries where sanitation conditions are poor; genotypes 3 and 4 (HEV-3 and HEV-4) are present in humans and some animals, and are the main cause of autochthonous cases of hepatitis E in developed countries (Doceul et al., 2016; Brayne et al., 2017). Thus far, genotypes 1–4 HEV are recognized to be the causative agents of human hepatitis E, among them genotypes 3 and 4 HEV are able to transmit from animals to humans and recognized as zoonotic.

Both genotype 3 and genotype 4 HEVs are highly prevalent in pigs, and pigs are the main natural reservoir of the two genotypes (Meng, 2013; Nan et al., 2017). While the exact transmission routes from pigs to humans remain unclear, foodborne transmission through consumption of raw or uncooked pork has been suspected to be an important route (Meng, 2013). The presence of HEV in retail pig liver has been confirmed in the United States of America (Feagins et al., 2007), Japan (Okano et al., 2014) and many European countries (Wenzel et al., 2011; Berto et al., 2012; Di Bartolo et al., 2012). Sausages containing raw pig liver were also found to be contaminated by HEV (Colson et al., 2010; Martin-Latil et al., 2014; Di Bartolo et al., 2015). Some cases with genotypes 3 and 4 have been linked to consumption of raw pork or undercooked pig liver and liver sausages (Yazaki et al., 2003; Colson et al., 2010, 2012).

Mainland China has been considered as a high endemic region of hepatitis E. However, in recent years, hepatitis E mainly occurs as sporadic cases in middle aged and elderly men, and most of the HEV strains isolated from patients are genotype 4 or 3 rather than genotype 1 (Liu et al., 2012). A transition in pattern from high-endemicity to low-endemicity has been observed (Liu et al., 2016; Wang et al., 2016; Sridhar et al., 2017). Consequently, zoonotic transmission from animal to human has attracted more and more attention as a public health problem (Nan et al., 2017).

China is both the largest pork producing and consuming country in the world. Unlike in most Western countries where pork goes through chilling and freezing process before consumption, in China it is more common to consume hot pork, that is, pork delivered directly from slaughterhouse to retail markets in fresh condition without being frozen. In addition to consuming pork muscle and ribs, the Chinese also consume pork viscera as popular food materials. These consumption habits of pork bring great challenges to food safety. This study aims to evaluate the prevalence of HEV in retail raw pork, pig livers, pig kidney, and blood curd collected from slaughterhouse and retail market in Hebei province, China to assess the risk of human contamination through consumption of pig products.

Materials and Methods

Sample collection and processing

From October 2017 to April 2018, swine samples were collected from the slaughterhouses and retail markets in Hebei Province, China. At the slaughterhouses, blood samples were collected individually at the bleeding stage, while pork, liver, and kidney samples were collected after the pigs were slaughtered. Retail pork, livers, kidneys, and blood curd were purchased from retail markets. The pigs were examined by quarantine inspection as they enter the slaughterhouse, and the retail pig pork and viscera were approved for consumption by the Animal Health Supervision Authority. The collected samples were transported to the testing laboratory in cold boxes with ice.

Swine blood samples were centrifuged at 3000 rpm for 10 min, and the sera were collected respectively and stored at −70°C until further use for anti-HEV antibody detection or HEV RNA detection. About 25 g of each sample of the pork muscle, liver, kidney, and blood curd was sliced, crushed to ground, mixed randomly, weighted respectively, and stored at −70°C until further use for HEV RNA detection.

Approximately 0.2 g of each above tissue was lysed and homogenized in 1 mL tissue lysis buffer supplied in the TRIzol Up plus RNA Extraction Kit (TransGen Biotech, Beijing, China), and clarified by centrifugation at 12,000 rpm for 15 min at 4°C. The supernatants from this centrifugation were harvested for HEV RNA detection.

Anti-HEV antibody detection in serum samples

The sera were tested for anti-HEV antibody by using a sandwich ELISA kit (Wantai Biopharmaceutical, Beijing, China) based on recombinant ORF2 antigens. This enzyme-linked immunosorbent assay allowed the detection of all antibody classes (immunoglobulin G, immunoglobulin M, and immunoglobulin A). The performing procedures were according to the manufacturer's instruction.

