International Journal of Food Microbiology 193 (2015) 29–33
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Short communication
Detection of hepatitis E virus in pork liver sausages Ilaria Di Bartolo a,⁎,1, Giorgia Angeloni a,1, Eleonora Ponterio a, Fabio Ostanello b, Franco Maria Ruggeri a a b
Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy Department of Veterinary Medical Sciences, University of Bologna, Ozzano Emilia, BO, Italy
a r t i c l e
i n f o
Article history: Received 21 March 2014 Received in revised form 30 September 2014 Accepted 4 October 2014 Available online 13 October 2014 Keywords: Hepatitis E Zoonosis Foodborne Murine norovirus Porcine adenovirus Real-time RT-PCR
a b s t r a c t Hepatitis E infection is regarded as an emerging public-health concern. The disease is normally self-limiting (mortality rate 1%), but chronic infections have recently been observed in transplanted patients. The etiological agent HEV is a small RNA virus infecting both humans and animals. In humans, the disease may be food-borne and pig is a main reservoir for zoonotic strains. In the present study, we evaluated the presence of HEV and swine fecal cross-contamination in pork liver sausages sold at a grocery store in Italy. HEV genome detection was performed by RT-qPCR, using harmonized protocols that included a process control (murine norovirus) and an internal amplification control. Swine fecal cross-contamination was assessed by determination of the ubiquitous porcine adenovirus. Overall, HEV genome belonging to genotype 3 was detected in both raw (10 out of 45 slices, 250 mg each, 22.2%) and dry (1 of 23 slices, 4.3%) liver sausages, but infectivity of the virus was not demonstrated. This pilot study fosters more investigations on HEV presence in pork-derived food, to assess the possible risk for the consumers. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Hepatitis E is a usually acute human disease caused by the hepatitis E virus (HEV) (Aggarwal and Naik, 2009). In industrialized countries, the infection was initially considered associated to travel in low-income countries, where the disease is endemic. Conversely, in the last 10 years an increasing number of autochthonous cases have been reported worldwide, including Europe (Aggarwal and Jameel, 2011). The disease is usually self-limiting and presents low mortality, but it can become chronic in transplanted patients and may be highly lethal among pregnant women (Aggarwal and Jameel, 2011). HEV infects humans and several animal species. Four mammalian HEV genotypes are recognized, among which genotypes 1 and 2 are restricted to humans, whereas genotypes 3 and 4 infect both humans and many animal species (pig, deer, wild boar, and rabbit) and circulate in developed countries (Meng, 2013). These two latter genotypes are considered zoonotic, and pigs and less frequently other animal species (wild boar, deer) act as reservoir. Genotype 3 was the first HEV strain detected in animals, specifically pigs (Meng et al., 1997), and it circulates mostly in US and Europe (Meng, 2013). Food-borne transmission of HEV has been increasingly reported in sporadic cases and small outbreaks associated with the consumption of raw or undercooked boar or deer
⁎ Corresponding author. Tel.: +39 06 4990 2787; fax: +39 06 4938 7101. E-mail addresses: [email protected] (I. Di Bartolo), [email protected] (G. Angeloni), [email protected] (E. Ponterio), [email protected] (F. Ostanello), [email protected] (F.M. Ruggeri). 1 These authors contributed equally to this article.
http://dx.doi.org/10.1016/j.ijfoodmicro.2014.10.005 0168-1605/© 2014 Elsevier B.V. All rights reserved.
meat, livers and liver sausages found to contain HEV (Colson et al., 2010; Masuda et al., 2005; Tei et al., 2003; Yazaki et al., 2003). Most importantly, HEV was detected frequently in both commercial livers and sausages sampled in grocery stores. In Japan, US and the Netherlands, 2–4.3%, 11% and 6.5% of commercial liver were positive for swine HEV RNA (Bouwknegt et al., 2007; Feagins et al., 2007; Okano et al., 2014; Yazaki et al., 2003) as were almost 31% of porkderivatives such as sausage (Martin-Latil et al., 2014). More recently, a study in UK showed that consumption of some pork-derived food was a risk factor for HEV infection (Said et al., 2014), and a small outbreak reported in France was linked to consumption of Corse pig liver sausage figatellu, found to be contaminated by genotype 3 HEV (Colson et al., 2010). In Italy, genotype 3 HEV was detected in swine and wild boar belonging to different age classes (Di Bartolo et al., 2008; Martinelli et al., 2013). Hepatitis E infection is widespread in pig herds affecting animals of different ages, including those doomed to slaughterhouse (Di Bartolo et al., 2011). HEV genomic RNA was detected in both swine feces and liver, and recently it was found along the pork production chain in Italy, specifically in two meat samples (6%) but not in sausage (Di Bartolo et al., 2012). In the same investigation, 6% and 10% of pork sausages at point of sale were positive for HEV genome in Spain (Di Bartolo et al., 2012) and in UK (Berto et al., 2012). In the last few years, an increase in the number of autochthonous cases of human hepatitis E has been reported also in Italy. These were associated mostly to genotype 3 and more rarely to genotype 4 (Garbuglia et al., 2013; La Rosa et al., 2011). The role of consumption of pork-derived food should be investigated to assess the risk of foodborne HEV transmission. The main site of HEV replication is the liver, suggesting that the presence
