Role of TLR gene expression and cytokine profiling in the immunopathogenesis of viral hepatitis E

Journal of Clinical Virology 73 (2015) 8–13

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Role of TLR gene expression and cytokine profiling in the immunopathogenesis of viral hepatitis E Manasi Majumdar a , R.K. Ratho a,∗ , Yogesh Chawla b , Mini P. Singh a a b

Department of Virology, Postgraduate Institute of Medical Education & Research, Chandigarh 160012, India Department of Hepatology, Postgraduate Institute of Medical Education & Research, Chandigarh 160012, India

a r t i c l e

i n f o

Article history: Received 18 June 2015 Received in revised form 31 August 2015 Accepted 29 September 2015 Keywords: Cytokine Hepatitis E virus HEV-ORF2 peptide Peripheral blood mono-nuclear cell Toll like receptor

a b s t r a c t Background: The clinical manifestations of Hepatitis E virus (HEV) range from self-limiting acute viral hepatitis (AVH) to acute liver failure (ALF). The varied clinical course is thought to be immune-mediated. Toll-like receptors (TLRs) play a central role in sensing and initiating innate antiviral-response and downstream signaling of TLRs modulates cytokine production, thereby playing an important role in determining the disease course. Objectives: The present study was designed to elucidate the role of TLRs and cytokine production in the immunopathogenesis of HEV. Study design: Peripheral blood mono-nuclear cells were separated from 50 AVH-HEV, 30 ALF-HEV patients and 50 healthy-controls. One-part of the PBMC was processed for RNA-extraction another pulsed with HEV-ORF2-peptide. Gene-expression levels of TLR (2–4, 7, and 8) were checked using semi-quantitative Real-time-PCR. Cytokine levels were analyzed using Cytokine-Bead-Array. TLR3-silencing experiments were performed and post-silencing cytokine levels were estimated. Results: TLR3 gene-expression in AVH was significantly higher than ALF (202.4 ± 36.36 Vs 13.71 ± 5.01; p < 0.0001). Higher amount of both anti-and pro-inflammatory cytokines; IFN␥, TNF-␣, IL10 and TGF␤ were detected in the PBMC culture-supernatant of AVH Vs ALF (p < 0.0001, p = 0.0008, p = 0.0002, p < 0.0001 respectively). Post-silencing TLR3, significant decrease in IFN␥ level was observed in the PBMC culture-supernatant (4.08 ± 1.06 Vs 23.20 ± 12.51; p =N0.0213). Conclusions: TLR3 and IFN␥ were found to play an important role in HEV disease pathogenesis. Patients capable of expressing high levels of TLR 3 and robust IFN␥ response are able to limit the disease and recover uneventfully; while the patients with lower expression of TLR3 and IFN␥ progress to ALF. © 2015 Published by Elsevier B.V.

1. Background Cloning and sequencing of the novel agent of enterically transmitted viral hepatitis has revolutionized the understanding of Hepatitis E virus (HEV) infection [1–3]. Annually 3.3 million cases of acute hepatitis E with 56,600 deaths have been reported by World health organization (WHO) [4].The disease ranges from asymp-

Abbreviations: AVH, acute viral hepatitis; ALF, acute liver failure; CTLs, cytotoxic T lymphocytes; DCs, dendritic cells; HEV, hepatitis E virus; MODS, multiple organ dysfunction syndromes; ODN, oligodeoxynucleotide; ORF2, open reading frame 2; PBMCs, peripheral blood mononuclear cells; PRRs, pattern-recognition receptors; PAMPs, pathogen associated molecular patterns; SIRS, systemic inflammatory response syndrome; TLRs, toll like receptors; WHO, World health organization. ∗ Corresponding author. Fax: +91 0172 2744401. E-mail addresses: [email protected] (M. Majumdar), virology [email protected] (R.K. Ratho), [email protected] (Y. Chawla), [email protected] (M.P. Singh). http://dx.doi.org/10.1016/j.jcv.2015.09.011 1386-6532/© 2015 Published by Elsevier B.V.

