Surveillance of hepatitis E virus contamination in shellfish in China

Abstract

Background: Hepatitis E virus (HEV) has been confirmed to be a zoonotic virus of worldwide distribution. HEV contamination in the water environment has not been well examined in China. The objective of this study was to evaluate HEV contamination in shellfish in a coastal area of China. Such contamination would be significant for evaluating public health risks.

Methods: samples of three species shellfish were collected from thirteen points of estuarine tidal flats around the Bohai Gulf and screened for HEV RNA using an in-house nested RT-PCR assay. The detected HEV-positive samples were further verified by gene cloning and sequencing analysis.

Results: the overall HEV-positive detection rate is approximately 17.5% per kilogram of shellfish. HEV was more common among S. subcrenata (28.2%), followed by A. granosa (14.3%) and R. philippinarum (11.5%). The phylogenetic analysis of the 13 HEV strains detected revealed that gene fragments fell into two known 4 sub-genotypes (4b/4d) groups and another unknown group.

Conclusions: 13 different sub-genotype 4 HEVs were found in contaminated shellfish in the Bohai Gulf rim. The findings suggest that a health risk may exist for users of waters in the Bonhai area and to consumers of shellfish. Further research is needed to assess the sources and infectivity of HEV in these settings, and to evaluate additional shellfish harvesting areas.

Figure 1

Locations of shellfish sampling and phylogenetic trees of hepatitis E virus. (a) Locations of shellfish collected in the Bohai gulf rim. The site of HEV RNA positive detection is marked by a positive sign within red circle and that of negative detection’s with a only a red circle . (b) Phylogenetic trees of hepatitis E virus (HEV) were constructed based on partial genomes. Each partial ORF3 (287basepairs) of 13 different clones of JZ12-1, JZ12-2, HLD12-1, HLD12-2, PJ12-1, DY12-1, SZ12-1, JZ13-1, JZ13-2, TJ13-1, TJ13-2, YK13-1and JZ13-3 (with GenBank accession No. KJ816338, KJ816339, KJ816336, KJ816337, KJ816343, KJ816335, KJ816344, KJ816340, KJ816341, KJ816345, KJ816346, KJ816347 and KJ816342, respectively.) was analyzed by the neighbor-joining method. The bootstrap value correspond to 1,000 replications of avian HEV was used as an outgroup. All nucleotide sequences determined in this study were marked by ▲. Other HEV sequences were retrived from GenBank.

Figure A1

Comparison of two methods for the recovery of HEV inoculated into shellfish digestive tissue using qRT-PCR. Recoveries with the in-house method in comparison with the reference method at gradient initial viral quantity in 10 mL supernatant samples of shellfish digestive tissue. Recoveries by reference method with the same gradient initial viral quantity in individual shellfish digestive tissue directly. The detailed procedure of the reference method (based on reports by Jothikumar et al. in 2006 [21,22]) with some slight modifications conducted in this test is as follows: Step 1. To prepare the test initial virus sample by qRT-PCR. The stored original previous HEV-positive supernatant detected by RT-PCR was further quantified by reference qRT-PCR assay [22]. The virus supernatant was diluted by gradient of n × Log10 (n = 1, 2, 3, 4, 5 and 6), respectively, as an initial virus sample for the test. Step 2. To prepare HEV contaminated shellfish samples in a similar way. The frozen shellfish were removed for thawing 20–40 min on ice. The individual shells were opened one by one with a sterile shucking knife, ensuring that the hand holding the animal is protected with a heavy-duty safety glove. The digestive glands were dissected from the animals using scissors and forceps and transfered to a clean Eppendorf tube. The weight of the individual gland mass (2.0 ± 0.2) g was made up by adding sterile water. Step 3. The digestive gland is finely chopped up with an equivalent homogenizer to a paste-like consistency. Step 4. Add (2.0 ± 0.2) mL of proteinase K solution and mix. Incubate at (37 ± 1.0) °C with shaking at approximately 320 min⁻¹ in a shaking incubator for (60 ± 5) min. Step 5. Carry out a secondary incubation by placing the tube in a water bath at (60 ± 2.0) °C for (15 ± 1) min. Step 6. Centrifuge at 3000× g for (5.0 ± 0.5) min, decant the supernatant into a clean tube, measure and record the volume of supernatant, in milliliters, and retain for RNA extraction. As shown in Figure A3, samples without results mean that the obtained Ct values were below the limitation of quantitation of the qRT-PCR test, and recoveries could not be calculated. The reference viral extract method was conducted according to ISO/TS 15216–2:2013 procedure with some slight modifications and the screening qRT-PCR assay. The results indicated that the efficiency of in-house virus extract method was comparable to that of reference method, but both of them had very low efficiency.

Figure A2

Comparison of the sensitivity of the in-house nRT-PCR and reference qRT-PCR methods for the detection of HEV RNA. The detection limit (2.5 × 10² copies/reaction) of the in-house nRT-PCR was less sensitive (10-fold) than that of reference qRT-PCR [21]. This results reflected that the real presence of HEV in shellfish might be higher than the results described in this study.

Figure A3

Specificity analysis of in-house nRT-PCR. M: DNA marker 2000 bp; 1: HEV positive control; 2: ddH₂O as negative control; 3–6 are porcine reproductive and respiratory syndrome virus, hepatitis A virus, Norovirus and porcine epidemic diarrhea virus, respectively. This result indicated that the detection specificity of in-house nRT-PCR was acceptable, though the number of trials in current system is limited.

Figure A4

Output of shellfish products of China from 1978 to 2012. Source: China Statistical Yearbook 2013 (http://www.stats.gov.cn/tjsj/ndsj/2013/html/Z1320e.htm).

Figure A5

Output of shellfish products of China in 2012 by percentage. Source: China Statistical Yearbook 2013 (http://www.stats.gov.cn/tjsj/ndsj/2013/html/Z1320e.htm).