Detection and analysis of HEV RNA

HEV RNA was extracted from 200 μL serum or supernatants of tissue homogenates and recovered in 40 μL of elution buffer using TRIzol Up plus RNA Kit (TransGen Biotech) according to the instructions. Aliquots of 8 μL of the RNA extracts were used to perform reverse transcription polymerase chain reaction (RT-PCR) in a volume of 20 μL reaction mixture of TransScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech) with oligo (dT) as primer. A 10 μL volume of the cDNA product was for nested PCR. The nested PCR was performed as previously described by using two sets of primers: the external primers are SEBO1 (5′-AAY TAT GCW CAG TAC CGG GTT G-3′) and SEEO1 (5′-CCC TTA TCC TGC TGA GCA TTC TC-3′); and the internal primers are SEBI1 (5′-GTY ATG YTY TGC ATA CAT GGC T-3′) and SEEI1 (5′-AGC CGA CGA AAT YAA TTC TGT C-3′) (Geng et al., 2013). A final amplification product of 348 bp was then analyzed by electrophoresis on a 1.0% (w/v) agarose gel and visualized by ethidium bromide staining. The final amplicons were sequenced with the internal PCR primers commercially by Sangon Biotech Co., Ltd. (Shanghai, China). The final 304 nt sequences obtained by omitted sequences of the primers were aligned with the corresponding regions of reference sequences (Smith et al., 2016) available in GenBank. Phylogenetic analysis was performed with a neighbor-joining algorithm (Molecular Evolutionary Genetics Analysis, MEGA7.0).

Levels of HEV RNA in positive samples were determined by limiting dilution analysis. Aliquots of 10 μL of the undiluted or half-log (10^0.5) fold serially diluted RNA extracts in duplicate was used to perform real-time RT-PCR with primers derived from the ORF1 region as previously described (Zhao et al., 2015). The HEV RNA load in samples of liver, kidney, and blood curd was calculated in genome equivalents per gram (GE/g), while in the serum was calculated in GE/mL. The sensitivity of real-time RT-PCR was 250 GE/g [2.4 (log GE/g)] or 50 GE/mL [1.7 (log GE/g)].

Statistical and spatial analyses

The statistical analyses were performed using SPSS 22 (version 22.0; SPSS, Inc., Chicago, IL). The χ ² test was used to determine whether there is a difference between expected prevalence/detection of HEV and the observed. Results having p-values of <0.05 were considered significant.

Results

HEV seroprevalence in slaughtered pigs

The swine serum samples collected at slaughterhouse were tested for anti-HEV antibody. Of the 115 samples 104 were anti-HEV antibody positive. Thus, the seroprevalence of HEV in pigs at the slaughter level was 90.4% [95% confidence interval (CI) 85.1–95.8)].

The presence of HEV RNA in raw pig liver, kidney, and blood curd

All collected samples undergone HEV RNA detection individually by RT-nested PCR. Of the samples collected from slaughterhouses, one blood, five liver, and two kidney samples were positive for HEV RNA. Of the samples collected from retail markets, two liver and two kidney samples were positive for HEV RNA. None of pork samples from either slaughterhouses or retail markets were positive (Table 1).

Table 1.

Hepatitis E Virus RNA Detection in Raw Pork and Pig Visceral for Consumption

Sample type	Slaughterhouse		Market
Sample type	Sample no.	Positive no.	Sample no.	Positive no.
Pork	117	0	41	0
Liver	79	5	35	2
Kidney	90	2	39	2
Blood^a	115	1	55	1

Samples from slaughterhouses were whole blood, and HEV RNA was detected in serum samples; samples purchased from markets were blood curd made by adding salt to fresh blood.

HEV, hepatitis E virus.

Since there was no significant difference in detection rates of livers or kidneys between samples collected from slaughterhouses and retail markets, the overall prevalence of HEV RNA in terms of sample was calculated. The overall HEV RNA prevalence with 95% CIs of pig liver, kidney, and blood was 6.1% (7/114) [1.6–10.3], 3.1% (4/129) [0.1–6.1], and 1.2% (2/170) [0–2.8] respectively (Table 2). The HEV RNA prevalence in both liver and kidney were statistically higher than in pork (Table 2). The prevalence of HEV RNA was not significantly different between kidney and liver (χ ² = 1.29. p > 0.25).

Table 2.

Comparison of Hepatitis E Virus RNA Positive Rates Between Pork and Viscera

Sample type	Tested no.	Positive no.	Positive rate % (95% CI)	p Value
Pork	158	0	0 (0–1.9)	Reference
Liver	114	7	6.1 (1.7–10.5)	<0.01
Kidney	129	4	3.1 (0.1–6.1)	<0.05
Blood^a	170	2	1.2 (0–2.8)	>0.25

Samples from slaughterhouses were whole blood, and HEV RNA was detected in serum samples; samples purchased from markets were blood curd made by adding salt to fresh blood.

