Pathogenesis of Hepatitis E Virus and Hepatitis C ... - Journal of Virology

JOURNAL OF VIROLOGY, Nov. 2010, p. 11264–11278 0022-538X/10/$12.00 doi:10.1128/JVI.01205-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Vol. 84, No. 21

Pathogenesis of Hepatitis E Virus and Hepatitis C Virus in Chimpanzees: Similarities and Differences䌤† Claro Yu,1‡ Denali Boon,1‡ Shannon L. McDonald,1 Timothy G. Myers,2 Keiko Tomioka,1 Hanh Nguyen,1 Ronald E. Engle,1 Sugantha Govindarajan,3 Suzanne U. Emerson,1 and Robert H. Purcell1* Laboratory of Infectious Diseases1 and Research and Technologies Branch,2 Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 50 South Drive, Building 50, Bethesda, Maryland 20892-8009, and Department of Pathology, Rancho Los Amigos Hospital, Downey, California 902413 Received 4 June 2010/Accepted 18 August 2010

The chimpanzee is the only animal model for investigating the pathogenesis of viral hepatitis types A through E in humans. Studies of the host response, including microarray analyses, have relied on the close relationship between these two primate species: chimpanzee samples are commonly tested with human-based reagents. In this study, the host responses to two dissimilar viruses, hepatitis E virus (HEV) and hepatitis C virus (HCV), were compared in multiple experimentally infected chimpanzees. Affymetrix U133 ⴙ 2.0 human microarray chips were used to assess the entire transcriptome in serial liver biopsies obtained over the course of the infections. Respecting the limitations of microarray probes designed for human target transcripts to effectively assay chimpanzee transcripts, we conducted probe-level analysis of the microarray data in conjunction with a custom mapping of the probe sequences to the most recent human and chimpanzee genome sequences. Time points for statistical comparison were chosen based on independently measured viremia levels. Regardless of the viral infection, the alignment of differentially expressed genes to the human genome sequence resulted in a larger number of genes being identified when compared with alignment to the chimpanzee genome sequence. This probably reflects the lesser refinement of gene annotation for chimpanzees. In general, the two viruses demonstrated very distinct temporal changes in host response genes, although both RNA viruses induced genes that were involved in many of the same biological systems, including interferoninduced genes. The host response to HCV infection was more robust in the magnitude and number of differentially expressed genes compared to HEV infection. Hepatitis E virus (HEV) and hepatitis C virus (HCV) are both positive-sense, single-stranded RNA viruses; however, they differ in classification, size, viral structure, and composition. HCV, a member of the family Flaviviridae, is an enveloped virus, approximately 60 nm in diameter, with a 9.5-kb genome (30). HEV is a nonenveloped virus, approximately 33 nm in diameter, with a genome length of 7.2 kb, and it is a member of the family Hepeviridae (30). Both viruses can infect the liver and cause clinical disease, which is indistinguishable; however, the epidemiology and natural history of these two viruses are strikingly different. HCV is transmitted parenterally, while HEV is transmitted via the fecal-oral route, primarily through contaminated water or food. The incubation period ranges from 15 to 64 days for HEV compared to 14 to 180 days for HCV. Approximately 3.3% of the world’s population has been infected with hepatitis C virus, and 3% are chronically infected because the majority (75 to 85%) of infections persist, putting patients at risk for sequelae such as cirrhosis and hepatocellular carcinoma (HCC) (2, 10). In contrast, it has been * Corresponding author. Mailing address: Laboratory of Infectious Diseases, 50 South Drive, Bldg. 50, Rm. 6523, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-8009. Phone: (301) 496-5090. Fax: (301) 402-0524. E-mail: [email protected]. ‡ C. Yu and D. Boon contributed equally to this work. † Supplemental material for this article may be found at http://jvi .asm.org/. 䌤 Published ahead of print on 25 August 2010.

estimated that approximately 33% of the world’s population has been exposed to HEV (1). Infection with this virus is generally self-limiting, without long-term sequelae; however, increased disease severity and mortality have been reported in pregnant women (1, 9) and in those with underlying liver disease (13). Antibodies to HEV appear early (approximately 4 weeks after infection), and prior exposure (by either infection or vaccination) can protect against reinfection, while antibodies to HCV appear much later (approximately 10 to 12 weeks after infection) (30, 38) and do not protect against reinfection (18). The differences in the disease manifestation of these two hepatotropic RNA viruses suggest dissimilar host responses, which might influence the course of the infections. Published microarray studies of viral hepatitis caused by hepatitis B virus (HBV) and HCV have described the host response at the transcriptome level and have utilized liver tissue or cell culture to study the changes in gene expression that result from viral infection or transfection. Although the chimpanzee (Pan troglodytes) is the only animal model for studying all five human hepatitis viruses, published reports of their use in studying the intrahepatic global response to viral hepatitis have been limited. Using chimpanzees to study the pathogenesis of viral hepatitis by microarray has several advantages: (i) human and chimpanzee genome sequences are highly similar; (ii) the inoculation and inoculum are controlled by the research design; (iii) preinfection samples serve as negative controls; and (iv) temporal changes and the progression of the disease throughout the time course of infection can be

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PATHOGENESIS OF HEV AND HCV IN CHIMPANZEES

studied in serially collected samples, including liver biopsies. In contrast, studies using human subjects are usually hampered by lack of homologous preinoculation samples and liver biopsies from the different phases of the infection, especially the acute phase. The study of gene expression by microarray analysis of RNA from the liver of HCV-infected chimpanzees was reported in three previous studies (5, 6, 40). Although these studies differed in experimental design, hybridization platform, data normalization, and analyses, there were four consistent observations. (i) The number and magnitude of induction of type I interferon-stimulated genes (ISGs) indicated that HCV induced a robust innate immune response. (ii) The adaptive immune response played a crucial role in host outcome, as indicated by the differential expression of cell surface markers and a number of genes that are expressed in T cells. (iii) Nonimmune genes contributed to the host response to HCV, including genes involved in cell structure, death, and proliferation. (iv) A large number of genes that were differentially expressed in these microarray studies have not yet been characterized. In contrast to HCV, the host response of chimpanzees to HEV infection has not been studied by microarray technology. In the present study, we used the most recent Affymetrix microarray technology for gene expression profiling to compare the global host responses to HCV and HEV infections in liver biopsies from frequently sampled chimpanzees. To accomplish this, probe sequences were independently mapped to the most recent versions of the human and chimpanzee genomes, resulting in the exclusion of probes having less than 100% sequence identity to the respective genome. The results from the genome-specific analyses confirmed the validity of using microarray chips designed for one species (humans) in studies of a closely related species (chimpanzees) (26). Our study identified similarities as well as differences in the host responses to infection with these two hepatotropic, positivesense, single-stranded RNA viruses. MATERIALS AND METHODS Chimpanzees and sample collection. Three chimpanzees were inoculated intravenously with the hepatitis C virus: chimp 1628 was infected with ⬍100 infectious doses (ID) of the H77 strain (genotype 1a) (31), chimp 1417 was infected with an unknown titer, in serum, of the HC-J6 strain (genotype 2a) (32), and chimp 1422 was infected with an unknown titer, in serum, of the T.N. strain (genotype 1a) (36). Four chimpanzees (1616, 1618, 1374, and 1375) were inoculated intravenously with 0.5 ml of the HEV SAR55 strain (genotype 1) at approximately 106 ID. Multiple preinoculation and weekly postinoculation serum samples and percutaneous needle liver biopsies were collected for all chimpanzees. Pieces of the biopsies were frozen in optimal cutting temperature compound (OCT), stored at ⫺80°C, and subsequently processed for microarray analysis. Serum samples were tested for alanine aminotransferase (ALT) (Analytics, Gaithersburg, MD) and virus-specific antibodies: anti-HCV IgG, using a commercial enzyme-linked immunosorbent assay (ELISA) kit (ELISA 2.0; Abbott, Chicago, IL); and anti-HEV IgG, based on a procedure described initially by Tsarev et al. and modified by Engle et al. (15, 46). The housing and care of the chimpanzees were in compliance with all relevant guidelines and requirements, and the animals were housed in facilities that are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. All animal study protocols involving chimpanzees were approved by the NIAID Animal Care and Use Committee, as well as by the Animal Care and Use Committee of the facility housing the animals. Liver histology. Hematoxylin and eosin staining was performed on formalinfixed, paraffin-embedded samples of the biopsies. The liver biopsy specimens were blinded with regard to the clinical and virological data, and histologic