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of endogenous virus in liver sausages may be a main risk factor. However, the virus is massively shed with swine feces (Di Bartolo et al., 2008), which may represent a possible source of HEV contamination during slaughtering and pork product processing. In the present study, we investigated the presence of HEV in both fresh and dry pork liver sausages in Italy, where they are frequently consumed raw or undercooked. Moreover, we evaluated the presence of porcine adenovirus in sausages as a relevant indicator of swine fecal contamination, since this virus is normally detected between 90 and 98% of swine fecal samples (Di Bartolo et al., 2012; Maluquer de Motes et al., 2004). 2. Materials and methods 2.1. Sampling Pork liver sausages, fresh and dry, were purchased in a butcher shop in Rome, Italy, where both processing and packaging were finished. Dry sausages were desiccated in the air during a 10 to 15 day maturation period. Four packs containing a total of 15 fresh liver sausages (about 80 g per sausage) and four packs containing 14 dry sausages were bought. To ensure the sampling of different production lots, the packs were bought in four different weeks between January and February 2013, when these products are more typically consumed. Each sausage was chopped in three slices, obtaining a total of 45 and 42 slices for fresh and dry liver sausages, respectively. Fat was discarded manually from slices, 250 mg of which was collected and subjected to RNA extraction, individually. 2.2. Sample preparation and nucleic acid extraction Before RNA/DNA extraction, all samples were artificially contaminated with 5 μl of a suspension of murine norovirus (MNV-1; 105 TCID50), which was used as sample process control. The virus was kindly provided by Dr. Virgin from Washington University, USA, and was prepared as described previously (Di Bartolo et al., 2012). Spiked samples were homogenized in 2.5 ml of lysis buffer (RTL) with 1 sterile stainless steel bead (5 mm, QIAGEN), using a mechanical disruptor (Tissue Lyser– QIAGEN) for three runs of 2 min at 46 oscillations s−1. After centrifugation at 5000 ×g per 20 min, the supernatant was used to extract nucleic acids by the RNeasy Midi kit (QIAGEN), according to the manufacturer's instructions and it was eluted in 100 μl of ddH2O. 2.3. Real time-PCR All reaction mixtures included an Internal Amplification Control IAC (Diez-Valcarce et al., 2011) and were in duplex format, targeting specific viruses (Murine NoV, HEV, Porcine Adenovirus) and IACs labeled with FAM (6-carboxy fluorescein) and VIC (5′-VIC-CCATACACATAGGTC AGG-MGB-NFQ-3′; Lifetechnologies) probes, respectively. All experiments included negative and positive controls, with and without IACs. 2.3.1. PAdV real time-PCR Ten microliters of extracted nucleic acids were analyzed for PAdV by duplex real-time PCR including IACs, with primers PAdV-F (5′-AACGGC CGCTACTGCAAG-3′) and PAdV-R (5′-AGCAGCAGGCTCTTGAGG-3′) and probe PAdV-P (5′-FAM-CACATCCAGGTGCCGC-BHQ1-3′) within the hexon region, using the TaqMan Universal PCR Master-Mix (Lifetechnologies) reaction kit, as previously described (Di Bartolo et al., 2012; Hundesa et al., 2009). 2.3.2. RT-qPCR for detection of MNV-1 and HEV Ten microliters of RNA was analyzed for detection of MNV-1 or HEV by a duplex one step reverse-transcription qPCR detecting both the IAC (VIC-labeled probe) (Diez-Valcarce et al., 2011) and the target viruses.
For the process control MNV-1, the overlapping ORF1/ORF2 region was amplified using primers Fw-ORF1/ORF2 (5′-CACGCCACCGATCTGT TCTG-3′), and Rv-ORF1/ORF2 (5′-GCGCTGCGCCATCACTC-3′), and the MNV-1 Probe ORF1/ORF2 (5′-FAM-CGCTTTGGAACAATG-MGB-NFQ3′). The primers used for HEV real-time detection were FHEV 5′-GGTG GTTTCTGGGGTGAC-3′, RHEV 5′-AGG GGTTGGTTGGATGAA-3′ together with the HEV Probe (5′-FAM-TGATTCTCAGCCCTT CGC-BHQ1-3′) annealing in the ORF3 gene (Baert et al., 2008; Jothikumar et al., 2006). Each sample was analyzed in duplicate and reactions were performed using RNA UltraSense reaction kit (Lifetechnologies), as previously described (Di Bartolo et al., 2012). For interpretation of results, a reaction with either a IACs Ct negative or with a value ≥40 was considered as inhibited or failed. If the Ct IAC value was as expected (a mean expected Ct value of 36) and the Ct value for the RNA extraction process control (MNV-1) or for the target virus was not detectable or was ≥40, the sample was considered as negative. In the absence of signals for MNV-1 (process control) and its IAC, the pre-amplification step (genome extraction) was concluded to have failed. If the FAM signals for MNV-1 or the target virus were detectable, independent on the IAC values, the reaction was considered to be positive. 2.3.3. Generation of standard curves for MNV-1 and HEV An RT-qPCR standard curve served as the basis for quantification of the viral load, expressed as genome equivalents (GE). For MNV-1, a 326 bp genomic fragment in the ORF1–ORF2 overlapping region was amplified using primers FMNV 5′-CTGCCATGCATGGT GAAAAG-′3 and RMNV 5′-GTCAATTTGGTTAATTTGCCCG-′3 (primer positions are 4888–4907 and 5191–5211, respectively, and are based on the reference strain acc. no. EU004678). The DNA amplicon obtained was cloned into the commercial vector pGEM-T Easy (Promega) and the resulting recombinant vector, named pMNV1, was used for in vitro transcription as described below. The synthetic RNA was also used to establish the limit of detection. For HEV, a plasmid named pGEMORF1-2-3HEV was built by Aoverhangs cloning in pGEM-T Easy (Promega) of an 890 bp genomic fragment spanning the ORF3 overlapping region extending from ORF1 to ORF2 of an Italian swine HEV genotype 3 strain (Acc. no. GU117636). The genomic fragment was amplified using primers ISP 5′-CCGTGTTTAT GGAGTTAGCCC-3′ (Zhai et al., 2006), annealing in the ORF1, and RSw28 5′-CTAGGATCCTAACSTC-3′, annealing in the ORF2 (primer positions are 4999–5020 and 5865–5881, respectively, based on the reference strain acc. no. JQ953665). The synthetic RNA was obtained by in vitro transcription of pMNV1 or pGEMORF1-2-3HEV by RiboMAX™ Large Scale RNA Production Systems (Promega) following the manufacturer's instructions. The RNA transcripts were subjected to DNase digestion (Sigma), phenol chloroform extraction, and precipitation with ethanol, and were resuspended in DEPC water. The RNA was quantitated using spectrophotometry, aliquoted and stored at −80 °C. The standard curves for murine norovirus or HEV were drawn by plotting the Cq values against the logarithm of the calculated copy numbers of the synthetic RNAs used in serial ten-fold dilutions. 2.4. Nested-RT-PCR for HEV and nested-PCR for PAdV detection Two different nested-RT-PCRs were performed for amplification of two HEV genomic regions in the ORF1: a 287 bp fragment in the methyltransferase coding gene (MTase) (Erker et al., 1999) and a 350 bp fragment in the RNA dependent RNA polymerase (RdRp) coding gene, as previously described (Zhai et al., 2006) with slight modification, using the one-step RT-PCR (QIAGEN). For PAdV, a PCR followed by nested PCR was performed as previously described, amplifying a 344 bp fragment in the hexon gene (Maluquer de Motes et al., 2004). All tests included positive and negative controls in every stage, from RNA extraction to nested-PCR.