HEV, hepatitis E virus; CI, confidence interval.

Levels of HEV RNA in the positive liver, kidney and blood curd

The positive samples by RT-nested PCR further underwent semi-quantification using real-time RT- PCR. The viral loads in the 13 positive samples ranged from 2.4 to 4.4 log GE/g (Fig. 1).

FIG. 1.

Hepatitis E virus RNA load (log GE/g) of the samples of different type. The detection limit of the protocol was 250 GE/g (2.4 log GE/g) or 50 GE/mL (1.7 log GE/mL). GE, genome equivalents.

Phylogeny of HEV sequences from pig viscera

The final amplicons of RT-nested PCR were conducted for sequences. Samples of the one serum, five livers, four kidneys were able to clearly get sequences. These sequence data have been submitted to the GenBank databases under accession numbers from MH807312 to MH807321. Sequence analysis showed that the 10 swine isolates had 83–98% nucleotide sequence identity. All 10 sequences clustered within genotype 4, and were further divided into subtypes 4a, 4b, 4d, and 4i (Fig. 2). Most the sequences were closely related with the known sequences from patients and (or) pigs (Fig. 2); one isolate ZS35 had 99.1% nucleotide sequence identity with a known isolate previously identified from a patient in this region (GenBank access number: KF691574).

FIG. 2.

Phylogenetic tree of the isolates based on 304 bp nucleotides sequences in ORF2 region. The tree was constructed using the Neighbor-Joining method with 1000 bootstrap replicates. Bootstrap values above a cutoff value of 80% are shown at nodes on the phylogenetic tree. The evolutionary distances were computed using the number of differences method. The scale bar indicates the number of nucleotide substitutions per site. Strains for which sequences were obtained in this study from the serum, livers, and kidneys of pigs are labeled with in diamond, circle dots and triangles respectively. Reference sequences are labeled with the GenBank accession number/host/subgenotype (or none). Evolutionary analyses were conducted in MEGA7.

Discussion

In this study, blood samples were collected individually as pigs were slaughtered. Of the sera, 90.4% (104/115) were anti-HEV antibody positive. Because slaughterhouses purchase live pigs from the surrounding farms, the result confirms that HEV is highly prevalent among pigs in this region. This result is consistent with previous studies, which found the seroprevalence of slaughter age pigs to be 91.4% in Beijing, China (Geng et al., 2011). In European countries, the individual seroprevalence in pigs over 6 months of age varied from 49% to 96% (Salines et al., 2017). The 114 serum samples also underwent HEV RNA detection, in which one sample was found to be positive; the positive rate was 0.9% (1/115) (Table 1). The pigs showed high seroprevalence and low RNA-positive rate in sera, reflecting that most pigs were slaughtered at the ending phase of the epidemic, while a few may still be under ongoing HEV infection. Previous reports in Japan indicates that HEV infects pigs at 2–4 months of age, the fecal shedding peak was in 3 to 4-month-old pigs, and HEV disappears from pigs at 6 months of age or later as a result of the biosynthesis of anti-HEV antibodies (Takahashi and Okamoto, 2014; Sasaki et al., 2018). In general, pigs were slaughtered at age of 6–7 months in China. However, the slaughter age may change as pork price fluctuates: when the pork prices are rising, pigs tend to be slaughtered at an age earlier than usual (Tan et al., 2017). In this case, the slaughter time is more likely to overlap with the peak of the infections, resulting in a higher positive rate of HEV RNA.

The study also found one blood curd sample to be HEV RNA positive, although with low viral load. Blood curd, a popular food in Chinese cuisine, is made from pig blood without undergoing any heating step in production. Since there are pigs slaughtered while still in the period of viremia, it is within expectation to detect contamination in blood curd as well. Note that, the pig blood for making blood curd is pooled from multiple pigs. If one of the pigs is in the period of viremia, the entire bunch of blood curd will become contaminated, while the concentration of virus becomes diluted. Consequently, there may be more blood curd samples that contain HEV but at levels under detection limit.