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evaluation was conducted by application of the Ishak modified histologic activity index (HAI) and fibrosis scoring system (22). Ishak activity was graded on a 0- to 18-point scale (22). RNA extraction and amplification. Frozen liver needle biopsies embedded in OCT were homogenized with a PRO 200 homogenizer with Multi-Gen 7 mm 316 stainless generators (Pro Scientific, Oxford, CT). Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA) and purified with an RNeasy minikit (Qiagen, Valencia, CA). Quantity and quality of total RNA were assessed with a Nanodrop 1000 (ThermoFisher Scientific, Waltham, MA) and a 2100 Bioanalyzer (Agilent, Santa Clara, CA). Total RNA was used for microarray analysis and quantitative reverse transcription-PCR (qRT-PCR) studies. Previous studies in our laboratory comparing 6 ng and 50 ng of input total RNA with two rounds of amplification demonstrated comparable results; therefore, only 6 ng of total RNA was amplified in order to conserve samples for future applications (data available upon request). cDNA was synthesized using a Genechip 2-cycle cDNA synthesis kit (Affymetrix, Santa Clara, CA). First-cycle cDNA was transcribed in vitro with a Megascript T7 kit (Ambion, Foster City, CA). First-cycle cRNA was purified with a Genechip IVT cRNA cleanup kit (Affymetrix, Santa Clara, CA). Second-cycle cDNA was purified with the Genechip sample cleanup module (Affymetrix, Santa Clara, CA) and transcribed with a Genechip 2-cycle target labeling and control reagents kit (Affymetrix, Santa Clara, CA), which incorporates biotinylated ribonucleotide analogs into the transcripts. Biotinylated cRNA was purified with a Genechip IVT cRNA cleanup kit (Affymetrix, Santa Clara, CA), and 15 ␮g of product was fragmented in the fragmentation buffer provided in the kit. Chip hybridization and data preprocessing. Biotin-labeled cRNA was hybridized to the Affymetrix U133 ⫹ 2.0 chip according to the manufacturer’s procedure by Science Applications International Corporation (SAIC) under a contract with the National Cancer Institute. Quantification of virus in blood and liver. RNA was extracted from 140 ␮l of serum or plasma with a QIAamp viral RNA minikit (Qiagen, Valencia, CA), and total RNA was eluted in 60 ␮l. The HCV titer in serum and plasma was determined by TaqMan using a procedure described by Engle et al. (14), with some modifications. Briefly, 10 ␮l of cDNA was mixed with 15 ␮l of TaqMan fast universal PCR master mix and primers (Applied Biosystems, Foster City, CA). The primers and probe were selected from a region of the 5⬘ untranslated region (UTR) that is highly conserved among the six major genotypes of HCV (7). These consisted of forward (AGY GTT GGG TYG CGA AAG) and reverse (CAC TCG CAA GCR CCC T) primers and a nonfluorogenic quencher (NFQ) minor groove binding (MGB) probe (CCT TGT GGT ACT GCC TGA) used at 500, 1,000, and 250 nM, respectively. TaqMan was performed with an ABI 7900HT real-time PCR system, following the manufacturer’s recommendations. A quantity standard line representing a 6-log range was constructed with the OptiQuant HCV RNA (Acrometrix, Benicia, CA) nucleic acid test reference panel. The dilution series is described in more detail elsewhere (14). Results were expressed as log10 international units (IU) per milliliter based on the WHO 96/790 reference standard (37). HEV viral titer in serum and plasma was determined by TaqMan in 10-␮l reactions. The primers and probe (a 112-base amplicon from positions 3355 to 3466) were selected from a region in open reading frame 1 (ORF1) of the HEV genome. The forward (GTT GGG CAG AAG CTA GTG TTC AC) and reverse (CCG TGG CAA TGA TGG TAG TCT) primers and probe (6-carboxyfluorescein [FAM]-ACC CCG GTT CAG TGA CGG TCC A–6-carboxytetramethylrhodamine [TAMRA]) were used at 900, 900, and 200 nM, respectively. TaqMan cycling conditions were based on the manufacturer’s recommendations. The quantification line was based on an in-house HEV standard that yielded a 6-log dynamic range. Results were expressed as log10 copies per reaction. HCV and HEV in liver samples were determined in a similar fashion, but the amount of input RNA was 50 ng per reaction and the viral titer was expressed as log10 genome equivalents (GE) per ng of total RNA. In addition, a housekeeping gene coding for GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was monitored in parallel with every sample to insure that similar amounts of RNA were used. Probe-level analyses. Three HCV-infected chimpanzees (1628, 1422, and 1417) and four HEV-infected chimpanzees (1616, 1618, 1374, and 1375) were used in our study. Grouping of the biological replicates was based on viremia: groups consisted of samples from the first positive week (and the second positive week for HCV), the peak positive week, the last positive week, the first negative week, and the fourth negative week (Fig. 1). The data from the last positive week for chimpanzee 1375 were not included in the analyses because of poor quality control results. The Genomatix ChipInspector software (http://www.genomatix .de/products/ChipInspector/) was used to map probes to transcripts in the human

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FIG. 1. HCV infection of three chimpanzees and HEV infection of four chimpanzees. Levels of alanine aminotransferase (ALT), antibodies, and virus in the blood and liver were assessed weekly. Microarrays were performed on preinoculation samples (B0) and postinoculation weekly time points relative to viremia: the first positive (F), second positive (S), peak positive (P), last positive (L), first negative (1N), and fourth negative (4N) weeks. HAI, histologic activity index using the Ishak scoring system. ND, no data available.

(version 37) and chimpanzee (version 2) genome sequences in ElDorado (Genomatix proprietary genome annotation database), in order to remove probes without 100% sequence identity, to perform a linear total intensity normalization of probes of each chip (.cel file), and to employ a standard permutational t test, similar to the significance analysis of microarrays (SAM) algorithm, at the singleprobe level to search for transcripts with differential expression (47). First, chip data (.cel files) from the pre- and postinoculation biopsies were imported into the ChipInspector software, filtered based on the genome mapping, and subsequently analyzed “exhaustively,” using all combinations of all biological replicates for each hepatitis virus. Then the statistical stringency, based on the falsediscovery rate, was set at approximately 5% after the normalization and the permutational t test were performed. Transcripts that were identified with at least three perfectly matched probes and exhibited a minimum linear fold change of 2 were considered differentially expressed. Finally, differentially expressed genes from each t test were manually assigned to functional categories. To determine the differential expression of genes in response to comparable levels of viremia, selected time points from two HCV-infected chimpanzees and two HEV-infected chimpanzees were tested using the ChipInspector software, as described above.

The correlation between independent results generated by Genomatix ChipInspector for humans and chimps was analyzed based on cross-comparison of available genome annotation for the two species. Similar genes between the human and chimpanzee genome alignments were first matched to alias or gene name, primarily from the NCBI databases, including Entrez Gene and Homologene. Second, genes were matched, based on presumed similarity, such as an annotation of “similar to” or “predicted,” in the absence of a known annotated gene. Finally, statistically significant probe sequences of remaining genes in both genome alignments were extracted and aligned to gene and probe sequences in the opposite genome to identify similar targets. Genes that could not be matched between the two species with any of the above criteria were counted as “unmatched.” IPA. Canonical pathways that were statistically overrepresented as differentially expressed genes in our data set were determined using the Ingenuity Pathway Analysis (IPA) software with a right-tailed Fisher’s exact test, using the entire IPA Knowledge Base as the reference set and a P value cutoff of 0.01. Additionally, network analyses of genes from each time point were performed in IPA to search specifically for transcription regulators that promote the differential expression of genes in our list. Only transcription regulators that interact

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FIG. 2. Histopathology. Acute hepatitis C is shown by multifocal lobular necrosis ranging from 5 to 10 to ⬎10 foci of necrosis per 10⫻ objective field. This change is pan-lobular and not accompanied by hepatocyte swelling. The portal inflammatory reaction is mild to moderate. Acute hepatitis E is shown by marked portal inflammatory reaction and periportal necrosis with spillover of inflammation. The lobular necrosis is mild, ranging from 0 to 2 foci per 10⫻ objective. There is no hydropic change of the hepatocytes. The acinar change usually seen in human acute hepatitis E is not identified in these biopsies.