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2.5. Sequencing Positive DNA amplicons were purified by ExoSap (Affymetrix) and sequencing reactions were carried out by Biofab (Biofab srl, Rome). Sequences were submitted to NCBI GenBank (Acc. no.: KF793829; KF793830; KF896811). Nucleotide sequences were analyzed using DNASIS Max software (Hitachisoft), and were compared with HEV sequences available in the NCBI GenBank (http://www.ncbi.nlm.nih. gov). Alignment and genotyping were carried out with the Bionumerics software packages v.6.0 (Applied Maths, Kortrijk, Belgium). The neighbor-joining tree was drawn by MEGA5.1 software (Tamura et al., 2011), using Kimura-2 as correction factor. 3. Results Eight packs of pork liver sausage were purchased in a butcher shop in Rome, Italy, where both processing and packaging are carried out. Three slices of 250 mg were sampled and analyzed independently from each of the 15 fresh and 14 dry sausages investigated. All the slices from fresh liver sausages (no. 45) but only 23 of 42 slices from dry sausages were found to be positive for the process control (MNV-1), and were subsequently tested also for HEV. Since at least one slice out of the three analyzed for each sausage had resulted positive for MNV-1, as a consequence every single pack was eventually included in the analyses for HEV and PAdV. The IAC gave positive signals (IAC Ct values ranging between 36 and 38 Ct for both dry and fresh sausages) in all MNV-1 RT-qPCR reactions, indicating that the lack of positive signal for MNV-1 in 19 dry liver sausage slices was not due to a presence of PCR inhibitors. Rather, MNV-1 negative results may suggest that some of the RNA extractions were unsuccessful. Subsequently, the recovery rate for RNA extraction was calculated as the ratio between the number of genome equivalents (GE) of MNV-1 that were recovered after RNA extraction from sausages and the GE copies of MNV-1 used to spike the samples. The mean recovery rate was 18% for fresh liver sausages, ranging between 4.5 and 31%, while the mean recovery rate for dry liver sausages was 5.72%, ranging between 0.43 and 16%. The Ct values for the MNV-1 ranged between 31–37 and 35–38 for fresh and dry liver sausages, respectively, the latter being closer to the theoretical detection limit of 39 Ct. All samples positive for MNV-1 were then analyzed for both HEV and porcine Adenovirus (PAdV) by RT-qPCR and real-time PCR, respectively. All slices were negative for PAdV by both real-time PCR and conventional PCR, suggesting the absence of direct fecal contamination. Five packages, four of fresh and one of dry liver sausage included at least one HEV positive sausage slice (Table 1). Ten out of 45 (22.2%) fresh and one of 23 (4.3%) dry liver sausage slices resulted positive for HEV RNA by RT-qPCR. Based on the real-time PCR results, the liver sausages examined were estimated to contain between 5 × 102 and 5 × 104 HEV GE per 250 mg. The lowest GE number was obtained twice, once in a fresh sausage slice and once in dry sausage (Table 1). All RNAs resulting positive for the process control MNV-1, including those negative for PAdV and HEV, were further analyzed by conventional RT-PCR and PCR assays for HEV and PAdV, respectively. All slices were confirmed as negative for PAdV. On the contrary, the HEV genome was detected by at least one of the two conventional RT-PCR performed, which amplify the MTase and RdRp (Table 1), in two slices from two fresh liver sausages out of 45 (4.4%) slices tested and in one dry liver sausage slice out of 23 (4.3%) (Table 1). Table 1 reports the results of conventional RT-PCRs conducted on the sausage slices previously found HEV-positive by RT-qPCR, showing that only three of 11 slices (from three different sausages) could be confirmed positive also by end-point RT-PCR. The strains detected were confirmed to belong to HEV genotype 3 by sequence analyses. Overall, three RdRp fragments were sequenced (sau15, sau32 and one dry sausage sau83). One of them (sau32) was too short for being submitted to GenBank. However, the alignment of the three sequences
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Table 1 Determination of genome equivalents of HEV and MNV-1 by real-time RT-PCR and detection of two HEV ORF1 target regions by conventional RT-PCR, in 11 HEV-positive sausage samples. Sample Pack
Sausage
A
3
B
4 5 9
C
10
D Ed
12 13
Slice sau8 sau9 sau12 sau14 sau15 sau25 sau27 sau30 sau31 sau32 sau83
Real-Time RTPCR Cta
RT-qPCR
MNV-1
HEV GE/250 mg
31.73 35.88 34.08 34.35 31.36 32.57 31.72 31.75 31.30 30.86 35.27
HEV 35.41 36.92 35.98 30.66 35.59 33.83 34.90 35.75 35.01 34.79 38.00
2.18 6.96 1.34 5.42 1.70 6.34 2.34 1.47 2.19 4.69 5.70
× × × × × × × × × × ×
RT-PCR
3
10 102 103 104 103 103 103 103 103 103 102
MTaseb
RdRpc
– – – – pos – – – – – pos
– – – – pos – – – – pos pos
Additional 57 fresh (35) or dry (22) sausage samples that resulted negative at any HEV test are not included. Genome equivalent (GE) for HEV calculated by RT-qPCR are reported. a Ct mean values for two replicates. b Reverse transcription-PCR (RT-PCR) amplifying a methyltransferase fragment. c RT-PCR amplifying an RNA-dependent RNA polymerase fragment. d Packs A through D refer to fresh sausages; pack E refers to dry liver sausages.
in the overlapping regions (110 nt) showed a nucleotide identity ranging from 72.7 to 88.3%, which indicates that three different HEV strains were detected. A similar value of nucleotide identity (89%) was also obtained by aligning the two available MTase fragment sequences (samples sau83 and sau15; the latter was not included in the tree because it is too short). A phylogenetic tree was drawn using both RdRp and MTase fragments (Fig. 1). Results confirmed a correlation between the HEV genomes detected in liver sausages in this study with swine HEV strains reported in Europe, including Italy, between 2009 and 2012. The highest nucleotide identity with previous Italian strains from swine was 91% and 86.7% in the RdRp and MTase, respectively.
4. Discussion Few quantitative data on HEV in food and clinical (animal and/or human) samples are available (Colson et al., 2010; Leblanc et al., 2010; Martin-Latil et al., 2014) and the infectious dose of the virus is still unknown. In the absence of efficient in vitro replication systems for HEV, only molecular detection data, such as quantitative real-time PCR data, can be presently used for a quantitative risk assessment. Proper assessment of molecular quantitative data should however take into account also the RNA recovery efficiency that is provided by the different extraction procedures available (Martin-Latil et al., 2014). As revealed using the murine norovirus as process control in this and in previous studies (Martin-Latil et al., 2012, 2014), the RNA recovery may be largely influenced by the matrix investigated and the specific extraction method used (Martin-Latil et al., 2014). In this study, all of the 87 samples tested were positive for the IAC, whereas no process control signal was obtained from 19 of 42 dry liver sausages tested. Consequently, the failure of RT-qPCR for the process control was most certainly due to a failed RNA extraction, possibly related to the different fat and salt concentrations in dry liver sausages and/or to the air drying process itself. We used an RNA extraction protocol suitable for an intracellular virus contamination, because most of infectious HEV can be expected to be inside the hepatocytes that form most part of the sausage matrix. The lower detection observed for MNV-1 in dry compared to fresh sausages suggests that the use of a process control may be recommendable to reduce the risk of false negative reactions when investigating RNA viruses in different food matrices (D'Agostino et al., 2012; Martin-Latil et al., 2012, 2014).
