Comparative Host Gene Transcription by Microarray
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病菌学杂志 2005年第10期
Department of Microbiology, Centre of Infection and Immunology, State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong
Division of Haematology
Division of Anatomical Pathology, Department of Pathology, The University of Hong Kong, Hong Kong
ABSTRACT
The pathogenesis of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) at the cellular level is unclear. No human cell line was previously known to be susceptible to both SARS-CoV and other human coronaviruses. Huh7 cells were found to be susceptible to both SARS-CoV, associated with SARS, and human coronavirus 229E (HCoV-229E), usually associated with the common cold. Highly lytic and productive rates of infections within 48 h of inoculation were reproducible with both viruses. The early transcriptional profiles of host cell response to both types of infection at 2 and 4 h postinoculation were determined by using the Affymetrix HG-U133A microarray (about 22,000 genes). Much more perturbation of cellular gene transcription was observed after infection by SARS-CoV than after infection by HCoV-229E. Besides the upregulation of genes associated with apoptosis, which was exactly opposite to the previously reported effect of SARS-CoV in a colonic carcinoma cell line, genes related to inflammation, stress response, and procoagulation were also upregulated. These findings were confirmed by semiquantitative reverse transcription-PCR, reverse transcription-quantitative PCR for mRNA of genes, and immunoassays for some encoded proteins. These transcriptomal changes are compatible with the histological changes of pulmonary vasculitis and microvascular thrombosis in addition to the diffuse alveolar damage involving the pneumocytes.
INTRODUCTION
Severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) is the etiological agent of SARS (26, 39). The disease is associated with significant mortality and morbidity (38). Such aggressive clinical behavior is very different from that of other known human coronaviruses, such as the group 1 coronaviruses 229E and NL63 or the group 2 coronavirus OC43. Although these viruses are generally associated with mild upper respiratory tract infection such as the common cold (47-49), these human coronaviruses can also cause pneumonia when the very young, elderly, or immunosuppressed hosts are affected (14, 15, 40). Animal models of SARS were established by infecting cynomolgus monkeys, ferrets, domestic cats, or mice (16, 27, 31, 33, 55), but there are questions about their reproducibility of the pathology mimicking human disease. Although the cellular receptors for attachment of SARS-CoV were found to be ACE2 (30) and, recently, L-SIGN (23), pathogenesis at the cellular level is largely unknown.
SARS-CoV is an enveloped, positive-sense, single-stranded RNA virus which can grow in embryonal monkey cell lines, including Vero E6 and fetal rhesus monkey kidney (FRhk-4) cells (26, 39). It can be subcultured onto other Vero cells and colonic carcinoma cell lines such as Caco-2 or LoVo (3, 10). Unlike other human coronaviruses, SARS-CoV proliferates rapidly and causes obvious cytopathic effects in Vero E6 cells within 48 h of inoculation (35). There are no other human cell lines known to be susceptible to infection by both SARS-CoV and other human coronaviruses. Recently there have been reports of a human hepatoma cell line, Huh7, which can be infected by pseudotyped lentiviral particles carrying the spike protein of SARS-CoV and wild-type replicative SARS-CoV (6, 20, 45). We report in this study the susceptibility of the cell line Huh7 to infection by both SARS-CoV and human coronavirus 229E (HCoV-229E). A comparative gene transcriptional profile at an early stage of infection of Huh7 cells by these two viruses was performed to elucidate their difference and its importance in the pathogenesis of disease.
MATERIALS AND METHODS
Cell lines and virus. The human hepatoma cell line Huh7 (courtesy of David Ho, Aaron Diamond AIDS Research Center) was used throughout this study. The cells were incubated at 37°C in minimal essential medium supplemented with 10% fetal calf serum and 100 IU/ml of penicillin and 100 μg/ml of streptomycin. Our prototype virus (SARS-CoV HKU-39849) was isolated from the lung tissue biopsy of the brother-in-law of the index patient who traveled from Guangzhou and started a superspreading event in the Hong Kong Special Administrative Region leading to the pandemic (37). The HCoV-229E strain (ATCC VR-740) was used in this study. The SARS-CoV and HCoV-229E strains used in our experiments had undergone three passages in the FRhk-4 cell and MRC-5 cell lines, respectively, and were stored at –70°C. Viral titers were determined as median tissue culture infective dose (TCID50) per ml in confluent Huh7 cells in 96-well microtiter plates, which standardized the viral inoculum and measured the relative susceptibility of the Huh7 cell line to these two viruses. The relative susceptibilities of the Vero 1008, Vero 76, Vero, and Huh7 cell lines to SARS-CoV and HCoV-229E were also tested by TCID50. One hundred TCID50 was confirmed by plaque assays to be equivalent to 85 PFU. All work with infectious virus was performed inside a type II biosafety cabinet in a biosafety containment level III facility, and the personnel wore powered air-purifying respirators (HEPA Airmate; 3 M, Saint Paul, Minn.).
