Original Article  /  Liver

Detection of hepatitis E virus RNA in sera of patients with hepatitis E by polymerase chain reaction Zhen-Yu Zhao, Bing Ruan, Hui Shao, Zhen-Juan Chen and She-Lan Liu Hangzhou, China

BACKGROUND: The duration of viremia during hepatitis E virus (HEV) infection has rarely been reported. This study was undertaken to detect HEV RNA in sera of patients with hepatitis E and to understand the process of HEV infection more thoroughly.

KEY WORDS: hepatitis E;  v iremia;  HEV RNA;  duration

METHODS: HEV RNA was detected in the serum samples of hospitalized patients with acute hepatitis E by reverse transcriptase-nested polymerase chain reaction (RTnPCR) using two pairs of primers from open reading frame (ORF) 1 of the HEV genome.

Introduction

RESULTS: The serum samples from 44 (70%) of 62 patients were positive for HEV RNA. Thirty-two of these patients, with 288 serial serum specimens, were followed up for the whole process, and 24 patients (75%) were positive for HEV RNA. The positive rates declined with the course of the disease, serum HEV RNA persisting for 20.6 days on average after onset of illness. Serum HEV RNA remained positive in 36 (81.8%) of the 44 patients at the time their alanine aminotransferase (ALT) began to decrease. There was no difference in HEV RNA positivity between serum with high levels of HEV antibody (peak P/N ratio ≥ 4.0) and that with low levels (peak P/N ratio 0.05), respectively. CONCLUSIONS: There is a relatively long period of HEV viremia in patients with hepatitis E. The proportion of HEV viremia and its duration are not directly related to serum ALT values or HEV antibody levels. (Hepatobiliary Pancreat Dis Int 2007; 6: 38-42)

Author Affiliations: Department of Infectious Diseases, First Affiliated Hospital, Zhejiang University School of Medicine; Key Laboratory of Infectious Diseases, Ministry of Public Health, Hangzhou 310003, China (Zhao ZY, Ruan B, Shao H, Chen ZJ and Liu SL) Corresponding Author: Bing Ruan, MD, Department of Infectious Diseases, First Affiliated Hospital, Zhejiang University School of Medicine; Key Laboratory of Infectious Diseases, Ministry of Public Health, Hangzhou 310003, China (Tel: 86-571-87236713; Email: [email protected]) © 2007, Hepatobiliary Pancreat Dis Int. All rights reserved.

H

epatitis E, the major form of enterically transmitted non-A, non-B hepatitis, is caused by hepatitis E virus (HEV).[1] The disease is a serious public health problem in many developing countries in Asia, the Middle East, and Africa, as well as in Mexico, where sanitation conditions are suboptimal.[2] HEV is transmitted primarily by the fecal-oral route through contaminated water.[3] The overall mortality rate of hepatitis E is generally lower than 1%, but it can be as high as 20%-25% among pregnant women.[4] HEV is currently classified as the sole member of the genus hepavirus.[5] The genome of HEV is an approximately 7.2 kb positive-sense, singlestranded RNA. It contains a short 5'  untranslated region, three open reading frames (ORF; ORF1, ORF2 and ORF3), and a short 3' untranslated region terminated by a poly (A) tail.[1, 6-8] ORF1 encodes nonstructural protein, ORF2 encodes capsid protein, and ORF3 encodes a cytoskeleton-associated phosphoprotein. HEV isolates from several countries worldwide have been classified into eight genotypes based on phylogenetic analysis of the full-length genomes.[9] Generally, hepatitis E is endemic in developing countries, but it also occurs in industrialized countries among individuals with no history of travel to areas of endemicity.[10-12] A significant proportion of healthy individuals in industrialized countries where hepatitis E is not endemic are seropositive for HEV antibody.[13, 14] Recently, accumulating evidence indicates that hepatitis E is a zoonosis. Pigs and other animals may act as reservoirs for HEV infection in

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Detection of HEV RNA in sera of patients with hepatitis E by polymerase chain reaction

humans.[15-17] Cross-species infection of HEV has been experimentally demonstrated.[18, 19] Information on the duration of viremia in patients with acute hepatitis E, although crucial for understanding the transmission dynamics and pathogenesis of this disease, only includes data on one human volunteer who ingested HEV, and a few studies based predominantly on single samples from patients.[20-23] The data on sequential samples obtained from patients are particularly limited. To fill this gap, serial serum samples from patients with hepatitis E were tested for HEV RNA. We report the duration and proportion of hepatitis E patients with viremia, and the relation between serum HEV RNA positivity and alanine aminotransferase (ALT) values or HEV antibody levels.