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Fig. 1. Phylogenetic tree based on 270 nt fragment of the RdRp (panel A) and MTAse (panel B). The neighbor-joining tree was drawn by MEGA5.1 software, using Kimura-2 as correction factor. Bootstrap values N60 are shown. Strains identified in this study are in bold. Reference strains from GenBank are included, for all entries host (Sw, porcine; Hu, human; Sau liver sausage), country of origin (FR, France; ES, Spain; JP, Japan; DE, Germany; SE, Sweden; HU, Hungary; Br, Brazil; IT, Italy; NL the Netherland; US, USA), year of identification and accession number are reported. Scale bar represents nucleotide substitutions per site.
The real numbers of HEV GE in 250 mg of liver sausages might be higher than the 5 × 102–5 × 104 determined in our study if considering the overall low rate of MNV-1 RNA recovery attained. In fact, this might apply also to HEV RNA recovery resulting in a lower quantitative estimation of HEV in samples tested. Indeed, a higher amount of HEV copies has been reported in liver, i.e. 107 per gram (Leblanc et al., 2010), and in pig liver sausages (figatellu, 106 copies per slice), the consumption of which was linked to a small HEV outbreak (Colson et al., 2010). The observation that in no case the three slices tested for each sausage were all positive for HEV may reflect a low and not homogeneous HEV contamination of the sausage. This is in line with the fact that infection is focal in the swine liver, that sausages in Italy are “minced meat” probably deriving from several livers, and that not all of the different animals are positive for HEV, particularly those admitted to slaughtering. In addition to the real-time RT-PCR, we have also used conventional RT-PCR to detect the HEV genome. The use of real-time PCR assays may provide a higher sensitivity (Gyarmati et al., 2007), and in our study only three out of 11 positive samples were in fact confirmed positive also by conventional PCR. Overall, the two assays showed a poor agreement, and the differences observed in detection were not related to the viral amount (Table 1). Test sensitivities can vary depending on the different target regions, on the specific HEV genotypes or subtypes, and the genetic variability of HEV virus may also make it hard to detect some positive samples (Mokhtari et al., 2013). The present study confirms the presence of HEV genome in food, specifically in both fresh and dry liver sausages. The absence of PAdV in the present investigation confirms that the risks for cross-contamination of pork products with swine feces during processing are negligible in Italy, supporting our previous study in the pork food chain (Di Bartolo et al., 2012). However, our data indicate that processing procedures for pork liver sausage production do not substantially abate endogenous HEV, even though the detection of short genome sequences from HEV does not necessarily imply the presence of infectious virus. Nevertheless, experimental animal infection or 3D cell culture studies confirmed both the presence of infectious HEV in contaminated commercial pig liver (Barnaud et al., 2012; Berto et al., 2013; Feagins et al., 2008) and HEV resistance to the heat treatment used in industrial pork product processing (Barnaud et al., 2012). The three HEV strains detected in this study were closely related to other swine strains detected both in Italy and other
European countries. This finding is not surprising because import/export of both animals and meat (including liver, entrails) between Italy and other European and non-EU countries is common (National Institute for Statistics, http://en.istat.it/), which favors the circulation of both indigenous and foreign HEV strains in most countries. Despite the data on HEV presence in food, the number of cases of human hepatitis E reported in Italy and in other industrialized countries is low, suggesting either a low pathogenicity of swine genotype 3 strains for humans or an insufficient virus load in pork products, or the abating of the virus titer and risk of infection by proper cooking of the pork food consumed. Nevertheless, a recent study reported a new emerging phylotype of genotype 3 HEV in swine in UK together with an increased number of human cases, which might suggest that the simultaneous circulation of different virus variants in the population might favor genetic variation of HEV and eventually result in strains associated with an increased risk of infection and/or pathogenicity (Ijaz et al., 2014).
Acknowledgments We thank H.W. Virgin for providing murine norovirus isolates. We would like to acknowledge Edoardo Vignolo for his valuable technical support. This work was supported by the European Commission Framework Program 7 project “Integrated Monitoring and Control of Food-borne Viruses in European Food Supply Chains (VITAL)” (grant no. KBBE 213178). The research leading to these results has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no 278433-PREDEMICS.
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Jothikumar, N., Cromeans, T.L., Robertson, B.H., Meng, X.J., Hill, V.R., 2006. A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus. J. Virol. Methods 131, 65–71. La Rosa, G., Muscillo, M., Vennarucci, V.S., Garbuglia, A.R., La Scala, P., Capobianchi, M.R., 2011. Hepatitis E virus in Italy: molecular analysis of travel-related and autochthonous cases. J. Gen. Virol. 92, 1617–1626. Leblanc, D., Poitras, E., Gagne, M.J., Ward, P., Houde, A., 2010. Hepatitis E virus load in swine organs and tissues at slaughterhouse determined by real-time RT-PCR. Int. J. Food Microbiol. 139, 206–209. Maluquer de Motes, C., Clemente-Casares, P., Hundesa, A., Martin, M., Girones, R., 2004. Detection of bovine and porcine adenoviruses for tracing the source of fecal contamination. Appl. Environ. Microbiol. 70, 1448–1454. Martinelli, N., Pavoni, E., Filogari, D., Ferrari, N., Chiari, M., Canelli, E., Lombardi, G., 2013. Hepatitis E virus in wild boar in the central northern part of Italy. Transbound. Emerg. Dis. http://dx.doi.org/10.1111/tbed.12118. Martin-Latil, S., Hennechart-Collette, C., Guillier, L., Perelle, S., 2012. Comparison of two extraction methods for the detection of hepatitis A virus in semi-dried tomatoes and murine norovirus as a process control by duplex RT-qPCR. Food Microbiol. 