tomatic infection to self-limiting acute viral hepatitis (AVH) and acute liver failure (ALF). In India nearly 30–70% of sporadic hepatitis cases are due to Hepatitis E virus [5,6]. Further, evidence of HEV infection has been detected in 30–45% of ALF cases [7]. Detection of anti-HEV IgM remains the conventional method of diagnosis [8]. In contrast, information on cell mediated immune response against HEV is limited. Studies have reported that AVH patients can mount T-cell mediated interferon-␥ (IFN-␥) and tumor-necrosis factor-␣ (TNF-␣) production [9,10], whereas, significantly lower lymphocyte proliferation index has been documented in ALF patients on stimulation with HEV specific immunogenic peptides [11]. Pathogens have unique signature molecules (PAMPs) that are recognized by Toll like receptors (TLRs) which results in either activation of antigen presenting cells and/or co-stimulation of T-cells, inducing both innate and adaptive immunity [12,13]. The expression level of TLRs can have direct effects on T cells, for example, expression of TLR2 will enhance T cell proliferation and will gener-

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Fig. 1. Representing gene-expression profile of TLRs in HEV patient’s PBMCs, clinically presented with features of AVH or ALF in un-stimulated condition as compared to the expression in healthy controls. The gene-expression of TLRs in AVH patients was higher than ALF patients. TLR2, 3 and 4 were significantly up regulated in AVH as compared to ALF in un-stimulated condition and marked bold faced.

ate efficient memory T cells [14]. TLR3 mediates cellular response to double stranded RNA (dsRNA) and promote activated CD4+ T-cell survival [15]. Further,TLRs can shape the outcome of viral infections via producing an array of cytokines [16]. TLR signaling has been found to play a key role in HBV and HCV infections [17,18]. 2. Objectives The current study was undertaken to determine the gene expression profile of TLR 2–4, 7 and 8 in HEV patients clinically presenting with AVH or ALF. Further, the cytokine response on stimulation of patient PBMCs with HEV-ORF2 immunogenic peptide (452–617a.a.) and the effect of in-vitro silencing TLR3 was studied to understand the roles played by TLRs in HEV pathogenesis.

Enzyme linked immunosorbant assay (ELISA) kit, as per the manufacturer’s instructions. The patients who were positive for anti-HEV IgM and negative for anti-HAV IgM, HBsAg and anti-HCV antibodies by ELISA, were enrolled for the study proper.Age and sex matched, apparently healthy individuals were additionally tested for antiHEV IgG (DSI,Italy), and those found negative for HBsAg, anti-HCV, anti-HAV IgM, anti-HEV IgM and IgG were recruited in the healthy control (HC) group.The study group comprised of 50 AVH, 30 ALF and 50 healthy controls. Chronic alcoholics, patients with history of hepatotropic drug use, immunosuppressant medication, chronic liver disease, hepatitis due to non-infectious or known bacterial cause were excluded from the study. The study was commenced following the approval of the institute ethics committee as per the National guidelines.

3. Study design

3.1. Definitions

The patients with clinical presentation of AVH or ALF with suspected viral etiology were recruited in this study during April 2009–June 2012. They were tested for the presence of viral hepatitis markers i.e., hepatitis B surface antigen (HBsAg; J Mitra, India), anti-hepatitis C antibodies (anti-HCV; J Mitra, India), anti-hepatitis A IgM (anti-HAV IgM; Orgenics, Israel) and anti-hepatitis E IgM (anti-HEV IgM; ImmunoVision, USA) using commercially available

Self-limiting Acute viral hepatitis (AVH) due to HEV was defined as patients positive for anti-HEV IgM, with serum aspartate aminotransferase elevation of at least five-fold or clinical jaundice or both. ALF was considered when the patient develop evidence of coagulation abnormality with an INR ≥1.5 and develop encephalopathy with an illness of <26 weeks duration, without any history of preexisting liver disease [19].