directly with at least two differentially expressed genes were investigated. Additional information on the IPA network analysis algorithm may be found at http://www.ingenuity.com. Gene expression validation by TaqMan qRT-PCR. Eleven genes were tested by qRT-PCR in order to confirm their differential expression by microarray analysis. These genes were selected based on biological interests and representative range of expression levels. Transferrin was used as the housekeeping gene for normalization. Primers and probes were selected with ABI TaqMan gene expression assays (Applied Biosystems, Foster City, CA) after assessing the sequence similarity of the primers and probe regions to the chimpanzee transcripts with ClustalW2 (EBI) and Sequencher (Gene Codes Corporation, Ann Arbor, MI) (see Table S1 in the supplemental material). Two sets of primers and probe specific for gamma interferon (IFN-␥) and HLA-C were custom designed with the ABI Primer Express software (Applied Biosystems, Foster City, CA). Assays were optimized and validated based on standard curve efficiency (E% ⫽ 10(⫺1/slope)⫺1 ⫻ 100%, where E ⫽ 100% ⫾ 10%) and relative efficiency (threshold cycle [⌬⌬CT] slope ⬍ 0.1), as recommended and described by the manufacturer. cDNA was generated from total RNA extracted from chimpanzee liver biopsies (ABI high-capacity cDNA reverse transcription kit; Applied Biosystems, Foster City, CA). Ten nanograms of chimpanzee RNA was added to the cDNA RT solution and incubated in an ABI 9700 thermocycler under the following conditions: 25°C for 10 min, 37°C for 120 min and 85°C for 5 min. The cDNA was preamplified using the TaqMan preamplification master mix (Applied Biosystems, Foster City, CA), using the manufacturer’s protocol. Briefly, 10 ␮l of cDNA was mixed with 25 ␮l of ABI TaqMan preamplification master mix, 12.5 ␮l of pooled 0.2⫻ TaqMan primers and probe, and 2.5 ␮l of PCR-grade water. The mixtures were incubated in an ABI 9700 thermocycler under the following conditions: 95°C for 10 min, followed by 14 cycles of 95°C for 15 s and 60°C for 4 min. Five microliters of diluted preamplified cDNA was added to 15 ␮l of ABI gene expression master mix (Applied Biosystems, Foster City, CA) and loaded onto ABI 384-well plates precoated with gene-specific primers and probe (Applied Biosystems, Foster City, CA). The plates were incubated in an ABI 7900 HT fast real-time PCR system (Applied Biosystems, Foster City, CA) under the following conditions: 50°C for 2 min and 95°C for 10 min, followed by 45 cycles at 95°C for 15 s and 60°C for 1 min. Each sample was tested in triplicate, and the average CT was used to calculate the relative gene expression using the 2⫺⌬⌬CT method, as described by ABI. Preinoculation samples from each chimpanzee were averaged to determine a baseline. For each selected time point, the average fold change (2⫺⌬⌬CT) across the biological replicates by qRT-PCR was compared with the average fold change of each gene by ChipInspector analysis. Microarray data accession number. The microarray .cel files have been deposited in the Gene Expression Omnibus (GEO) website under series no. GSE22160.

RESULTS Virus levels and ALT. The three HCV-infected chimpanzees and the four HEV-infected chimpanzees exhibited very distinct pathogenesis profiles (Fig. 1). Although both viruses were detected in the blood and the liver 1 or 2 weeks after inoculation, HEV disappeared 4 to 5 weeks postinoculation, while HCV remained for over 9 weeks before the virus initially became undetectable, with reappearance and disappearance in the blood and the liver up to week 25. All chimpanzees cleared their respective infections and did not progress to chronicity. Increased serum ALT levels are an indicator of liver damage, and the peak ALT levels were generally higher in the HCV infections than in HEV infections (76, 192, and 744 U/liter; geometric mean [GM] ⫽ 221 U/liter in HCV-infected chimpanzees versus 57, 53, 111, and 87 U/liter [GM ⫽ 73 U/liter] in HEV-infected chimpanzees). HCV-infected chimpanzees became anti-HCV positive 1 to 4 weeks after the virus initially disappeared from the blood. In contrast, HEV-infected chimpanzees became anti-HEV positive 1 week before or during the week of the last positive viremia. Lastly, HCV reemerged in the blood and the liver after antibody seroconversion, while HEV remained undetectable after seroconversion. Histology. Among the HCV-infected chimpanzees, 1628 and 1422 had more severe hepatitis than 1417 (Fig. 1 and 2). The highest HAI score in the series for 1417 was only 3⫹, and focal necrosis in the lobules was minimal (ⱕ2⫹). Most of the biopsies from this chimpanzee were normal and had no necrosis. In contrast, the total HAI score was 6 to 7⫹, and the lobular necrosis component reached 3 to 4⫹ in chimpanzees 1422 and 1628. In 1628, on week 13, the lobules had ⬎10 foci per 10⫻ objective field. The variable histopathologic response resulting from the same dose and strain of HEV inoculum implies that the host response is dependent on more than the inoculum size. Among the HEV-infected chimpanzees, 1616 and 1618 had more severe hepatitis than 1374 and 1375. Chimpanzees 1616 and 1618 showed lobular necrosis ranging from 1 to 2 foci per 10⫻ field; interestingly, these chimpanzees also had portal inflammation

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FIG. 3. Cross comparison of the number of differentially expressed genes in HCV- and HEV-infected chimpanzees when the alignment of Affymetrix probes is with the human versus chimpanzee genomes. Permutational t tests between baseline samples and postinoculation samples were performed on four HEV-infected chimpanzees and three HCV-infected chimpanzees, using the Genomatix ChipInspector software. A false-discovery rate of 5% was the threshold for the perfectly matched probes on the Affymetrix U133 ⫹ 2.0. Transcripts with at least three significantly expressed probes and a 2-fold change were considered positive. Virus levels in the blood and the liver were measured by quantitative reverse transcription-PCR (TaqMan), and the geometric means (GM) across the biological replicates were determined for each postinoculation time point.

(included in the HAI score in Fig. 1). The lobular necrosis was not as severe as in two of the three acute HCV cases (1422 and 1628). Chimpanzees 1374 and 1375 had no or rare necrosis and no portal inflammation. In general, lobular necrosis was multifocal and more common in the HCV infections and portal inflammation was more marked in the HEV infections (Fig. 2). Probe-level analyses. The individual oligonucleotide probes on the Affymetrix U133 ⫹ 2.0 Genechip were independently aligned with both the human and chimpanzee genomes in the Genomatix ElDorado database. The alignment identified and selected probes with 100% sequence identity with a single transcript in each genome. After reducing the results to pergene summary of expression level and utilizing cross-species homology analysis, in general, the same pattern of gene expression resulted from the alignment of probes with the human or the chimpanzee genome, where the same gene was mapped to chip probes in both; however, the number of upregulated genes detected when probes were mapped to transcripts from the chimpanzee genome was lower than that when probes were mapped to the human genome (Fig. 3).

The alignment of differentially expressed probes with the human genome identified a larger number of well-annotated genes. In the HEV-infected chimpanzees, the majority of the differentially expressed genes that could be identified were similar, regardless of the genome sequence alignment: 93% (54/58) of the genes from the human genome alignment matched similar genes in the chimpanzee genome alignment, and 92% (49/53) of the genes from the chimpanzee genome alignment matched similar genes in the human genome alignment (data not shown). Only four genes in the chimpanzee list could not be matched to a similar gene in the human list. In HCV-infected chimpanzees 69% (149/215) of the genes identified in the human genome alignment could be matched to a similar gene in the chimpanzee genome alignment, and 89% (149/167) of the differentially expressed genes that mapped to the chimpanzee genome matched genes in the list from the human genome alignment. Out of 65 genes in the human genome alignment that could not be matched to a similar gene in the chimpanzee genome alignment, approximately 13 (20%) had no known or “similar to” counterpart in the chimpanzee