Methods Patients and samples Patients with acute hepatitis E, diagnosed on the basis of typical clinical and biochemical features, and the presence of serum IgG antibodies to HEV, were included in the study. The typical clinical signs and symptoms of hepatitis E included anorexia, nausea, malaise, abdominal pain, and jaundice. The presence of IgG antibody to HEV in sera was determined by a commercial HEV enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions (Genelabs Diagnostics, Singpore). All patients provided informed consent. 531 serum samples were collected from 62 patients between days 2 and 58 after the onset of illness. All events were measured with reference to the day of onset of the first symptom. HEV RNA was extracted from 100 μl of serum with a QIAamp Viral RNA Mini Kit (QIAGEN, Valencia, Calif) according to the manufacturer's instructions. Isolated RNA was stored at -80  ℃ until further analysis. To avoid artifacts due to contamination of reagents or samples, handling error, or amplification of RNA from non-HEV sources, serum samples obtained from voluntary blood donors and patients with acute hepatitis A were used as negative controls. The positive control was one sample of bile from a rhesus monkey experimentally infected with a pool of acute-phase stools from one female patient in the Xinjiang HEV epidemic.[24] Primer synthesis Two sets of primers for reverse transcriptasepolymerase chain reaction (RT-PCR) and reverse

transcriptase-nested polymerase chain reaction (RTnPCR) based on the highly conserved region of HEV ORF1 from the Burmese strain of HEV (GenBank accession no. M73218)[7] were synthesized. The external set of primers was designed to produce a 562bp PCR product. The nucleotide sequences of the external set of primers were: forward primer, 5'-CAT GGT CGA GAA GGG CCA GG-3' (nucleotide position numbers 4089-4108 nt); backward primer, 5'-GCG GAA GTC ATA ACA GTG GG-3' (4631-4650 nt). The expected size of the PCR product amplified with the nested set of primers was designed to be 218bp. Nucleotide sequences of the nested set of primers were: forward primer, 5'-ATG ACT TTG CTG AGT TTG ACT-3' (4403-4423 nt); and backward primer, 5'-CAT ATT CCA GAC AGT ATT CC-3' (4601-4620 nt).

RT-PCR and RT-nPCR RT-PCR was performed with a QIAGEN OneStep RT-PCR kit according to the manufacturer's instructions. Briefly, a reaction solution was adjusted to 50 μl of the reaction mixture, including 10 μl 5× QIAGEN OneStep RT-PCR buffer, 10 μl 5×QIAGEN solution, 2 μl deoxynucleotide triphosphate (dNTP) mixture (containing 10 mmol/μl of each dNTP), 2 μl external forward primer (100 pmol/μl), 2 μl external backward primer (100 pmol/μl), 2 μl QIAGEN OneStep RT-PCR enzyme mixture, 1 μl RNase inhibitor (10 U/μl; GIBCO), 10 μl template RNA, and 11 μl RNase-free water. The thermal cycling conditions were: one step of reverse transcription for 30 minutes at 50  ℃ and an initial PCR activation step for 15 minutes at 95  ℃. This step was followed by 40 cycles of denaturation for 1 minute at 94  ℃, annealing for 1 minute at 55  ℃, and extension for 1 minute 30 seconds at 72  ℃, as well as a final incubation for 10 minutes at 72  ℃. The remaining RNA was removed with 2 μl RNase H (Invitrogen, Carlsbad, CA, USA) by incubation for 20 minutes at 37  ℃. Nested PCR was conducted with the following components: 3 μl RT-PCR product, 5 μl 10×Ex Taq PCR buffer (Mg2+ -free), 5 μl MgCl2 (25 mg/ml), 4 μl dNTP mixture (containing 10 mmol of each dNTP), 1 μl nested forward primer (100 pmol/μl), 1 μl nested backward primer (100 pmol/μl), 0.5 μl Takara Ex Taq polymerase (5 U/μl), and 30.5 μl triple-distilled H2O. The thermal cycling conditions for the nested PCR included 5 cycles of denaturation for 1 minute at 94  ℃, annealing for 1 minute at 45  ℃, and extension for 1 minute and 30 seconds at 72  ℃. This was followed by 35 cycles of denaturation for 1 minute at 94  ℃, annealing for 1 minute at 55  ℃, and extension