Table 1 Serum biochemical parameters of AVH and ALF patients included in this study. Variables

AVH (n = 50) (Mean ± SD)

(95% CI)

ALF (n = 30) (Mean ± SD)

(95% CI)

p-Value

Bilirubin mg/dl ALT IU/ml AST IU/ml ALP IU/ml PT in seconds PTI

13.21 ± 7.74 764.2 ± 703.2 779.4 ± 833.1 388.9 ± 261.9 16.09 ± 8.47 78.55 ± 24.78

(10.67–15.76) (496.7–1032) (468.3–1091) (287.4–490.5) (11.88–20.31) (66.95–90.14)

18.85 ± 9.66 710.4 ± 1064 326.4 ± 325 256.4 ± 144.6 33.66 ± 15.88 47.00 ± 20.91

(15.49–22.22) (350.4–1070) (213.0–439.9) (211.3–301.4) (28.12–39.20) (39.33–54.67)

0.0054 0.2376 0.0570 0.0607 p < 0.0001 p < 0.0001

Results are expressed as mean ± standard deviation. p < 0.05 was considered significant. Values which significantly differed in the two groups were marked in bold. ALT, alanine transferase; AST, aspartate transferase; ALP, alkaline phosphatase; PT, prothrombine time; PTI,prothrombine time index; IU/ml, international unit/ml. AVH and ALF patients were infected with HEV genotype 1.

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Fig. 2. (a & b): This figure shows the paired observations in the gene expression profile in un-stimulated Vs stimulated PBMC of ALF and AVH patients (2a,b) represented as fold increase as compared to healthy controls. In ALF patients (2a) a significant up-regulation in the TLR expression of TLR 2, 3 and 7 was observed on stimulation with HEV-ORF2 peptide. While, the TLR expression pattern in the AVH PBMCs remained constitutively high both in the stimulated and un-stimulated condition. On applying ANOVA both in the un-stimulated and stimulated counterparts TLR3 emerged as the significantly up-regulated TLR in AVH and ALF patients (p < 0.0001) as compared to TLR2, 4, 7 and 8 in any random pairing.

3.2. Sample collection and PBMC separation Approximately 10 ml of venous blood was collected aseptically by trained health care personnel. Serum was separated from 3 ml of clotted blood and used for serological and molecular testing for viral diagnosis. The rest was processed for isolation and culture of PBMC as described previously [20]. Finally 2 × 105 cells were stimulated with 2 ␮g of HEV ORF-2 protein (452–617 amino acid, immunogenic epitope) (ProSpec, USA) and incubated for three days. Post-stimulation the plates were centrifuged briefly, the supernatant was kept at −70 ◦ C for cytokine analysis and the PBMC pellet was processed for RNA extraction and TLR expression studies.

3.3. RNA extraction and semi-quantitative real-time PCR for gene-expression study of TLRs Total RNA was extracted from both un-stimulated and stimulated PBMCs of AVH (n = 50), ALF (n = 30) and HC (n = 50) using

TRIzol reagent (Life technologies; USA) and quantitated using NanoDrop (Thermo Fisher Scientific,USA). One microgram of total RNA was reverse transcribed to cDNA using random hexameric primers and RevertAidTM First Strand cDNA Synthesis Kit (Fermentas, Glen Burnie,MD, USA). Semi-quantitative real-time PCR (RT-PCR) was performed in the Roche Light Cycler® 480 (Roche, Switzerland) using MaximaTM SYBR Green qPCR Master Mix (Fermentas, USA). All samples were amplified in duplicate; non-template reactions were included as negative control. Briefly 2.5 ␮l cDNA template was used for each PCR with 200 nM forward and reverse primers [21] in a total reaction volume of 25 ␮l. All the genes were standardized to run at a single thermal profile.The thermal cycling profile was standardized to 10 min at 95 ◦ C, followed by 40 cycles of 95 ◦ C for 15 s, 60 ◦ C for 30 s, and 72 ◦ C for 15 s. Melting curve analysis was performed after each run to determine the specificity of the PCR products. Further, gel electrophoresis of the PCR products revealed one distinct band.Validation of 2−Ct method was performed by checking the amplification efficiencies of the tar-

Table 2 Representing the levels of cytokines (pg/ml) in the PBMC culture supernatants and the statistical significance between the estimated levels using Mann–Whitney’s U -Test. Clinicalpresentation