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genome. In summary, the majority of the differentially expressed genes identified by the human and the chimpanzee genome alignments were similar, but a major portion of genes that were unique only to the chimpanzee genome alignment lacked informative annotation. Additionally, the chimpanzee genome alignment may have removed positive probes with less than 100% sequence similarity, reducing the number of probes, and therefore genes, used for the analysis. As a result, the output from the human genome alignment was used for further analysis. Although the HEV-infected chimpanzees were inoculated with a large dose of virus, their transcriptional response was smaller in the number of genes and magnitude of differential expression compared with the HCV-infected chimpanzees, of which at least two received lower doses of virus. The majority of differentially expressed genes were upregulated, including over 91% of the genes in HCV-infected chimpanzees and 100% of the genes in HEV-infected chimpanzees. In general, HCV infection induced the expression of over three times as many genes (215 genes) as HEV infection (58 genes). In addition, the number of genes that were induced by HCV infection gradually increased, peaking during the last week of viremia (Fig. 3). A significant number of genes remained differentially expressed 1 month later, probably resulting from a continuing occult infection. In contrast, in HEV-infected chimpanzees, the number of upregulated genes peaked during the first week of viremia and returned to baseline with the cessation of viremia (Fig. 3 and 4). Table S2 in the supplemental material contains a comprehensive list of differentially expressed genes, whereas Fig. 4 contains only genes with at least 1 log2 fold change of 1.2 or greater. Almost all of the differentially expressed genes in HEVinfected chimpanzees were differentially expressed in HCVinfected chimpanzees at higher magnitudes, suggesting that hepatitis E induced an attenuated response compared with hepatitis C. Only 2 of the 58 differentially expressed genes were exclusive to the HEV-infected chimpanzees (see Table S2 in the supplemental material). The two upregulated genes that were exclusive to HEV-infected chimpanzees were the FOS gene, which encodes a leucine zipper protein that dimerizes with JUN to form transcription factor complex AP1, and the LCN2 gene, which codes for a neutrophil gelatinase-associated lipocalin precursor (NGAL) (National Center for Biotechnology Information). Therefore, there were no immune-associated genes that were differentially expressed exclusively in HEV-infected chimpanzees. In order to compare the host responses to infection with the two viruses and understand the biological phenomena, differentially expressed genes were grouped into mutually exclusive functional categories (Fig. 4). Of the three genes that encoded important immune transcription factors, two, the IRF7 and STAT1 genes, were induced in both HCV- and HEV-infected chimpanzees (Fig. 4); however, the IRF1 gene was induced only in HCV-infected chimpanzees (see Table S2 in the supplemental material). Not surprisingly, the majority of the genes that were upregulated in chimpanzees infected by either virus were associated with the immune response by the induction of interferons. HCV-infected chimpanzees demonstrated a larger number and higher magnitude of expression of interferoninduced genes, implying that HCV was a more robust inducer

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of interferon than HEV. Furthermore, the expression of adaptive immune-associated genes, such as major histocompatibility complex (MHC) molecules and immune-specific cell markers, was dramatically lower in HEV-infected chimpanzees than in HCV-infected chimpanzees. Some of the more consequential significantly expressed genes are described by functional category below. Unless otherwise stated, the genes (represented by their products) appear in Fig. 4. Chemokines. Chemokines and their respective cell receptors orchestrate the movement of cells in the liver. Three wellknown IFN-␥-induced chemokines involved in neutrophil and T-cell trafficking were induced in chimpanzees by both viruses: CXCL9, CXCL10, and CXCL11 (25; National Center for Biotechnology Information). HCV-infected chimpanzees expressed additional chemokines whose expression increased shortly before the first disappearance of viremia. These included three Cys-Cys motif (CC) chemokines that have chemotactic activity for specific cells: CCL8 for monocytes, lymphocytes, basophils, and eosinophils; CCL18 for naïve T cells, CD4⫹ and CD8⫹ T cells, and nonactivated lymphocytes (see Table S2 in the supplemental material); and CCL5 for monocytes, memory T helper cells, and eosinophils (National Center for Biotechnology Information). Interestingly, CCL5 was only upregulated after viremia became undetectable, at the first and fourth negative time points. This gene was also identified in a previous study on HCV infection in chimpanzees as a late responder or outcome predictor that correlated with IFN-␥ in HCV infection (40). The difference in the number, magnitude, and diversity of chemokines in HCV-infected chimpanzees indirectly demonstrates the greater complexity of the cellular infiltration during infection. One of the most differentially expressed genes was the gene coding for ISG15. The protein encoded by this gene performs multiple functions. For example, ISG15 has been shown to attract and activate neutrophils, which have been documented to infiltrate the liver during HCV and HEV infections in humans (24, 28, 33; National Center for Biotechnology Information). In addition, this gene’s product can function as a ubiquitin-like protein, with targets that include STAT1, JAK1, mitogen-activated protein kinase 3 (MAPK3), EIF2AK2, and MX1 (17). Lastly, this gene has been shown to induce the production of IFN-␥ in T cells (12). Transcription and RNA modification. The products of three classical antiviral genes were differentially expressed in infections by both viruses: OAS, MX1, and EIF2AK2. The genes coding for these proteins control viral replication by cleaving RNA, inhibiting transcription, and inhibiting translation, respectively. Additionally, both viruses induced ISG20, which has been shown to reduce the levels of hepatitis B surface and e antigens in HepG2 cells and, more relevantly, the replication of HCV in Huh7 cells (20, 23). Both viruses exhibited differential expression of transcription factor STAT1, which implies the activation of the Jak/Stat pathway. The RIG-I pathway is another pathway involved in the induction of interferon-induced genes when it is activated by a double-stranded RNA (dsRNA) intermediate in the cytoplasm. In both infections, differentially expressed helicases that are involved in this pathway included IFIH1, DHX58, and two hypothetical proteins with helicase superfamily C-terminal domain, including DDX60L and DDX60. Interestingly,

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DDX58 (RIG-I) was differentially expressed only in HCVinfected chimpanzees. Interferon inducible (uncategorized). Interferon-induced genes that were placed in the “interferon inducible” category had indeterminate or putative function(s) or had functions that were not included in any of the other main categories. Two genes were upregulated in both HCV- and HEV-infected chimpanzees. IFI27 is a putative highly hydrophobic protein that had a 33% overall sequence similarity to IFI6, which inhibits mitochondrially mediated apoptosis, as described below. RSAD2 has been shown to inhibit HCV replication in cell culture (23), possibly by impairing virus budding through the disruption of lipid rafts at the plasma membrane (UniProt). Amino acid and translation. Besides the differential expression of EIF2AK2, which is known to inhibit translation through the phosphorylation and inactivation of the eukaryotic initiation factor 2 alpha (eIF2a), two other interferon-induced genes that inhibit the initiation pathway of translation were induced by both HCV and HEV infections: the IFIT1 and IFIT2 genes. Inactivation of translation is caused by the binding of IFIT1 and IFIT2 to eIF3c and IFIT2 to eIF3e (42). MHC and immunoglobulin-associated receptors. Compared with HCV-infected chimpanzees, the expression of MHC molecules in HEV-infected chimpanzees was much lower in number and magnitude. More specifically, HEV infection of chimpanzees induced six type 1 MHCs along with ␤2-microglobulin (B2M) (see Table S2 in the supplemental material). HCVinfected chimpanzees induced one additional type 1 MHC (HLA-E) and 13 type 2 MHC molecules (Table S2). In addition, HCV-infected chimpanzees induced TAP1 and TAP2, which are ATP-binding cassette transporters that pump degraded cytosolic peptides across the endoplasmic reticulum into class I molecules for presentation to CD8⫹ T cells (National Center for Biotechnology Information). Lastly, HCVinfected chimpanzees induced genes containing immunoglobulin domains: three coding for products belonging to the butyrophilin protein family, which are type 1 receptor glycoproteins that are involved in lipid, fatty acid, and sterol metabolism; and a gene coding for polymeric immunoglobulin receptor (PIGR), which mediates transport of polymeric immunoglobulin molecules (National Center for Biotechnology Information). Cell markers and immune cells. The group of genes coding for cell markers represented an indirect measure of infiltration and activity of immune-associated cells in the liver. HEVinfected chimpanzees and HCV-infected chimpanzees expressed two genes in this group: the BST2 gene, which is highly expressed in T cells, monocytes, NK cells, and dendritic cells and functions in pre-B-cell growth; and the LGALS3 gene, which is involved in apoptosis, innate immunity, cell adhesion,