Numerous studies have reported the presence of HEV RNA in retail pig livers and sausages containing pig liver, demonstrating the transmission risk related to the consumption of pig liver. In this study, we found 6.1% (7/114) liver samples to be HEV RNA positive with the viral load up to 10^4.4 GE/g, further confirming the presence of HEV in livers for food consumption in this region. Although the reported prevalence of HEV RNA in pig livers at slaughter age varies in a wide range from 0.8% to 21% worldwide (Salines et al., 2017), the positive rate we determined appears to be consistent with those previously reported of 3.5% (4/114) in Beijing (Li et al., 2009) and 1.5% (7/479) in Hong Kong (Chan et al., 2017) in China, where genotype 4 HEV is prevalent as the predominant genotype. Moreover, we found HEV RNA to be prevalent at 3.1% (4/129) (Table 2) in pig kidneys, another popular Chinese food item. It is not surprising to detect HEV in pig kidneys, considering that there are pigs with ongoing infection at the slaughterhouse and that extrahepatic HEV dissemination is common in naturally infected pigs (de Deus et al., 2007). The replication of HEV in extrahepatic tissues has been reported in animals both experimental infected and natural infected with the virus. While there is no previous report of HEV RNA in pig kidneys for food at retail market, the presence of HEV RNA in pig intestine purchased from markets has been reported in Hong Kong (Chan et al., 2017).

Consistent with most previous studies (Leblanc et al., 2010; Berto et al., 2012; Feurer et al., 2018), we were unable to detect HEV RNA in pork samples collected from either slaughterhouse or retail market. As a result, the prevalence of HEV RNA in pig liver and kidney are both statistically higher than that of pork (Table 2). Since the pork samples were collected at same time as liver and kidney samples, the difference in prevalence may be attributed to the different dissemination of the virus in the pig body. Compared to blood, liver and other organs are more likely to host prolonged periods of HEV replication. It should also be noted that the pork muscle contains little blood after the slaughter processing, during which the pork is bled and rinsed, while the viscera still contains much blood. This may also result in finding more virus in the viscera than pork, if the slaughtered pig was at viremia period. In this case, although there was no detectable HEV RNA in pork, HEV may still remain in pork at a negligible viral titer. Of note, pig liver and kidney harbored a higher viral load than other pig muscle, indicating a higher risk of transmission associated with the consumption of pig viscera.

In the study, HEV positivity in pig liver and kidney did not markedly decrease from slaughterhouse to market. A recent study demonstrated that HEV virions obtained from cell culture maintained infectivity for up to 21 days at 37°C and for 28 days at room temperature (Johne et al., 2016), which indicates that HEV viral particles can remain highly stable in such environments. Due to the popularity of consuming fresh hot pork in China, pork and pig viscera generally takes less than a day to go from the slaughterhouse to market and from market to kitchen. Thus, the harboring HEV could remain active and present risks to consumers. This is supported by reports of association between hepatitis E seroprevalence and pork consumption (Gonwong et al., 2014). Prevention of zoonotic HEV infection could benefit from enhanced monitoring of the presence of infectious HEV in pork-derived foods. Although pig products for sale are derived from quarantined pigs that are in general considered safe, they may still contain HEV as the current quarantine procedures do not include HEV testing. Further, additional warnings and recommendations could be provided to consumers, urging consumers to properly handle and cook pig products to reduce risks relevant to HEV.

Eight different genotypes have been identified for Orthohepevirus A in Hepeviridae family (Purdy et al., 2017). Among the eight genotypes, genotypes 1–4 are well recognized as human pathogens, and genotype 7 may also have the potential to infect humans; genotypes 3–8 are zoonotic (Purdy et al., 2017). In this study, all HEV sequences detected in pig blood, liver, and kidney belong to genotype 4 and could be further identified as subtypes 4a, 4b, 4d, and 4i (Fig. 2). These subtypes have been previously shown to circulate in pigs and humans in China (Liu et al., 2012). The sequences also showed close relationship to strains derived from human hepatitis E cases from this region, thus indicating a zoonotic transmissibility of the strains to humans.

In conclusion, our study showed that a small percentage of pigs at slaughter age in Hebei province, China may still be under ongoing viremia, and confirmed the presence of genotype 4 HEV RNA in pig viscera sold to end consumers. Importantly, we found pig liver and kidney are more likely to contain HEV than pork, suggesting a higher potential HEV transmission risks related to consuming pig viscera.

Footnotes

Acknowledgments

This study was supported by Natural Science Foundation of Hebei Province, China (grant: H2017201198); Science and Technology Research Project of Hebei Colleges and Universities, China (ZD2018229); and National Science Foundation of China (grant: 81371830).

Disclosure Statement

No competing financial interests exist.

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