IFN-␥ Mean ± S.E

IL4 Mean ± S.E

TNF-␣ Mean ± S.E

IL10 Mean ± S.E

TGF-␤ Mean ± S.E

IL12 Mean ± S.E

HC AVH ALF AVH Vs HC p value ALF Vs HC p value AVH Vs ALF p value

0.7 ± 0.29 292.3 ± 88 3.24 ± 0.8 <0.0001***

1.74 ± 0.32 3.25 ± 0.76 5.49 ± 2.45 0.2214

0.93 ± 0.34 113.4 ± 51.7 9.05 ± 3.26 <0.0001***

1.71 ± 0.47 443.6 ± 76.2 12.59 ± 17.4 <0.0001***

0.78 ± 0.52 86.23 ± 12.68 12.59 ± 5.03 <0.0001***

0.59 ± 0.24 2.55 ± 0.76 1.89 ± 0.53 0.0067**

0.0314*

0.1060

0.0159*

0.0009***

0.2371

0.0794

<0.0001***

0.4848

0.0008***

0.0002***

<0.0001***

0.6112

AVH patients mounted significantly elevated levels of both pro-and anti-inflammatory cytokines, where as in ALF patients a generalized low cytokine response was observed.

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FCS and further stimulated with 2 ␮g of HEV pORF2 peptide. Three days post stimulation the culture supernatants were harvested and stored at −70 ◦ C for CBA. 3.6. Statistical analysis TLRs expression data and levels of cytokine produced were statistically analyzed by non-parametric Mann-Whitney’s U-test for unpaired and Wilcoxon ranked sum test for paired observations. Multi-parametric analysis using ANOVA was performed to determine the relationship between TLR gene-expressions. The spearman’s correlation analysis was used to determine whether impaired cytokine production was correlated with gene-expression levels of various TLRs. Results were considered significant at a p value less than 0.05. 4. Results 4.1. Demographic profile

Fig. 3. Histogram representing IFN␥ concentration in the culture supernatant of PBMCs treated with 40, 60 and 80 pico-moles (pmoles) of TLR3 siRNA with a documented 58.77 ± 8.76 fold down regulation of TLR3 as compared to paired nonsilenced PBMCs and healthy controls on stimulation with HEV-ORF2-peptide (n = 5). Experiment was performed in triplicate and represented as mean ± standard deviation.

get and reference genes, which was found to be approximately equal and then applied for calculating relative gene expression of sample of interest (patient group) as compared to calibrators (healthy controls) normalized to the corresponding endogenous housekeeping GAPDH gene.

The mean age of the AVH subjects in this study was 29.14 ± 12.69 years and ALF was 28.38 ± 11.67 years with a male to female ratio of 5.25:1 and 0.88:1 respectively. Serum biochemical parameters were significantly altered, where in bilirubin levels (p = 0.0054) and prothrombin time (p < 0.0001) was found to be significantly higher in ALF group as compared to AVH group (Table 1). 4.2. TLR gene-expression ex-vivo (un-stimulated) A significant up-regulation in the gene-expression level of TLR2 (15.26 ± 4.39 Vs 2.84 ± 0.79; p = 0.022), TLR3 (202.4 ± 36.36 Vs 13.71 ± 5.01; p < 0.0001) and TLR4 (10.25 ± 2.58 Vs 0.69 ± 0.51; p <0.0001) was observed in the un-stimulated PBMCs of AVH patientsas compared to ALF patients (Fig. 1). 4.3. TLR gene-expression post-stimulation

3.4. Cytometric bead array (CBA) Harvested PBMC culture supernatants stored at −70 ◦ C were thawed. Undiluted supernatants were used for simultaneous detection of IFN␥, TNF␣, IL12, IL4, IL10 and TGF␤ using the multiplexed cytometric bead array (CBA Flex, BD Biosciences, USA) according to the manufacturer’s instructions. The concentration range for detection using this assay was 10–2,500 pg/ml for each of the six cytokines. 3.5. Transfection of siRNA and gene silencing Small interfering RNA (siRNA) against TLR3 and fluorescently labeled non-silencing control sequence without homology to known mammalian genes BLOCK-iTTM (Life technologies; USA) were obtained for standardizing transfection experiments. The sequence for TLR3 siRNA was; sense CAACGACUGAUGCUCCGAAtt and antisense UUCGGAGCAUCAGUCGUUaa (Cat no: 4,392,420, siRNA ID: s234, Life technologies; New York, USA). PBMCs were plated overnight before transfection in opti-MEM medium and then transfected with 1 ␮gm of Lipofectamine® 2000 (Life technologies; New York, USA) and 40, 60 and 80 picomole of TLR3 siRNA. According to the previously standardized protocol after addition of siRNA-lipid complexes into each well, culture plates were centrifuged at 1000 rpm for 30 min and incubated in 5% CO2 at 37 ◦ C atmosphere for 18 h [22]. One part of the silenced PBMC was subjected to RNA extraction and TLR3 gene expression studies using custom made TLR3silencing specific primers (Life technologies; New York, USA) by SYBR green semi-quantitative real-time PCR. The other part was washed, maintained in RPMI-1640 with 10%