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and T-cell regulation (National Center for Biotechnology Information). Remarkably, in HCV-infected chimpanzees, the differential expression of the majority of the genes of this group appeared the week before viremia first disappeared. Interestingly, KLRK1, which is a marker for natural killer cells, was upregulated after HCV was initially cleared from the blood (see Table S2 in the supplemental material). Lastly, HCV-infected chimpanzees expressed transcripts related to secreted proteins: GZMA, a serine protease that is secreted by cytolytic T lymphocytes and natural killer cells to lyse target cells; and LYZ, an antimicrobial agent whose natural substrate is the bacterial cell wall peptidoglycan (National Center for Biotechnology Information). Signal transduction/kinases/GTPases. GBP1 was differentially expressed in both the HCV- and HEV-infected chimpanzees. Guanylate-binding proteins (GBPs) are classified as a subfamily within the protein family of large GTPases, and they are generally induced by IFN-␥, although GBP1 is also induced by type 1 interferon (45; National Center for Biotechnology Information). The higher induction of GBP1 and two additional guanylate-binding proteins (GBP2 and GBP5) in HCVinfected chimpanzees was consistent with the difference in the levels of both type I and II interferons between the two viruses (see Table S2 in the supplemental material). Interestingly, GBP1 has been shown to exhibit antiviral activity (3). Cell membrane and structure. The highest differentially expressed genes in the cell membrane and structure category in HCV and HEV infections were interferon-induced genes: the RTP4, IFI44, and IFI44L genes. IFI44 has been upregulated in other studies that investigated HCV infection, and its overexpression is thought to lead to binding and depletion of intracellular GTP, resulting eventually in cell cycle arrest (19). Ubiquitination and proteasome components. The main function of the ubiquitin and proteasome system is to tag proteins for structural modification or degradation. Some of the proteins in this system are induced by interferon; however, their role in hepatitis pathogenesis has not been completely determined. The induction of genes whose products represent major components in ISGylation, including ubiquitin (UBE2L6 and UBD), ligases (HERC5 and HERC6), and proteosome components (PSMB9), as well as ISG15, which was mentioned in the “chemokine” category, highlighted the active role of ubiquitination and proteasomal modification of proteins in HCV and HEV infections. In addition, both HCV and HEV induced the TRIM22 gene, which is a type I interferoninduced gene that is expressed in leukocytes and other tissues. TRIM22 is involved in multiple cellular processes, including transcription regulation and E3 ubiquitin ligase activity. Cell damage and apoptosis. Antiapoptotic and proapoptotic genes were induced in both infections; however, antiapoptotic

FIG. 4. Fold change (log2) of the differentially expressed genes in functional categories. Only genes with an average fold change (log2) of greater than 1.2 are shown. (Refer to Table S2 in the supplemental material for the complete list.) Permutational t tests were performed on three HCV-infected chimpanzees and four HEV-infected chimpanzees, comparing preinoculation samples and postinoculation samples, which were grouped based on the viremia (Fig. 1). More specifically, baseline samples were compared with the first, peak, and last positive weeks of viremia. In addition, baseline samples were compared with the first and fourth weeks postviremia. For the HCV-infected chimpanzees, the second week of positive viremia was also compared with baseline samples. A false-discovery rate (FDR) cutoff of 5% was applied to the permutational t test analyses for each probe in each group. Significant probes were mapped to the human genome, and transcripts with at least three significant probes and a linear 2-fold change or greater were selected. Lastly, transcripts for the same genes were averaged and reported as mean fold change (log2).

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FIG. 5. Network analysis. Transcription regulators and the number of direct interactions with significant genes were determined with the Ingenuity Pathway Analysis (IPA) software, and only transcription regulators with at least two interactions (edges) with genes from the permutational t test analyses of HCV- and HEV-inoculated chimpanzees were included in the table. Transcription regulators with asterisks were added by the IPA software in the generation of networks.

genes were induced to a higher magnitude and for a longer duration. The IFI6 antiapoptotic gene, which inhibits mitochondrially mediated apoptosis and the activation of caspase-3, was differentially expressed in infections by both viruses. Conversely, HEV induced, at a lower level than HCV, one proapoptotic gene, the SHISA5 gene. Only HCV infections were associated with other expressed genes that are proapoptotic, including the TNFSF10 gene, the CASP1 gene, which plays a central role in the execution of cell apoptosis and proteolytically cleaves and activates interleukin 1, a cytokine involved in inflammation; and the OPTN gene, which utilizes tumor necrosis factor alpha and Fas-ligand pathways to mediate apoptosis (see Table S2 in the supplemental material). Cell cycle, DNA, and histones. Genes categorized in the “cell cycle, DNA, and histones” group were involved in gene regulation and cell replication. In both HCV- and HEV-infected chimpanzees, there were two differentially expressed genes with antiproliferative effects: the SAMD9 gene (National Center for Biotechnology Information) and the gene coding for IFITM1, an interferon (types I and II)-induced multipass membrane protein involved in the transduction of antiproliferative and homotypic adhesion signals (UniProt). HCV-infected chimpanzees induced a larger number of upregulated genes that code for proteins that bind DNA, including ZNFX1, BATF2, and ZBP1, which bind and enhance the association of double-stranded DNA with IRF3 and induce innate immune genes (see Table S2 in the supplemental material) (National Center for Biotechnology Information), and genes that were indirectly involved in DNA metabolism and cell cycle, including the KIF20A, RRM2, TYMS, and NT5C3 genes (National Center for Biotechnology Information). Lipid-associated and metabolism genes. There were no lipidassociated genes that were differentially expressed in HEVinfected chimpanzees. In HCV-infected chimpanzees, however, three apolipoprotein-associated genes were differentially expressed, including those coding for two members of the apolipoprotein L family: APOL3 and APOL6 are cytoplasmic proteins involved in the movement and binding of lipids to organelles (National Center for Biotechnology Information). The third upregulated gene was the APOBEC3Gb gene.

Lastly, three genes regulated by the SREBP pathway were differentially expressed in the HCV-infected chimpanzees: the SQLE, HMGCS1, and SCD genes (see Table S2 in the supplemental material). Miscellaneous. Genes with known functions that are not in the categories above were placed in the “miscellaneous” category. The LCN2 gene, which encodes a protein identified in the granules of granulocytes, was upregulated exclusively in HEV-infected chimpanzees (48); its induction (along with the ISG15 gene as described above) in hepatitis E-infected chimpanzees suggested the potential role of neutrophils in hepatitis. IPA. Over 90% of differentially expressed genes in the HEVand HCV-infected chimpanzees were mapped in the IPA Knowledge Base and used in the analysis. The 17 canonical pathways, some representing the innate immune response, protein ubiquitination, and antigen presentation, were significantly represented in both HEV- and HCV-infected chimpanzees (see Table S3 in the supplemental material). Additionally, other canonical pathways were identified in the HCV-infected chimpanzees; these included some that represented the adaptive immune response, such as the T-cell response, B-cell development, and cell cycle. Not surprisingly, the transcription regulators with high interconnectedness that were identified by the IPA network analysis software included genes from the immune response: the IRF7 and STAT1 genes in HCV- and HEV-infected chimpanzees and the IRF1, STAT3, and IRF9 genes only in HCVinfected chimpanzees (Fig. 5). Both virus infections were associated with a common and constitutively active transcription factor in the liver: HNF4A. The relative expression level of HNF4A fluctuated and displayed relatively lower magnitude of differential expression (⬍3-fold) by qRT-PCR analysis over the entire time course of two HCV-infected chimpanzees (1628 and 1422) and two HEV-infected chimpanzees (1616 and 1374) (data available upon request). Lastly, HCV infections were associated with five additional transcription regulators: NMI, TRIM25, CCNE1, CDKN2A, and ZBTB16. The results of an additional permutational t test for the two HCV- and two HEV-infected chimpanzees with comparable levels of viremia (⬃5 log10 genomes/ml) demonstrated a

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FIG. 6. Differential expression of genes with relatively similar levels of HCV and HEV viremia. A permutational t test was performed on time points from two HCV-infected chimpanzees and two HEV-infected chimpanzees with similar viremia levels of approximately 5 logs. Correlation analysis was performed on the fold changes (log2) of probes that were in common between the HCV- and HEV-inoculated chimpanzees.