31, 246–253. Martin-Latil, S., Hennechart-Collette, C., Guillier, L., Perelle, S., 2014. Method for HEV detection in raw pig liver products and its implementation for naturally contaminated food. Int. J. Food Microbiol. 176, 1–8. Masuda, J., Yano, K., Tamada, Y., Takii, Y., Ito, M., Omagari, K., Kohno, S., 2005. Acute hepatitis E of a man who consumed wild boar meat prior to the onset of illness in Nagasaki, Japan. Hepatol. Res. 31, 178–183. Meng, X.J., 2013. Zoonotic and foodborne transmission of hepatitis E virus. Semin. Liver Dis. 33, 41–49. Meng, X.J., Purcell, R.H., Halbur, P.G., Lehman, J.R., Webb, D.M., Tsareva, T.S., Haynes, J.S., Thacker, B.J., Emerson, S.U., 1997. A novel virus in swine is closely related to the human hepatitis E virus. Proc. Natl. Acad. Sci. U. S. A. 94, 9860–9865. Mokhtari, C., Marchadier, E., Haim-Boukobza, S., Jeblaoui, A., Tesse, S., Savary, J., RoqueAfonso, A.M., 2013. Comparison of real-time RT-PCR assays for hepatitis E virus RNA detection. J. Clin. Virol. 58, 36–40. Okano, H., Takahashi, M., Isono, Y., Tanaka, H., Nakano, T., Oya, Y., Sugimoto, K., Ito, K., Ohmori, S., Maegawa, T., Kobayashi, M., Nagashima, S., Nishizawa, T., Okamoto, H., 2014. Characterization of sporadic acute hepatitis E and comparison of hepatitis E virus genomes in acute hepatitis patients and pig liver sold as food in Mie, Japan. Hepatol. Res. 44, E63–E76. Said, B., Ijaz, S., Chand, M.A., Kafatos, G., Tedder, R., Morgan, D., 2014. Hepatitis E virus in England and Wales: indigenous infection is associated with the consumption of processed pork products. Epidemiol. Infect. 142, 1467–1475. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. Tei, S., Kitajima, N., Takahashi, K., Mishiro, S., 2003. Zoonotic transmission of hepatitis E virus from deer to human beings. Lancet 362, 371–373. Yazaki, Y., Mizuo, H., Takahashi, M., Nishizawa, T., Sasaki, N., Gotanda, Y., Okamoto, H., 2003. Sporadic acute or fulminant hepatitis E in Hokkaido, Japan, may be foodborne, as suggested by the presence of hepatitis E virus in pig liver as food. J. Gen. Virol. 84, 2351–2357. Zhai, L., Dai, X., Meng, J., 2006. Hepatitis E virus genotyping based on full-length genome and partial genomic regions. Virus Res. 120, 57–69.
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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Short communication
Detection of hepatitis E virus in pork liver sausages Ilaria Di Bartolo a,⁎,1, Giorgia Angeloni a,1, Eleonora Ponterio a, Fabio Ostanello b, Franco Maria Ruggeri a a b
Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy Department of Veterinary Medical Sciences, University of Bologna, Ozzano Emilia, BO, Italy
a r t i c l e
i n f o
Article history: Received 21 March 2014 Received in revised form 30 September 2014 Accepted 4 October 2014 Available online 13 October 2014 Keywords: Hepatitis E Zoonosis Foodborne Murine norovirus Porcine adenovirus Real-time RT-PCR
a b s t r a c t Hepatitis E infection is regarded as an emerging public-health concern. The disease is normally self-limiting (mortality rate 1%), but chronic infections have recently been observed in transplanted patients. The etiological agent HEV is a small RNA virus infecting both humans and animals. In humans, the disease may be food-borne and pig is a main reservoir for zoonotic strains. In the present study, we evaluated the presence of HEV and swine fecal cross-contamination in pork liver sausages sold at a grocery store in Italy. HEV genome detection was performed by RT-qPCR, using harmonized protocols that included a process control (murine norovirus) and an internal amplification control. Swine fecal cross-contamination was assessed by determination of the ubiquitous porcine adenovirus. Overall, HEV genome belonging to genotype 3 was detected in both raw (10 out of 45 slices, 250 mg each, 22.2%) and dry (1 of 23 slices, 4.3%) liver sausages, but infectivity of the virus was not demonstrated. This pilot study fosters more investigations on HEV presence in pork-derived food, to assess the possible risk for the consumers. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Hepatitis E is a usually acute human disease caused by the hepatitis E virus (HEV) (Aggarwal and Naik, 2009). In industrialized countries, the infection was initially considered associated to travel in low-income countries, where the disease is endemic. Conversely, in the last 10 years an increasing number of autochthonous cases have been reported worldwide, including Europe (Aggarwal and Jameel, 2011). The disease is usually self-limiting and presents low mortality, but it can become chronic in transplanted patients and may be highly lethal among pregnant women (Aggarwal and Jameel, 2011). HEV infects humans and several animal species. Four mammalian HEV genotypes are recognized, among which genotypes 1 and 2 are restricted to humans, whereas genotypes 3 and 4 infect both humans and many animal species (pig, deer, wild boar, and rabbit) and circulate in developed countries (Meng, 2013). These two latter genotypes are considered zoonotic, and pigs and less frequently other animal species (wild boar, deer) act as reservoir. Genotype 3 was the first HEV strain detected in animals, specifically pigs (Meng et al., 1997), and it circulates mostly in US and Europe (Meng, 2013). Food-borne transmission of HEV has been increasingly reported in sporadic cases and small outbreaks associated with the consumption of raw or undercooked boar or deer
⁎ Corresponding author. Tel.: +39 06 4990 2787; fax: +39 06 4938 7101. E-mail addresses: [email protected] (I. Di Bartolo), [email protected] (G. Angeloni), [email protected] (E. Ponterio), [email protected] (F. Ostanello), [email protected] (F.M. Ruggeri). 1 These authors contributed equally to this article.
http://dx.doi.org/10.1016/j.ijfoodmicro.2014.10.005 0168-1605/© 2014 Elsevier B.V. All rights reserved.