Stimulated PBMCs of ALF patients with virus specific HEV ORF2 peptide (452–617a.a) showed nearly 3 fold increase in the geneexpression of TLR 2, 10 fold increase of TLR 7 and 100 fold increase in TLR3 (Fig. 2a). The expression of TLR3 in AVH patients constitutively remained higher both in un-stimulated and stimulated condition (Fig. 2b). On applying the multi-parametric statistical analysis (ANOVA) considering all the different TLRs tested both in un-stimulated and stimulated condition,the TLR3 emerged as the most significantly up-regulated TLR in the AVH and ALF patients (p < 0.0001) in any random pairing. 4.4. Cytokine levels in the culture supernatant in response to HEV pORF2 stimulation IFN-␥ levels in the culture supernatant of stimulated PBMCs of AVH patients were significantly higher than ALF and HC. The ALF patients were unable to mount a robust IFN-␥ production as compared to AVH and healthy controls. Levels of TNF-␣, IL10 and TGF-␤ were significantly higher in AVH as compared to ALF. Whereas, the level of IL12 remained comparable in both the groups. IFN-␥:IL 4 ratio was found to be 89.76 in AVH and 0.59 ALF showing a Th2 bias in ALF patients (Table 2). 4.5. Effect of TLR3 gene silencing The TLR3 siRNA concentration of 40, 60 and 80 picomoles were tested for performing gene silencing experiments as documented by our previous experiments [22]. Eighteen hours

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post-transfection, the confirmation of TLR3 gene-down regulation was performed by semi-quantitative real-time PCR. On comparing TLR3 with house-keeping gene-expression (GAPDH) 58.77 ± 8.76 fold down regulation was observed. After silencing of TLR3, when the PBMCs were stimulated with HEV pORF2 a significant decrease in the IFN-␥ level was observed in the culture supernatant (Fig. 3). 4.6. Significant correlation between TLR gene-expression and cytokine production A positive correlation was observed between the gene expression levels of TLR3 and IFN␥ (0.896); TLR3 and TNF-␣ (0.462) in AVH patients. However,a negative correlation was observed between TLR3 and IL12 production in ALF patients (−0.596). 5. Discussion Toll like receptors are components of pattern recognition receptors (PRRs), which recognizes the PAMPs present on various viruses [23,24]. Several lines of evidence have shown that TLR signaling plays key role in the elimination of invading viruses. Rhinovirus infection has been shown to up-regulate TLR3 gene-expression in human bronchial epithelial cells [25]. Hepatitis C virus infection increases TLR4 expression in Raji B cells, leading to enhanced antiviral activity [26]. Investigation of hepatocytes and kupffer cells (KCs) from fresh liver biopsies of chronic hepatitis B (CHB) patients has demonstrated reduced expression of TLR2 in HBeAg positive patients as compared to HBeAg negative patients [27,28]. Further, Injection of ligands for TLR 3–5, 7 and 9 has been shown to suppress HBV replication in transgenic mice [18,29]. The present study documents that HEV-AVH patients have significantly up-regulated gene expression of TLR 2, 3 and 4 in PBMCs as compared to HEV-ALF patients. On multi-parametric analysis TLR3 was found to be significantly up regulated as compared to all other TLRs tested i.e. TLR 2, 4, 7 and 8 both in stimulated and unstimulated PBMCs. Further, the low level of TLR3 gene expression in ALF patients was significantly up-regulated following in-vitro stimulation with HEV pORF2 peptide. (Fig. 2) On evaluating the cytokine response, IFN-␥ and TNF-␣ levels in the culture supernatant of AVH patients were significantly higher than the ALF patients. In addition, an elevated level of antiinflammatory cytokines (IL10 and TGF␤) was also observed in AVH patients. This might play an important role in establishing a balance between pro and anti-inflammatory cytokines in AVH patients. On the other hand, in ALF patients a generalized low cytokine response and Th2 bias was observed. Studies have reported ALF patients had a marked expansion of B cells that can secret immunoglobulin G [30]. The clinical correlation with respect to the cytokine levels in the present study may be substantiated by the theory of immunological dissonance in systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndromes (MODS) [31], which states that; when the balance between pro and antiinflammatory response is lost, over whelming immune reaction develops. However, the minimal reactivity to immunogenic HEV ORF2 peptide as observed in ALF patients included in this study, might be a consequence of exhaustive immune response followed by anergy. Interferons play a central role in the induction of antiviral responses, enhance dendritic cell (DC) maturation, natural killer (NK) cell cytotoxicity, and differentiation of virus-specific cytotoxic T lymphocytes [32]. In the present study, a positive correlation (r2 ; 0.896) between TLR3 gene expression of AVH patients and the levels of IFN␥ in the PBMC culture supernatant was observed. Negishi et al. have proved a critical role of TLR3 dependent IFN␥ (Type II IFN) signaling pathway in antiviral innate immune response against