greater number of differentially expressed genes in HCV infection compared to HEV infection; however, between the two infections there were 81 genes in common that had similar magnitudes of expression (Fig. 6), and they consisted largely of innate immune genes. TaqMan qRT-PCR. qRT-PCR was more sensitive than microarray for measuring expression of genes (Fig. 7A and B). The coefficient of determination (R2) was higher for genes with greater magnitude of differential expression (i.e., the IFI6, CXCL9, and ISG15 genes) than that for genes with low-level differential expression (i.e., the HLA-C, HLA-DPB1, and B2M genes). However, qRT-PCR validated the positive microarray results for all genes that were selected for analysis based on a fold change of 2, and the comparison of all nine genes resulted in an R2 of 0.75, which is comparable with those of other studies that used the Affymetrix platform to analyze liver tissues (11, 29). For two HCV-infected chimpanzees (1628 and 1422) and two HEV-infected chimpanzees (1616 and 1374) the relative expression of IFN-␥ was measured by qRT-PCR as an indicator of the adaptive immune response (Fig. 8). Although the pattern of IFN-␥ expression in both infections demonstrated an inverse correlation with viremia, the dramatically lower magnitude of differential expression in HEV-infected chimpanzees validated the reduced expression of adaptive immune-associated genes detected by microarray. DISCUSSION Microarray technology has been invaluable in studying the pathogenesis of viral hepatitis because it assesses the host response to infection across the entire transcriptome. However, the ability to assess thousands of transcripts has posed challenges in data analysis. In addition, the continuous evolution of sequence and annotation information in major databases poses a great challenge in comparing studies that utilize

microarrays designed from different versions or builds of the genome. The sequences of the oligonucleotide probes used on Affymetrix arrays were set at the time the chip version was released; thus, annotation changes and any other discrepancies between the actual genome sequence and information available at the time of array design will interfere with the accuracy of gene expression estimates. Affymetrix estimates the abundance of biological transcripts by hybridization of approximately 11 different probe sequences and their associated singlebase-mutated “mismatch” probe in each probe set, then applies an algorithm that robustly estimates expression of a transcript based on all probes in the probe set. An early report by Carter et al. (8) demonstrated the utility of redefining probe sets to include only those probes that match the current build of the genome. The Irizarry lab has contributed an exhaustive review of the variety of algorithms that also redefine the Affymetrix array design in conjunction with various statistically robust methods for estimating probe abundance (21). Genomatix ChipInspector uses a distribution-free method of probelevel analysis, whereby each probe is tested for statistical significance and any gene having 3 or more significant probes associated with it will be called a “significant” gene. We tested for differential expression using the human-matched probes (NCBI genome Build 37) and the chimpanzee-matched probes (NCBI genome Build 2) independently. Our study validated the use of human-based probes for chimpanzee samples, although it eliminated a probably small number of genes that were divergent between the two species. Results from probelevel sequence analyses confirmed the high homology between the two genomes, but also highlighted the advanced maturity in the sequencing and annotation of the human genome (Fig. 3). For this reason, we used the human genome data for further analysis. Transcriptional changes from microarray studies in general provide an incomplete picture of the overall biological events

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FIG. 7. (A) Comparison between qRT-PCR analysis and microarray data. Fold changes on probes in the Affymetrix U133 ⫹ 2.0 chip that were significant based on the permutational t test were averaged across each transcript and then across each gene. Quantitative PCR was performed on nine selected genes and compared with the microarray data of the same chimpanzees that were used for the permutational t test, including HCV-infected chimpanzees 1628 and 1422 and HEV-infected chimpanzees 1616 and 1374. Transferrin was used as the housekeeping gene in the qPCR procedure. (B) Correlation between microarray data and qPCR on individual genes. These genes were plotted together in panel A.

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FIG. 8. qRT-PCR of IFN-␥ in chimpanzees infected with HEV or HCV. IFN-␥ was tested by primer- and probe-specific TaqMan qPCR (see Table S1 in the supplemental material). Total RNA from liver biopsies was converted to cDNA, amplified, and tested on plates that were precoated with primers and probes, as described in Materials and Methods. The relative quantities were calculated for each time point by subtracting the CT values of the housekeeping gene (coding for transferrin) and the average of the preinoculation samples from the CT value of each sample.

in the liver, due to the inability of the technology to capture alterations of proteins whose activation is a result of posttranslational modifications, such as phosphorylation or ubiquitination. Other limitations include the incomplete representation of the genes in the genome and the quality of probe sequences. The IPA software was used to address these issues by identifying pathways and building networks associated with genes that were significant by microarray analysis (Fig. 5). IPA resulted in mostly immune-associated pathways and networks; however, a common liver transcription factor (HNF4A) identified by IPA, but not in our microarray analyses, was evaluated by qRT-PCR. The results indicated low-level and variable expression profiles between the biological replicates, validating its lack of differential expression by microarray analysis. Previous microarray-based studies provided significant insight into the pathogenesis of hepatitis C virus. Advances in the platform, the genomic databases, the tools for analyses, and the use of true biological replicates in the analyses we performed, permitted us not only to confirm, but also to expand on previous microarray studies of the pathogenesis of hepatitis C virus in chimpanzee liver, although direct comparison between studies is not practical due to differences in study design and analysis algorithms. Although the chimpanzees in our study completely resolved their HCV infections, chimpanzees from a previous study progressed to chronic HCV infection (40). Many of the genes that were induced in both studies were the same, and therefore, the same biological pathways were overrepresented in acute resolving and chronic HCV infections in chimpanzees. It is worth noting that differentially expressed genes identified in our study represented the same biological systems as previous studies; however, our analyses

captured additional chemokines and immune genes, compared with previous studies (5, 40). For example, chemokines CCL8 and CCL18 and genes coding for immune-associated products DHX58, DDX60, IRF1, and B2M were not identified as differentially expressed in previous studies. Parallel studies using the same reagents, platforms, and technologies that we performed on the pathogenesis of hepatitis E virus in chimpanzee liver permitted a direct comparison between the two RNA virus infections. The differences in the pathogenesis profiles of HCV and HEV infections (duration of viremia, etc.) (Fig. 1) confirm that the two viruses have dissimilar life cycles. Additionally, differences in the severity of hepatitis and in the magnitude and duration of elevations in ALT between the two infections suggested that the two viruses differ in their abilities to stimulate the adaptive immune response and related mechanisms to clear the virus. Although it has been previously demonstrated that the inoculum size poorly correlates with the level of hepatitis in HCV-infected chimpanzees, it is important to note that the chimpanzees were inoculated with 106 ID of HEV compared to ⬍100 ID of HCV for chimpanzee 1628 (16). Interestingly, the comprehensive global probe-level analyses demonstrated a robust innate immune response correlating with viremia for both viral infections, while the adaptive immune response in HEVinfected chimpanzees was attenuated compared to that in HCV infections, paralleling differences in ALT levels and the overall HAI scores (40). Indeed, hepatitis E in humans often presents as mild disease or subclinical infection, as evident in cohorts (e.g., blood bank donors) with high seropositive rates and no history of hepatitis (44). While HCV is

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also often subclinical in its presentation, our data indicate that in mild HCV infections in chimpanzees the transcriptional response is greater than the transcriptional response in mild HEV infections in chimpanzees. A major component of the host response to both HCV and HEV infections was the expression of interferon-induced genes. The induction of similar type I interferoninduced genes suggested that both RNA viruses activate the innate immune response through similar mechanisms. However, viremia levels reflect the magnitude of the expression of immune genes in both infections regardless of the inoculum size (Fig. 6). Therefore, the attenuation in the magnitude of the innate and, more strikingly, the attenuation in the number and magnitude of the adaptive immune response genes in HEV infection, compared to HCV infection, along with the shorter duration of viremia and the earlier seroconversion associated with full protection, suggested that HEV may be more susceptible to the potent effects of the immune response. RNA viruses induce the innate immune response through the RIG-I, Toll-like receptor 3 (TLR3), and TLR7 pathways, resulting in the transcription of IFN-␤ and subsequently innate immune genes. However, the repertoire of genes that are induced by type I interferons and their roles in viral infection have not been completely elucidated. Here, as in previous microarray studies, expression of type I, II, or III interferons was not detectable by microarray analysis (5, 40); however, both the HCV- and HEV-infected chimpanzees provided evidence of the innate immune response by the differential expression of genes involved in the RIG-I and Jak/Stat pathways as well as by expression of genes known to be induced by type I interferon. It is worth noting that there are multiple pathways involved in the induction of interferon-induced genes. Therefore, we cannot conclusively determine which specific pathways were activated in our study. Although HCV infection resulted in a more robust induction based on the larger number of genes, higher magnitude of differential expression, and longer duration of expression, the virus was not readily eradicated. In contrast, HEV infection induced a somewhat less robust response of shorter duration but which resulted in a complete resolution of infection with no recurrence of the virus. These findings are consistent with a scenario in which both HCV and HEV stimulate a robust innate immune response but only HEV is highly susceptible to the effects of ISGs. Therefore, in the case of HCV infections, the adaptive immune response must compensate for an ineffective innate response; in the case of HEV infections, the adaptive immune response need only supplement an effective innate response and could be less robust. The outcome of HCV infection has been shown to depend greatly on IFN-␥ induction and the resulting adaptive immune response (40, 43). Similar to other microarray studies, differential expression of IFN-␥ was too low to be considered significant in our microarray analysis (5, 40); however, in both HCV and HEV infections, expression of IFN-␥ induction was detected by qRT-PCR with chimpanzee-specific primer and probe sequences, with a differential expression approximately 10-fold higher in HCV-infected chimpanzees than in HEV-infected animals (Fig. 8). Lower