meat, livers and liver sausages found to contain HEV (Colson et al., 2010; Masuda et al., 2005; Tei et al., 2003; Yazaki et al., 2003). Most importantly, HEV was detected frequently in both commercial livers and sausages sampled in grocery stores. In Japan, US and the Netherlands, 2–4.3%, 11% and 6.5% of commercial liver were positive for swine HEV RNA (Bouwknegt et al., 2007; Feagins et al., 2007; Okano et al., 2014; Yazaki et al., 2003) as were almost 31% of porkderivatives such as sausage (Martin-Latil et al., 2014). More recently, a study in UK showed that consumption of some pork-derived food was a risk factor for HEV infection (Said et al., 2014), and a small outbreak reported in France was linked to consumption of Corse pig liver sausage figatellu, found to be contaminated by genotype 3 HEV (Colson et al., 2010). In Italy, genotype 3 HEV was detected in swine and wild boar belonging to different age classes (Di Bartolo et al., 2008; Martinelli et al., 2013). Hepatitis E infection is widespread in pig herds affecting animals of different ages, including those doomed to slaughterhouse (Di Bartolo et al., 2011). HEV genomic RNA was detected in both swine feces and liver, and recently it was found along the pork production chain in Italy, specifically in two meat samples (6%) but not in sausage (Di Bartolo et al., 2012). In the same investigation, 6% and 10% of pork sausages at point of sale were positive for HEV genome in Spain (Di Bartolo et al., 2012) and in UK (Berto et al., 2012). In the last few years, an increase in the number of autochthonous cases of human hepatitis E has been reported also in Italy. These were associated mostly to genotype 3 and more rarely to genotype 4 (Garbuglia et al., 2013; La Rosa et al., 2011). The role of consumption of pork-derived food should be investigated to assess the risk of foodborne HEV transmission. The main site of HEV replication is the liver, suggesting that the presence
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of endogenous virus in liver sausages may be a main risk factor. However, the virus is massively shed with swine feces (Di Bartolo et al., 2008), which may represent a possible source of HEV contamination during slaughtering and pork product processing. In the present study, we investigated the presence of HEV in both fresh and dry pork liver sausages in Italy, where they are frequently consumed raw or undercooked. Moreover, we evaluated the presence of porcine adenovirus in sausages as a relevant indicator of swine fecal contamination, since this virus is normally detected between 90 and 98% of swine fecal samples (Di Bartolo et al., 2012; Maluquer de Motes et al., 2004). 2. Materials and methods 2.1. Sampling Pork liver sausages, fresh and dry, were purchased in a butcher shop in Rome, Italy, where both processing and packaging were finished. Dry sausages were desiccated in the air during a 10 to 15 day maturation period. Four packs containing a total of 15 fresh liver sausages (about 80 g per sausage) and four packs containing 14 dry sausages were bought. To ensure the sampling of different production lots, the packs were bought in four different weeks between January and February 2013, when these products are more typically consumed. Each sausage was chopped in three slices, obtaining a total of 45 and 42 slices for fresh and dry liver sausages, respectively. Fat was discarded manually from slices, 250 mg of which was collected and subjected to RNA extraction, individually. 2.2. Sample preparation and nucleic acid extraction Before RNA/DNA extraction, all samples were artificially contaminated with 5 μl of a suspension of murine norovirus (MNV-1; 105 TCID50), which was used as sample process control. The virus was kindly provided by Dr. Virgin from Washington University, USA, and was prepared as described previously (Di Bartolo et al., 2012). Spiked samples were homogenized in 2.5 ml of lysis buffer (RTL) with 1 sterile stainless steel bead (5 mm, QIAGEN), using a mechanical disruptor (Tissue Lyser– QIAGEN) for three runs of 2 min at 46 oscillations s−1. After centrifugation at 5000 ×g per 20 min, the supernatant was used to extract nucleic acids by the RNeasy Midi kit (QIAGEN), according to the manufacturer's instructions and it was eluted in 100 μl of ddH2O. 2.3. Real time-PCR All reaction mixtures included an Internal Amplification Control IAC (Diez-Valcarce et al., 2011) and were in duplex format, targeting specific viruses (Murine NoV, HEV, Porcine Adenovirus) and IACs labeled with FAM (6-carboxy fluorescein) and VIC (5′-VIC-CCATACACATAGGTC AGG-MGB-NFQ-3′; Lifetechnologies) probes, respectively. All experiments included negative and positive controls, with and without IACs. 2.3.1. PAdV real time-PCR Ten microliters of extracted nucleic acids were analyzed for PAdV by duplex real-time PCR including IACs, with primers PAdV-F (5′-AACGGC CGCTACTGCAAG-3′) and PAdV-R (5′-AGCAGCAGGCTCTTGAGG-3′) and probe PAdV-P (5′-FAM-CACATCCAGGTGCCGC-BHQ1-3′) within the hexon region, using the TaqMan Universal PCR Master-Mix (Lifetechnologies) reaction kit, as previously described (Di Bartolo et al., 2012; Hundesa et al., 2009). 2.3.2. RT-qPCR for detection of MNV-1 and HEV Ten microliters of RNA was analyzed for detection of MNV-1 or HEV by a duplex one step reverse-transcription qPCR detecting both the IAC (VIC-labeled probe) (Diez-Valcarce et al., 2011) and the target viruses.
For the process control MNV-1, the overlapping ORF1/ORF2 region was amplified using primers Fw-ORF1/ORF2 (5′-CACGCCACCGATCTGT TCTG-3′), and Rv-ORF1/ORF2 (5′-GCGCTGCGCCATCACTC-3′), and the MNV-1 Probe ORF1/ORF2 (5′-FAM-CGCTTTGGAACAATG-MGB-NFQ3′). The primers used for HEV real-time detection were FHEV 5′-GGTG GTTTCTGGGGTGAC-3′, RHEV 5′-AGG GGTTGGTTGGATGAA-3′ together with the HEV Probe (5′-FAM-TGATTCTCAGCCCTT CGC-BHQ1-3′) annealing in the ORF3 gene (Baert et al., 2008; Jothikumar et al., 2006). Each sample was analyzed in duplicate and reactions were performed using RNA UltraSense reaction kit (Lifetechnologies), as previously described (Di Bartolo et al., 2012). For interpretation of results, a reaction with either a IACs Ct negative or with a value ≥40 was considered as inhibited or failed. If the Ct IAC value was as expected (a mean expected Ct value of 36) and the Ct value for the RNA extraction process control (MNV-1) or for the target virus was not detectable or was ≥40, the sample was considered as negative. In the absence of signals for MNV-1 (process control) and its IAC, the pre-amplification step (genome extraction) was concluded to have failed. If the FAM signals for MNV-1 or the target virus were detectable, independent on the IAC values, the reaction was considered to be positive. 2.3.3. Generation of standard curves for MNV-1 and HEV An RT-qPCR standard curve served as the basis for quantification of the viral load, expressed as genome equivalents (GE). For MNV-1, a 326 bp genomic fragment in the ORF1–ORF2 overlapping region was amplified using primers FMNV 5′-CTGCCATGCATGGT GAAAAG-′3 and RMNV 5′-GTCAATTTGGTTAATTTGCCCG-′3 (primer positions are 4888–4907 and 5191–5211, respectively, and are based on the reference strain acc. no. EU004678). The DNA amplicon obtained was cloned into the commercial vector pGEM-T Easy (Promega) and the resulting recombinant vector, named pMNV1, was used for in vitro transcription as described below. The synthetic RNA was also used to establish the limit of detection. For HEV, a plasmid named pGEMORF1-2-3HEV was built by Aoverhangs cloning in pGEM-T Easy (Promega) of an 890 bp genomic fragment spanning the ORF3 overlapping region extending from ORF1 to ORF2 of an Italian swine HEV genotype 3 strain (Acc. no. GU117636). The genomic fragment was amplified using primers ISP 5′-CCGTGTTTAT GGAGTTAGCCC-3′ (Zhai et al., 2006), annealing in the ORF1, and RSw28 5′-CTAGGATCCTAACSTC-3′, annealing in the ORF2 (primer positions are 4999–5020 and 5865–5881, respectively, based on the reference strain acc. no. JQ953665). The synthetic RNA was obtained by in vitro transcription of pMNV1 or pGEMORF1-2-3HEV by RiboMAX™ Large Scale RNA Production Systems (Promega) following the manufacturer's instructions. The RNA transcripts were subjected to DNase digestion (Sigma), phenol chloroform extraction, and precipitation with ethanol, and were resuspended in DEPC water. The RNA was quantitated using spectrophotometry, aliquoted and stored at −80 °C. The standard curves for murine norovirus or HEV were drawn by plotting the Cq values against the logarithm of the calculated copy numbers of the synthetic RNAs used in serial ten-fold dilutions. 2.4. Nested-RT-PCR for HEV and nested-PCR for PAdV detection Two different nested-RT-PCRs were performed for amplification of two HEV genomic regions in the ORF1: a 287 bp fragment in the methyltransferase coding gene (MTase) (Erker et al., 1999) and a 350 bp fragment in the RNA dependent RNA polymerase (RdRp) coding gene, as previously described (Zhai et al., 2006) with slight modification, using the one-step RT-PCR (QIAGEN). For PAdV, a PCR followed by nested PCR was performed as previously described, amplifying a 344 bp fragment in the hexon gene (Maluquer de Motes et al., 2004). All tests included positive and negative controls in every stage, from RNA extraction to nested-PCR.