Monitoring of virus-induced cytopathic effect, antigen detection, and semiquantitative and quantitative RT-PCR. Huh7 cells and culture supernatants infected with either SARS-CoV or HCoV-229E at a multiplicity of infection of 100 TCID50 per cell were collected at 2, 4, 12, and 24 h postinfection. A washing step was performed 1 h postinoculation. The percentages of cells developing cytopathic effects were counted by inverted light microscopy at 24 and 48 h. The rate of viral replication was measured by reverse transcription-quantitative PCR (RT-qPCR) on the culture filtrate. The amount of coronavirus antigen expression in infected cells was measured by indirect immunofluorescence with convalescent-phase sera of patients with SARS-CoV or HCoV-229E infection, as reported previously (2, 8). Briefly, harvested cells were prepared and fixed in ice-cold acetone for 10 min. Convalescent-phase serum at a dilution of 1 in 100 was used to react with the infected cells harvested at various time points. After 30 min incubation, the cells were washed twice in phosphate-buffered saline for 5 min each, and then goat anti-human fluorescein isothiocyanate conjugate (INOVA Diagnostics, Inc., San Diego, CA) was added and the cells were further incubated for 30 min at 37°C. The cells were washed again as described above, and the percentage of positive cells was manually estimated under UV microscopy.
Reverse transcription-PCR (RT-PCR) for SARS-CoV and HCoV-229E was done directly on culture filtrate according to our previous protocol (2). Briefly, total RNA extracted from culture filtrate with the QIAamp virus RNA minikit (Qiagen) as instructed by the manufacturer was reverse transcribed with random hexamers. cDNA was amplified with SARS-CoV primers (forward, 5'-TACACACCTCAGCGTTG-3'; reverse, 5'-CACGAACGTGACGAAT-3') and HCoV-229E primers (forward, 5'-GGTACTCCTAAGCCTTCTTCG-3'; reverse, 5'-GACTATCAAACAGCATAGCAGC-3'). Using real-time RT-qPCR assays, cDNA was amplified in SYBR Green I fluorescence reactions (Roche, Mannheim, Germany). For the RT-qPCR of SARS-CoV, a 20-μl reaction mixtures containing 2 μl cDNA, 3.5 mmol/liter magnesium chloride, and 0.25 μmol/liter of the same forward and reverse primers as in the reaction mixtures were thermal cycled with a Light Cycler (Roche) (95°C for 10 min, followed by 50 cycles of 95°C for 10 s, 57°C for 5 s, and 72°C for 9 s with ramp rates of 20°C/s). For the RT-qPCR of HCoV-229E, the conditions were similar to those described above except that 45 cycles of 95°C for 10 s, 65°C for 3 s, and 72°C for 12 s were used. Plasmids with the target sequences were used to generate the standard curve. At the end of the assay, the PCR products of SARS-CoV and HCoV-229E (182 bp and 295 bp, respectively) were subjected to a melting curve analysis (65 to 95°C, 0.1°C/s) to determine the specificity of the assay. All assays were performed in replicates.