coxsackie virus B serotype 3 (CVB3) by demonstrating that the mice deficient in TLR3 are more vulnerable to CVB3 than wild type mice in terms of higher mortality and acute myocarditis [33]. Furthermore, Schulz et al. have documented that the murine DCs are activated to mature by phagocytosis of apoptotic bodies from virally infected cells which produce cytokines in a TLR3-dependent manner [34]. DCs are the innate immune system’s principal cell involved in cross-presentation which either leads to, cross-priming resulting in virus specific cytotoxic T cell (CTL) response, or in cross-tolerance, resulting in T-cell inactivation. Immunization with virally infected cells or cells loaded with poly(I:C) led to a significant increase in TLR3-dependent CTL cross-priming. In the present study we observed, a significantly higher TLR3 gene-expression in the PBMCs of AVH patients as compared to ALF patients that might have facilitated robust cross-priming of virus-associated antigen and helped mounting a strong HEV specific-cytotoxic T-cell activity and cytokine production. Recent research documents regulating cytokine production by modulating the TLR function for therapeutic purposes. TLR7 ligand has therapeutic potential in the treatment of HCV [35]. Synthetic ODN, a TLR9 stimulator, has been used commercially in the HBV vaccine [36]. In the present context the study provides novel insight into the mechanism of TLR mediated differential cytokine production playing an important role in determining the course of viral hepatitis E. In conclusion, the author’s have shown that the patients expressing high levels of TLR3 mount a robust IFN␥ response and are able to limit the disease and recover uneventfully; on the other hand patients with lower expression of TLR3, IFN␥ production have a marked Th2 bias, progress to severity and might develop acute liver failure. Further, this study documents that silencing TLR3 leads to a significant decrease in IFN-␥ in the PBMC culture supernatant of HEV patients, suggesting important role played by the TLR3-IFN␥ axis in HEV disease pathogenesis. Conflict of interest None declared Funding The study was partly funded by Indian council of medical research Ad-hoc research scheme vide the sanction number 5/7/21/2009-ECD-I. Ethical approval Ethical approval was given by Institute Ethics Committee—PGIMER, Chandigarh. Judgment’s reference number: 7926-PG-1Trg-08/23116. Acknowledgements Manasi Majumdar is a recipient of senior research fellowship from the Council of Scientific and Industrial Research, Govt. of India. References [1] A.W. Tam, M.M. Smith, M.E. Guerra, C.C. Huang, D.W. Bradley, K.E. Fry, G.R. Reyes, Hepatitis E virus (HEV): molecular cloning and sequencing of the full-length viral genome, Virology 185 (1991) 120–131. [2] G.J. Dawson, K.H. Chau, C.M. Cabal, P.O. Yarbough, G.R. Reyes, I.K. Mushahwar, Solid-phase enzyme-linked immunosorbent assay for hepatitis E virus IgG and IgM antibodies utilizing recombinant antigens and synthetic peptides, J. Virol. Methods 38 (1992) 175–186. [3] M.S. Khuroo, Discovery of hepatitis E: the epidemic non-A, non-B hepatitis 30 years down the memory lane, Virus Res. 161 (2011) 3–14.

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