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2.5. Sequencing Positive DNA amplicons were purified by ExoSap (Affymetrix) and sequencing reactions were carried out by Biofab (Biofab srl, Rome). Sequences were submitted to NCBI GenBank (Acc. no.: KF793829; KF793830; KF896811). Nucleotide sequences were analyzed using DNASIS Max software (Hitachisoft), and were compared with HEV sequences available in the NCBI GenBank (http://www.ncbi.nlm.nih. gov). Alignment and genotyping were carried out with the Bionumerics software packages v.6.0 (Applied Maths, Kortrijk, Belgium). The neighbor-joining tree was drawn by MEGA5.1 software (Tamura et al., 2011), using Kimura-2 as correction factor. 3. Results Eight packs of pork liver sausage were purchased in a butcher shop in Rome, Italy, where both processing and packaging are carried out. Three slices of 250 mg were sampled and analyzed independently from each of the 15 fresh and 14 dry sausages investigated. All the slices from fresh liver sausages (no. 45) but only 23 of 42 slices from dry sausages were found to be positive for the process control (MNV-1), and were subsequently tested also for HEV. Since at least one slice out of the three analyzed for each sausage had resulted positive for MNV-1, as a consequence every single pack was eventually included in the analyses for HEV and PAdV. The IAC gave positive signals (IAC Ct values ranging between 36 and 38 Ct for both dry and fresh sausages) in all MNV-1 RT-qPCR reactions, indicating that the lack of positive signal for MNV-1 in 19 dry liver sausage slices was not due to a presence of PCR inhibitors. Rather, MNV-1 negative results may suggest that some of the RNA extractions were unsuccessful. Subsequently, the recovery rate for RNA extraction was calculated as the ratio between the number of genome equivalents (GE) of MNV-1 that were recovered after RNA extraction from sausages and the GE copies of MNV-1 used to spike the samples. The mean recovery rate was 18% for fresh liver sausages, ranging between 4.5 and 31%, while the mean recovery rate for dry liver sausages was 5.72%, ranging between 0.43 and 16%. The Ct values for the MNV-1 ranged between 31–37 and 35–38 for fresh and dry liver sausages, respectively, the latter being closer to the theoretical detection limit of 39 Ct. All samples positive for MNV-1 were then analyzed for both HEV and porcine Adenovirus (PAdV) by RT-qPCR and real-time PCR, respectively. All slices were negative for PAdV by both real-time PCR and conventional PCR, suggesting the absence of direct fecal contamination. Five packages, four of fresh and one of dry liver sausage included at least one HEV positive sausage slice (Table 1). Ten out of 45 (22.2%) fresh and one of 23 (4.3%) dry liver sausage slices resulted positive for HEV RNA by RT-qPCR. Based on the real-time PCR results, the liver sausages examined were estimated to contain between 5 × 102 and 5 × 104 HEV GE per 250 mg. The lowest GE number was obtained twice, once in a fresh sausage slice and once in dry sausage (Table 1). All RNAs resulting positive for the process control MNV-1, including those negative for PAdV and HEV, were further analyzed by conventional RT-PCR and PCR assays for HEV and PAdV, respectively. All slices were confirmed as negative for PAdV. On the contrary, the HEV genome was detected by at least one of the two conventional RT-PCR performed, which amplify the MTase and RdRp (Table 1), in two slices from two fresh liver sausages out of 45 (4.4%) slices tested and in one dry liver sausage slice out of 23 (4.3%) (Table 1). Table 1 reports the results of conventional RT-PCRs conducted on the sausage slices previously found HEV-positive by RT-qPCR, showing that only three of 11 slices (from three different sausages) could be confirmed positive also by end-point RT-PCR. The strains detected were confirmed to belong to HEV genotype 3 by sequence analyses. Overall, three RdRp fragments were sequenced (sau15, sau32 and one dry sausage sau83). One of them (sau32) was too short for being submitted to GenBank. However, the alignment of the three sequences
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Table 1 Determination of genome equivalents of HEV and MNV-1 by real-time RT-PCR and detection of two HEV ORF1 target regions by conventional RT-PCR, in 11 HEV-positive sausage samples. Sample Pack
Sausage
A
3
B
4 5 9
C
10
D Ed
12 13
Slice sau8 sau9 sau12 sau14 sau15 sau25 sau27 sau30 sau31 sau32 sau83
Real-Time RTPCR Cta
RT-qPCR
MNV-1
HEV GE/250 mg
31.73 35.88 34.08 34.35 31.36 32.57 31.72 31.75 31.30 30.86 35.27
HEV 35.41 36.92 35.98 30.66 35.59 33.83 34.90 35.75 35.01 34.79 38.00
2.18 6.96 1.34 5.42 1.70 6.34 2.34 1.47 2.19 4.69 5.70
× × × × × × × × × × ×
RT-PCR
3
10 102 103 104 103 103 103 103 103 103 102
MTaseb
RdRpc
– – – – pos – – – – – pos
– – – – pos – – – – pos pos
Additional 57 fresh (35) or dry (22) sausage samples that resulted negative at any HEV test are not included. Genome equivalent (GE) for HEV calculated by RT-qPCR are reported. a Ct mean values for two replicates. b Reverse transcription-PCR (RT-PCR) amplifying a methyltransferase fragment. c RT-PCR amplifying an RNA-dependent RNA polymerase fragment. d Packs A through D refer to fresh sausages; pack E refers to dry liver sausages.