Microarray analysis. Human genome-wide gene expression was examined with the GeneChip system HG-U133A microarray (Affymetrix Inc., Santa Clara, CA), which is composed of more than 22,000 oligonucleotide probe sets interrogating approximately 18,400 unique transcripts, including 14,500 well-characterized human genes. Quality control, GeneChip hybridization, and data acquisition and analysis were performed at the Genome Research Centre, The University of Hong Kong, according to the standard protocols available from Affymetrix. In brief, total RNAs of the infected or uninfected cell lines at different time points were extracted using the RNeasy minikit (Qiagen, Valencia, CA). Double-stranded cDNA was synthesized from 10 μg of total RNA with the GeneChipT7-Oligo (dT) Promoter Primer Kit (Affymetrix, Inc.) and the SuperScript Choice System (Invitrogen). Biotin-labeled cRNA was then synthesized by in vitro transcription using the BioArray High Yield RNA Transcript Labeling Kit (Affymetrix, Inc). After fragmentation, 15 μg of labeled cRNA was hybridized to the oligonucleotide microarray. The chips were washed and stained using the GeneChip Fluidics Station 400 (Affymetrix) and then scanned with the GeneChip Scanner 3000 (Affymetrix). Data analysis was performed using the Microarray Suite Expression Analysis software (version 5.1; Affymetrix). For comparison across different arrays, the data for each array were normalized by a global scaling strategy, using a scaling target intensity of 500. By using the Affymetrix-defined comparison mathematical algorithms, a fold change in expression between each of the infected samples in comparison to the uninfected mock control was calculated, log2 transformed, and further classified as not changed, increased (signal log ratio change P value of <0.005), decreased (signal log ratio change P value of >0.995), or marginally increased or decreased. To classify a gene as significantly upregulated or downregulated after infection at a specific time point, two additional criteria were used: (i) the fold change must be greater than or equal to 2 (signal log ratio of 1 if upregulated or 1 if downregulated) to be classified as increased or decreased, and (ii) genes that were classified as upregulated must be flagged as present in the infected samples, while genes that were classified as downregulated must be flagged as present in the uninfected control sample. All gene chip procedures were performed in replicates.
Gene expression analysis by semiquantitative PCR, RT-qPCR, and immunoassays. Genes with significant transcriptional changes known to be associated with biological significance were selected for further analysis by semiquantitative PCR, RT-qPCR, and immunoassays. RT-qPCR was performed according to our previous protocol (56). The extracted RNA was pretreated with DNase. Primers which specifically amplified nine genes related to apoptosis, inflammation, and coagulation were designed (Table 1). First-strand cDNA was synthesized from the total RNA by reverse transcription with random hexamers. Semiqualitative comparison was performed using simple gel electrophoresis and ethidium bromide staining (10). Quantitative PCR was performed using the SYBR Green I fluorescence reactions in a Light Cycler as described above. Detailed PCR conditions are available upon request. Serial dilutions of a reference cDNA derived from the SARS-CoV-infected sample were used to generate the standard curve. Melting curve analysis was performed for each primer pair at the end of the reaction to confirm the specificity of the assay. The housekeeping porphobilinogen deaminase gene was used for standardization of the initial RNA content of a sample. Experiments were performed in duplicate, and the result for an individual sample was expressed as the mean expression level of a specific gene/porphobilinogen deaminase gene relative to the reference cDNA. The relative expression between each infected sample and the uninfected control was then calculated and expressed as fold change.
Three sets of immunoassays (human interleukin-8 [IL-8] from BD Biosciences and PAI1 [serpine 1] and TFPI2 from Diagnostica Stago) were performed according to our previous protocols (5, 28) and the manufacturers' instructions. Briefly, for IL-8 assay, 100 μl culture supernatant or standard was added to IL-8 monoclonal antibody-coated wells and incubated for 2 h at room temperature. After washing five times, 100 μl a biotinylated anti-human IL-8 monoclonal antibody was added to each well. After five washings, 100 μl of tetramethyl benzidine substrate was added, followed by incubation for 30 min at room temperature. The reaction was stopped by adding 50 μl stop solution. The absorbance was read at 450 nm within 30 min. The experimental conditions and procedure were similar for the immunoassays for PAI1 and TFP1. All experiments were done in replicates to ensure accuracy and reproducibility.
Statistical analysis. The fold change of the target gene expressions and the difference in the concentrations of expressed proteins between SARS-CoV and HCoV-229E at different postinoculation time points were compared by Student's t test. A P value of <0.05 was considered significant. A statistical package (SPSS 10.0; SPSS Hong Kong, Hong Kong) was used for all analyses.
RESULTS
Susceptibility of the Huh7 cell line to SARS-CoV and HCoV-229E. With a multiplicity of infection of 100, a cytopathic effect was visible in Huh7 cells at 24 and progressed to about 50% cell death at 48 h with both viruses. SARS-CoV but not HCoV-229E grew well in the Vero cell line. The viruses produced comparable TCID50 of around 107 per ml in the culture supernatant of Huh7 cells at 48 h (Table 2). In terms of viral load, a 1-log-unit increase of viral genome copy was noted at 12 h postinoculation with both viruses, which was followed by a peak at 24 h (Table 3). Antigen expression could be observed by indirect immunofluorescence in over 50% of the cells at 24 h postinoculation for both viruses.