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relative levels of IFN-␥ in HEV-infected chimpanzees can be partially attributed to the lesser induction of CXC chemokines and the absence of CC chemokines, which affect the hepatic infiltration of cells that produce IFN-␥, including NK, NKT, CD4, and cytotoxic T cells (CTL). Additionally, lower levels of IFN-␥ in HEV-infected chimpanzees may also reflect the inability to detect the immune transcription factor IRF-1 (see Table S2 in the supplemental material), which is a potent transactivator involved in its induction (27). The robustness of the adaptive immune response in HCV-infected chimpanzees may result from the increased transcription of type I and II HLA genes, which encode MHC molecules that present viral antigens to immune cells (Fig. 4). Stoichiometric analyses of HCV replication complexes have demonstrated that the amount of viral protein in Huh-7 cells far exceeded the viral RNA by over 1,000-fold and that only a minor fraction (⬍5%) was involved in RNA synthesis (34). The observation of excess viral proteins and the induction of HLA genes in our study could explain the robustness of the adaptive immune response in HCV-infected chimpanzees. In contrast, HEV-infected chimpanzees demonstrated lower levels of adaptive immune response genes. HEV-infected chimpanzees induced, at lower levels, only type I MHC along with ␤2-microglobulin, suggesting that type II MHC, and possibly intrahepatic CD4⫹ T cells, might contribute minimally to the cellular control of the infection. Srivastava et al. demonstrated that the increase of CD4⫹ cells in the blood of patients with acute HEV infection were not helper T cells of types 1 or 2, suggesting IFN-␥ may have been induced by natural killer T cells (39). In addition, the expansion of CD8⫹ T cells in the blood was not evident in patients with acute HEV infection (39). The induction of CD4⫹ and CD8⫹ cell markers was not detected by our microarray analysis, implying that these cells were not sequestered in the liver and providing further evidence of the minimal adaptive immune response in HEV infection. Lastly, preliminary microarray analysis on peripheral blood mononuclear cells (PBMCs) from HEV-infected chimpanzees demonstrated lower levels of induction of both innate and adaptive immune response compared to liver tissues, and TaqMan qRT-PCR provided no evidence of HEV in these cells (data not shown). While upregulation of transcripts is not entirely predicative of the immune response at the functional protein level, the larger number of upregulated immune cell-associated genes in HCV-infected chimpanzee liver provided strong evidence that the hepatic cellular response to the infection was more substantial, supporting further a more robust adaptive immune response to HCV than to HEV. The differential expression of lipid-associated genes in HCV-infected chimpanzees was consistent with the role of lipid in the life cycle of this enveloped virus. For example, HCV has been shown to associate with very-low-density and low-density lipoproteins in plasma (4), and in chronic infections, intracellular lipids accumulate in infected hepatocytes as steatosis (35). Previously, Su et al. identified genes involved in lipid metabolism by the SREBP pathway, including ATP-citrate lyase and fatty acid synthase using correlationbased analyses on an earlier Affymetrix chip (40). Our data substantiated the perturbation of the SREBP signaling path-

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way by the differential expression of SQLE, HMGCS1, and SCD. Interestingly, HEV-infected chimpanzees had no differentially expressed genes associated with lipid metabolism and function, even though HEV virions are transiently complexed with lipids (41). In summary, we validated the use of the human Affymetrix chip for analyzing chimpanzee samples by first aligning probes to the most recent builds of human and chimpanzee genomes; thus, we were able to focus on those that matched either genome perfectly and use only those probes for statistical analyses. Because of the high sequence homology between the two species and the more complete annotation of the human genome, it was chosen for identification of genes with the assumption that nonannotated chimpanzee genes that reacted with the same vetted probes were indeed the same as the human genes. Results from the probe-level analyses in HCV- and HEV-infected chimpanzees highlighted very distinct temporal changes in the onset and duration of host response to each virus. Based on both the number and the magnitude of significantly expressed genes from the global analysis, HCV infection induced a broader and more robust overall immune response than HEV infection in chimpanzees. ACKNOWLEDGMENTS This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases. We thank Stacy Ricklefs and Karin Bok for providing guidance on developing the qRT-PCR procedure. We are grateful to Jun Yang, Brandie Fullmer, Marjorie Bosche, and Wuxing Yuan, the Laboratory of Immunopathogenesis and Bioinformatics at SAIC-Frederick, Inc., for their support and for performing the chip hybridization; and Kathleen Meyer for assisting with the GEO submission. Lastly, we thank Marisa St. Claire, Max Shapiro, and Charlene Shaver for the care and handling of the chimpanzees in this study. The authors have no conflicts of interest to disclose.

12.

13. 14.

15.

16.

17.

18. 19.

20.

21. 22.

23.

24.

25. 26.

27. REFERENCES 1. Aggarwal, R., and S. Jameel. 2008. Hepatitis E vaccine. Hepatol. Int. 2:308– 315. 2. Alter, M. J. 2007. Epidemiology of hepatitis C virus infection. World J. Gastroenterol. 13:2436–2441. 3. Anderson, S. L., J. M. Carton, J. Lou, L. Xing, and B. Y. Rubin. 1999. Interferon-induced guanylate binding protein-1 (GBP-1) mediates an antiviral effect against vesicular stomatitis virus and encephalomyocarditis virus. Virology 256:8–14. 4. Andre, P., F. Komurian-Pradel, S. Deforges, M. Perret, J. L. Berland, M. Sodoyer, S. Pol, C. Brechot, G. Paranhos-Baccala, and V. Lotteau. 2002. Characterization of low- and very-low-density hepatitis C virus RNA-containing particles. J. Virol. 76:6919–6928. 5. Bigger, C. B., K. M. Brasky, and R. E. Lanford. 2001. DNA microarray analysis of chimpanzee liver during acute resolving hepatitis C virus infection. J. Virol. 75:7059–7066. 6. Bigger, C. B., B. Guerra, K. M. Brasky, G. Hubbard, M. R. Beard, B. A. Luxon, S. M. Lemon, and R. E. Lanford. 2004. Intrahepatic gene expression during chronic hepatitis C virus infection in chimpanzees. J. Virol. 78:13779– 13792. 7. Bukh, J., R. H. Purcell, and R. H. Miller. 1992. Importance of primer selection for the detection of hepatitis C virus RNA with the polymerase chain reaction assay. Proc. Natl. Acad. Sci. U. S. A. 89:187–191. 8. Carter, S. L., A. C. Eklund, B. H. Mecham, I. S. Kohane, and Z. Szallasi. 2005. Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements. BMC Bioinformatics 6:107. 9. Chandra, V., S. Taneja, M. Kalia, and S. Jameel. 2008. Molecular biology and pathogenesis of hepatitis E virus. J. Biosci. 33:451–464. 10. Chen, S. L., and T. R. Morgan. 2006. The natural history of hepatitis C virus (HCV) infection. Int. J. Med. Sci. 3:47–52. 11. Conti, A., S. Scala, P. D’Agostino, E. Alimenti, D. Morelli, B. Andria, A.

28.

29.

30.

31.

32.

33.

34. 35. 36.