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for 1 minute and 30 seconds at 72  ℃, as well as a final incubation for 7 minutes at 72  ℃. The RT-nested PCR products were analyzed in a 1.0% agarose gel stained with ethidium bromide (10 mg/ml) under a UV transilluminator.

Results Specificity and sensitivity of RT-nPCR Fig. shows the electrophoresis of RT-nPCR products of HEV RNA from four patients with hepatitis E. All 4 products were 218 base pairs in size, as expected. Time course of viremia All 531 serum samples (mean 8.6 per patient)

Fig. Electrophoresis of RT-nPCR products of HEV RNA. M: the ladder of molecular weight markers (PBL); lanes 1-4: four serum specimens from patients with hepatitis E; lanes 5-7: negative controls from saline, blood donor, and serum of one patient with acute hepatitis A, respectively; lane 8: bile specimen from a rhesus monkey experimentally infected with a pool of acute-phase stools from one female patient in the Xinjiang HEV epidemic. Table. Detection of HEV RNA in sera from all 62 patients and serial sera from 32 patients with hepatitis E All sera from 62 patients Days HEV RNA post-onset n positive (%)

Serial sera from 32 patients

2-5 6-10 11-15 16-20 21-25 26-30 31-35 36-40 41-45 46-50 51-55 56-58

17 44 59 62 57 59 56 51 45 33 25 23

12 (70.5) 33 (75.0) 41 (69.4) 37 (59.6) 22 (38.5) 12 (20.3) 8 (14.2) 6 (11.7) 2 (4.4) 1 (3.0) 1 (4.0) 0 (0.0)

Total

531

175 (32.9)

32 24 (75.0) 32 22 (68.8) 32 18 (56.3) 32 12 (37.5) 32 6 (18.8) 32 4 (12.5) 32 3 (9.4) 32 0 (0) 32 0 (0) No follow-up No follow-up No follow-up 288 89 (30.9)

n

HEV RNA positive (%)

collected from 62 patients between day 2 and day 58 after onset of the first symptoms were tested for HEV RNA (Table). Only 4 of 103 serum samples collected on days 41-55 tested positive. None of the 23 serum samples collected after more than 56 days tested positive. The maximum duration at which a serum sample tested positive was 51 days after the onset of the symptoms. Forty-four (70%) of the 62 patients had HEV RNA in their serum samples. As shown in the Table, 288 serial serum samples collected from 32 patients between days 2 and 45 after the onset of disease were tested for HEV RNA. Twenty-four (75%) of the 32 patients had HEV RNA in serum samples. A difference in the positivity rate was found between days 6-10 and 21-25 (75% vs. 38.5%, P0.05), respectively.