in the overlapping regions (110 nt) showed a nucleotide identity ranging from 72.7 to 88.3%, which indicates that three different HEV strains were detected. A similar value of nucleotide identity (89%) was also obtained by aligning the two available MTase fragment sequences (samples sau83 and sau15; the latter was not included in the tree because it is too short). A phylogenetic tree was drawn using both RdRp and MTase fragments (Fig. 1). Results confirmed a correlation between the HEV genomes detected in liver sausages in this study with swine HEV strains reported in Europe, including Italy, between 2009 and 2012. The highest nucleotide identity with previous Italian strains from swine was 91% and 86.7% in the RdRp and MTase, respectively.
4. Discussion Few quantitative data on HEV in food and clinical (animal and/or human) samples are available (Colson et al., 2010; Leblanc et al., 2010; Martin-Latil et al., 2014) and the infectious dose of the virus is still unknown. In the absence of efficient in vitro replication systems for HEV, only molecular detection data, such as quantitative real-time PCR data, can be presently used for a quantitative risk assessment. Proper assessment of molecular quantitative data should however take into account also the RNA recovery efficiency that is provided by the different extraction procedures available (Martin-Latil et al., 2014). As revealed using the murine norovirus as process control in this and in previous studies (Martin-Latil et al., 2012, 2014), the RNA recovery may be largely influenced by the matrix investigated and the specific extraction method used (Martin-Latil et al., 2014). In this study, all of the 87 samples tested were positive for the IAC, whereas no process control signal was obtained from 19 of 42 dry liver sausages tested. Consequently, the failure of RT-qPCR for the process control was most certainly due to a failed RNA extraction, possibly related to the different fat and salt concentrations in dry liver sausages and/or to the air drying process itself. We used an RNA extraction protocol suitable for an intracellular virus contamination, because most of infectious HEV can be expected to be inside the hepatocytes that form most part of the sausage matrix. The lower detection observed for MNV-1 in dry compared to fresh sausages suggests that the use of a process control may be recommendable to reduce the risk of false negative reactions when investigating RNA viruses in different food matrices (D'Agostino et al., 2012; Martin-Latil et al., 2012, 2014).
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Fig. 1. Phylogenetic tree based on 270 nt fragment of the RdRp (panel A) and MTAse (panel B). The neighbor-joining tree was drawn by MEGA5.1 software, using Kimura-2 as correction factor. Bootstrap values N60 are shown. Strains identified in this study are in bold. Reference strains from GenBank are included, for all entries host (Sw, porcine; Hu, human; Sau liver sausage), country of origin (FR, France; ES, Spain; JP, Japan; DE, Germany; SE, Sweden; HU, Hungary; Br, Brazil; IT, Italy; NL the Netherland; US, USA), year of identification and accession number are reported. Scale bar represents nucleotide substitutions per site.
The real numbers of HEV GE in 250 mg of liver sausages might be higher than the 5 × 102–5 × 104 determined in our study if considering the overall low rate of MNV-1 RNA recovery attained. In fact, this might apply also to HEV RNA recovery resulting in a lower quantitative estimation of HEV in samples tested. Indeed, a higher amount of HEV copies has been reported in liver, i.e. 107 per gram (Leblanc et al., 2010), and in pig liver sausages (figatellu, 106 copies per slice), the consumption of which was linked to a small HEV outbreak (Colson et al., 2010). The observation that in no case the three slices tested for each sausage were all positive for HEV may reflect a low and not homogeneous HEV contamination of the sausage. This is in line with the fact that infection is focal in the swine liver, that sausages in Italy are “minced meat” probably deriving from several livers, and that not all of the different animals are positive for HEV, particularly those admitted to slaughtering. In addition to the real-time RT-PCR, we have also used conventional RT-PCR to detect the HEV genome. The use of real-time PCR assays may provide a higher sensitivity (Gyarmati et al., 2007), and in our study only three out of 11 positive samples were in fact confirmed positive also by conventional PCR. Overall, the two assays showed a poor agreement, and the differences observed in detection were not related to the viral amount (Table 1). Test sensitivities can vary depending on the different target regions, on the specific HEV genotypes or subtypes, and the genetic variability of HEV virus may also make it hard to detect some positive samples (Mokhtari et al., 2013). The present study confirms the presence of HEV genome in food, specifically in both fresh and dry liver sausages. The absence of PAdV in the present investigation confirms that the risks for cross-contamination of pork products with swine feces during processing are negligible in Italy, supporting our previous study in the pork food chain (Di Bartolo et al., 2012). However, our data indicate that processing procedures for pork liver sausage production do not substantially abate endogenous HEV, even though the detection of short genome sequences from HEV does not necessarily imply the presence of infectious virus. Nevertheless, experimental animal infection or 3D cell culture studies confirmed both the presence of infectious HEV in contaminated commercial pig liver (Barnaud et al., 2012; Berto et al., 2013; Feagins et al., 2008) and HEV resistance to the heat treatment used in industrial pork product processing (Barnaud et al., 2012). The three HEV strains detected in this study were closely related to other swine strains detected both in Italy and other
European countries. This finding is not surprising because import/export of both animals and meat (including liver, entrails) between Italy and other European and non-EU countries is common (National Institute for Statistics, http://en.istat.it/), which favors the circulation of both indigenous and foreign HEV strains in most countries. Despite the data on HEV presence in food, the number of cases of human hepatitis E reported in Italy and in other industrialized countries is low, suggesting either a low pathogenicity of swine genotype 3 strains for humans or an insufficient virus load in pork products, or the abating of the virus titer and risk of infection by proper cooking of the pork food consumed. Nevertheless, a recent study reported a new emerging phylotype of genotype 3 HEV in swine in UK together with an increased number of human cases, which might suggest that the simultaneous circulation of different virus variants in the population might favor genetic variation of HEV and eventually result in strains associated with an increased risk of infection and/or pathogenicity (Ijaz et al., 2014).
Acknowledgments We thank H.W. Virgin for providing murine norovirus isolates. We would like to acknowledge Edoardo Vignolo for his valuable technical support. This work was supported by the European Commission Framework Program 7 project “Integrated Monitoring and Control of Food-borne Viruses in European Food Supply Chains (VITAL)” (grant no. KBBE 213178). The research leading to these results has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no 278433-PREDEMICS.
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