11277

Tammaro, C. Attanasio, F. Della Ragione, V. Scuderi, F. Fabbrini, M. D’Esposito, E. Di Florio, L. Nitsch, F. Calise, and A. Faiella. 2007. Wide gene expression profiling of ischemia-reperfusion injury in human liver transplantation. Liver Transpl. 13:99–113. D’Cunha, J., E. Knight, Jr., A. L. Haas, R. L. Truitt, and E. C. Borden. 1996. Immunoregulatory properties of ISG15, an interferon-induced cytokine. Proc. Natl. Acad. Sci. U. S. A. 93:211–215. Dhawan, P. S., and H. G. Desai. 1998. Subacute hepatic failure: diagnosis of exclusion? J. Clin. Gastroenterol. 26:98–100. Engle, R. E., R. S. Russell, R. H. Purcell, and J. Bukh. 2008. Development of a TaqMan assay for the six major genotypes of hepatitis C virus: comparison with commercial assays. J. Med. Virol. 80:72–79. Engle, R. E., C. Yu, S. U. Emerson, X. J. Meng, and R. H. Purcell. 2002. Hepatitis E virus (HEV) capsid antigens derived from viruses of human and swine origin are equally efficient for detecting anti-HEV by enzyme immunoassay. J. Clin. Microbiol. 40:4576–4580. Feinstone, S. M., H. J. Alter, H. P. Dienes, Y. Shimizu, H. Popper, D. Blackmore, D. Sly, W. T. London, and R. H. Purcell. 1981. Non-A, non-B hepatitis in chimpanzees and marmosets. J. Infect. Dis. 144:588–598. Giannakopoulos, N. V., J. K. Luo, V. Papov, W. Zou, D. J. Lenschow, B. S. Jacobs, E. C. Borden, J. Li, H. W. Virgin, and D. E. Zhang. 2005. Proteomic identification of proteins conjugated to ISG15 in mouse and human cells. Biochem. Biophys. Res. Commun. 336:496–506. Grebely, J., D. L. Thomas, and G. J. Dore. 2009. HCV reinfection studies and the door to vaccine development. J. Hepatol. 51:628–631. Hallen, L. C., Y. Burki, M. Ebeling, C. Broger, F. Siegrist, K. OroszlanSzovik, B. Bohrmann, U. Certa, and S. Foser. 2007. Antiproliferative activity of the human IFN-alpha-inducible protein IFI44. J. Interferon Cytokine Res. 27:675–680. Hao, Y., and D. Yang. 2008. Cloning, eukaryotic expression of human ISG20 and preliminary study on the effect of its anti-HBV. J. Huazhong Univ Sci. Technolog. Med. Sci. 28:11–13. Irizarry, R. A., Z. Wu, and H. A. Jaffee. 2006. Comparison of Affymetrix GeneChip expression measures. Bioinformatics 22:789–794. Ishak, K., A. Baptista, L. Bianchi, F. Callea, J. De Groote, F. Gudat, H. Denk, V. Desmet, G. Korb, R. N. MacSween, et al. 1995. Histological grading and staging of chronic hepatitis. J. Hepatol. 22:696–699. Jiang, D., H. Guo, C. Xu, J. Chang, B. Gu, L. Wang, T. M. Block, and J. T. Guo. 2008. Identification of three interferon-inducible cellular enzymes that inhibit the replication of hepatitis C virus. J. Virol. 82:1665–1678. Johnson, K., A. Kotiesh, J. K. Boitnott, and M. Torbenson. 2007. Histology of symptomatic acute hepatitis C infection in immunocompetent adults. Am. J. Surg. Pathol. 31:1754–1758. Laing, K. J., and C. J. Secombes. 2004. Chemokines. Dev. Comp. Immunol. 28:443–460. Lin, L., S. Liu, H. Brockway, J. Seok, P. Jiang, W. H. Wong, and Y. Xing. 2009. Using high-density exon arrays to profile gene expression in closely related species. Nucleic Acids Res. 37:e90. Liu, J., X. Guan, and X. Ma. 2005. Interferon regulatory factor 1 is an essential and direct transcriptional activator for interferon ␥-induced RANTES/CCl5 expression in macrophages. J. Biol. Chem. 280:24347–24355. Malcolm, P., H. Dalton, H. S. Hussaini, and J. Mathew. 2007. The histology of acute autochthonous hepatitis E virus infection. Histopathology 51:190– 194. Mas, V. R., D. G. Maluf, K. J. Archer, K. Yanek, X. Kong, L. Kulik, C. E. Freise, K. M. Olthoff, R. M. Ghobrial, P. McIver, and R. Fisher. 2009. Genes involved in viral carcinogenesis and tumor initiation in hepatitis C virusinduced hepatocellular carcinoma. Mol. Med. 15:85–94. Mushahwar, I. K. 2008. Hepatitis E virus: molecular virology, clinical features, diagnosis, transmission, epidemiology, and prevention. J. Med. Virol. 80:646–658. Ogata, N., H. J. Alter, R. H. Miller, and R. H. Purcell. 1991. Nucleotide sequence and mutation rate of the H strain of hepatitis C virus. Proc. Natl. Acad. Sci. U. S. A. 88:3392–3396. Okamoto, H., S. Okada, Y. Sugiyama, K. Kurai, H. Iizuka, A. Machida, Y. Miyakawa, and M. Mayumi. 1991. Nucleotide sequence of the genomic RNA of hepatitis C virus isolated from a human carrier: comparison with reported isolates for conserved and divergent regions. J. Gen. Virol. 72: 2697–2704. Peron, J. M., M. Danjoux, N. Kamar, R. Missoury, H. Poirson, J. P. Vinel, J. M. Mansuy, C. Bureau, J. Izopet, P. Brousset, and J. Selves. 2007. Liver histology in patients with sporadic acute hepatitis E: a study of 11 patients from South-West France. Virchows Arch. 450:405–410. Quinkert, D., R. Bartenschlager, and V. Lohmann. 2005. Quantitative analysis of the hepatitis C virus replication complex. J. Virol. 79:13594–13605. Ramalho, F. 2003. Hepatitis C virus infection and liver steatosis. Antiviral Res. 60:125–127. Sakai, A., S. Takikawa, R. Thimme, J. C. Meunier, H. C. Spangenberg, S. Govindarajan, P. Farci, S. U. Emerson, F. V. Chisari, R. H. Purcell, and J. Bukh. 2007. In vivo study of the HC-TN strain of hepatitis C virus recovered from a patient with fulminant hepatitis: RNA transcripts of a molecular

11278

37.

38.

39.

40.

41.

YU ET AL.

clone (pHC-TN) are infectious in chimpanzees but not in Huh7.5 cells. J. Virol. 81:7208–7219. Saldanha, J., N. Lelie, and A. Heath. 1999. Establishment of the first international standard for nucleic acid amplification technology (NAT) assays for HCV RNA. WHO Collaborative Study Group. Vox Sang. 76:149–158. Schofield, D. J., J. Glamann, S. U. Emerson, and R. H. Purcell. 2000. Identification by phage display and characterization of two neutralizing chimpanzee monoclonal antibodies to the hepatitis E virus capsid protein. J. Virol. 74:5548–5555. Srivastava, R., R. Aggarwal, S. Jameel, P. Puri, V. K. Gupta, V. S. Ramesh, S. Bhatia, and S. Naik. 2007. Cellular immune responses in acute hepatitis E virus infection to the viral open reading frame 2 protein. Viral Immunol. 20:56–65. Su, A. I., J. P. Pezacki, L. Wodicka, A. D. Brideau, L. Supekova, R. Thimme, S. Wieland, J. Bukh, R. H. Purcell, P. G. Schultz, and F. V. Chisari. 2002. Genomic analysis of the host response to hepatitis C virus infection. Proc. Natl. Acad. Sci. U. S. A. 99:15669–15674. Takahashi, M., T. Tanaka, H. Takahashi, Y. Hoshino, S. Nagashima Jirintai, H. Mizuo, Y. Yazaki, T. Takagi, M. Azuma, E. Kusano, N. Isoda, K. Sugano, and H. Okamoto. 2010. Hepatitis E Virus (HEV) strains in serum samples can replicate efficiently in cultured cells despite the coexistence of HEV antibodies: characterization of HEV virions in blood circulation. J. Clin. Microbiol. 48:1112–1125.

J. VIROL. 42. Terenzi, F., D. J. Hui, W. C. Merrick, and G. C. Sen. 2006. Distinct induction patterns and functions of two closely related interferon-inducible human genes, ISG54 and ISG56. J. Biol. Chem. 281:34064–34071. 43. Thimme, R., C. Neumann-Haefelin, T. Boettler, and H. E. Blum. 2008. Adaptive immune responses to hepatitis C virus: from viral immunobiology to a vaccine. Biol. Chem. 389:457–467. 44. Thomas, D. L., P. O. Yarbough, D. Vlahov, S. A. Tsarev, K. E. Nelson, A. J. Saah, and R. H. Purcell. 1997. Seroreactivity to hepatitis E virus in areas where the disease is not endemic. J. Clin. Microbiol. 35:1244–1247. 45. Tripal, P., M. Bauer, E. Naschberger, T. Mortinger, C. Hohenadl, E. Cornali, M. Thurau, and M. Sturzl. 2007. Unique features of different members of the human guanylate-binding protein family. J. Interferon Cytokine Res. 27:44–52. 46. Tsarev, S. A., T. S. Tsareva, S. U. Emerson, A. Z. Kapikian, J. Ticehurst, W. London, and R. H. Purcell. 1993. ELISA for antibody to hepatitis E virus (HEV) based on complete open-reading frame-2 protein expressed in insect cells: identification of HEV infection in primates. J. Infect. Dis. 168:369–378. 47. Tusher, V. G., R. Tibshirani, and G. Chu. 2001. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. U. S. A. 98:5116–5121. 48. Xu, S., and P. Venge. 2000. Lipocalins as biochemical markers of disease. Biochim. Biophys. Acta 1482:298–307.