Discussion Hepatitis E is an important public health problem in many developing countries, and also occurs in developed countries among individuals with no history of travel to areas where HEV is endemic. The pathogenesis of this disease is not clear. HEV is transmitted primarily by the fecal-oral route. In most outbreak settings, HEV transmission is associated with fecally contaminated drinking water. HEV RNA has been detected in water by RT-PCR, but the sources of fecal contamination leading to hepatitis E outbreaks have not been determined.[25] In addition, the stability of HEV in the environment is unknown. So, other routes of transmission may exist. Information on the duration of viremia is very important to understand the transmission dynamics and pathogenesis of this

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Detection of HEV RNA in sera of patients with hepatitis E by polymerase chain reaction

disease, but related data are particularly limited. ORF1 encodes nonstructural protein in the HEV genome. The nucleotide sequences are most highly conserved in this region. In fact, the Burmese strain and other HEV strains have more than 96% coincidence of nucleotides in the ORF1 region.[9] Thus, this region is the best for genome amplification by RT-PCR to detect the virus. We designed two sets of primers based on the ORF1 region to detect HEV RNA in serum with RT-nPCR. No positive results were detected among the negative controls. The bile specimen of a rhesus monkey experimentally infected with HEV was positive for HEV RNA. The specificity and sensitivity of our assay were acceptably high. We tested 531 serum samples collected from 62 patients with acute hepatitis E for HEV RNA. This is the largest case series describing the course of viremia. HEV RNA was detected in the sera of 44 (70%) of 62 patients, showing that viremia occurs in most hepatitis E patients. The confirmation of viremia should broaden concepts of hepatitis E pathophysiology and epidemiology. In another study of sera from patients with acute hepatitis E, HEV RNA was detected in 71% and 91% of the samples obtained on days 0-3 and 8-11, respectively; however, insufficient numbers of specimens were collected after the second week of illness.[21] Viremia was reported to last for 16 days in a human volunteer.[20] In a study on four children with acute hepatitis E, serum HEV RNA was detectable in all of them on days 2-3 after the onset of the disease, but in none on days 10 and 20.[23] In our study, viremia persisted for 51 days after the onset of symptoms in one patient. The strength of our study lies in the larger number of patients than in previous reports. The finding that serum HEV RNA remained positive in most patients after the time when ALT activity began to decrease suggests an uncoupling of viral replication and liver injury. One possible explanation is that HEV virions may be released from hepatocytes by a mechanism other than cell lysis. Another possibility is that virus detected in serum after the decrease in ALT activity was released after replication in tissues other than the liver. HEV antigens have been detected in the cytoplasm of cells in the small intestine, spleen, and lymph nodes of experimentally infected rats, suggesting that HEV replicates in these cells.[26] Perhaps HEV replicates in tissues other than the liver in humans as well. The other finding in our study was that there was no relation between serum HEV RNA positivity and level of IgG antibody to HEV. IgG antibody to HEV seems

to have no protective effect. The fact of a relatively long period of viremia in HEV-infected persons suggests the possibility of parenteral transmission, much the same as hepatitis B virus, which is transmitted principally by blood and blood fractions, and causes persistent viremia.[27,  28] Administration of blood products manufactured from large plasma pools has also been suggested as a possible mode of HEV transmission because current methods of viral inactivation used for these products may not inactivate nonenveloped viruses.[29] Transfusion-transmitted HEV infection has been demonstrated.[30] Thought should be given as to whether the screening of donated blood for HEV is warranted in HEV-endemic areas to protect blood recipients from infection. At a minimum, the stability of HEV in appropriately stored blood or blood products should be investigated. In summary, our data show that viremia occurs in and lasts a relatively long period in most patients with acute hepatitis E. Thus, transmission of HEV by administration of blood or blood products is likely to occur. In areas endemic for hepatitis E, it may be prudent to screen donor blood for HEV to eliminate transfusion-transmitted HEV infection. The stability and infectivity of HEV in the environment should be explored.

Acknowledgement The authors acknowledge Professor Hui Zhuang, Beijing University, for providing the bile specimen of a rhesus monkey experimentally infected with a pool of acute-phase stools from one female patient in the Xinjiang HEV epidemic. Funding: This study was sponsored in part by a grant from the National Natural Science Foundation of China (NSFC, No. 39670666) and Zhejiang Provincial Natural Science Foundation of China (No. 394185). Ethical approval: Not needed. Contributors: ZZY wrote the main body of the article under the supervision of RB. RB proposed and prepared the study. SH analyzed the data. All authors contributed to the follow-up of patients and the interpretation of study. RB is the guarantor. Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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