Effects on gene expression of host cells determined by microarray. Two hundred twenty-four genes were significantly altered within 4 h postinoculation in the transcriptomal expression analysis. Only 21 genes were perturbed by HCoV-229E per se, whereas 164 genes were altered by SARS-CoV infection only, and the remaining 39 genes were altered by both coronaviruses. Out of the 164 genes with altered expression in SARS-CoV, 38 were upregulated and only one was downregulated at both 2 and 4 h postinoculation. Forty-three were upregulated and 16 were downregulated at 2 h postinoculation. Forty-nine were upregulated and 17 were downregulated at 4 h postinoculation. In contrast, for HCoV-229E infection, only one gene was upregulated and no gene was downregulated at both 2 and 4 h postinoculation. No gene was upregulated and two were downregulated at 2 h postinoculation. Fourteen genes were upregulated and four were downregulated at 4 h postinoculation. When multiple transcripts of the same gene were eliminated and analyzed, genes related to apoptosis (n = 23), inflammatory or immune response (n = 34), and coagulation (n = 5) were identified, in addition to the expected genes related to the stress response and metabolism and other unknown genes (Table 4). Of the 23 apoptotic genes affected, 13 of them are proapoptotic and 11 of them are upregulated in SARS-CoV infection, compared with only 3 in HCoV-229E infection (Table 5). For inflammation and immune response, 32 genes are upregulated in SARS-CoV, compared with only 3 in HCoV-229E. These include the genes for NFKB1A; NFKB2; IL-8; transforming growth factor ?2; chemokines CXCL1, -2, -3, -5, -6, and -10; ICAM1; and tumor necrosis factor alpha-induced proteins. Quite unexpectedly, genes of the procoagulation pathway were also affected by SARS-CoV infection, with upregulation of PLSCR1 (phospholipid scramblase 1), EGR1 (early growth response 1 gene), PAI1/SERPINE1 (plasminogen activator inhibitor 1), and THBS1 (thrombospondin 1). In terms of stress response, seven genes were upregulated in SARS-CoV infection, compared with only one in HCoV-229E infection. Overall there were far more changes in expression of genes related to the cell cycle, transcription, and metabolism and those with miscellaneous and unknown functions in SARS-CoV infection. When the Pathway Assist software (Ariadne Genomics Inc.) was used for linking the altered genes in cellular pathways for SARS-CoV, there were clear clusterings of altered genes related to apoptosis, inflammation, and coagulation (Fig. 1).
Confirmation of cellular gene and protein expression by semiquantitative PCR, RT-qPCR, and immunoassays. Since previous studies have documented the alterations of the apoptotic and inflammatory pathways in relation to SARS-CoV infection, only key genes related to inflammation and apoptosis were chosen to confirm the results of microarray analysis by semiquantitative RT-PCR and RT-qPCR. For the coagulation pathway, both procoagulation and anticoagulation genes were chosen. A similar trend of upregulation of gene expression was found by RT-qPCR, with SARS-CoV showing a 1.4- to 10.8-fold increase compared with HCoV-229E for coagulation (TFPI2, PAI1, and THBS1), inflammation (IL-8 and NFKB2), transcription (JUNB), and apoptotic (PHLDA1, CARD10, and BAX) pathways (Fig. 2 and 3). IL-8 was increased in both SARS-CoV and HCoV-229E infection, but the increase was much higher in the case of SARS-CoV. The enzyme immunoassay showed that SARS-CoV induced higher concentrations of PAI1 and IL-8 than HCoV-229E at 2, 4, 12, and 24 h postinoculation. Both SARS-CoV and HCoV-229E induced similar TFPI2 expression at 4, 12, and 24 h postinoculation, but at 2 h postinoculation SARS-CoV induced a lower concentration of this predominately anticoagulation protein (Fig. 4).
DISCUSSION
Of the four coronaviruses known to infect human, HCoV-229E, HCoV-OC43, and NL63 are generally associated with mild upper respiratory tract infection such as the common cold in immunocompetent hosts (47-49). In contrast, SARS-CoV causes respiratory failure in over 60% of affected persons, with a mortality rate of 15% (37, 38). Besides pneumonia, SARS is also clinically manifested as watery diarrhea without enterocolitis (48.6%) (8), hepatitis without liver failure (49.4%) (22), lymphopenia (75%) (37), impaired coagulation (63%) (51), and occasionally pulmonary vasculitis and thrombosis in the lungs of those who died (36, 13). Much is already known about many aspects of SARS, including the virology (18, 26, 39, 53), genomics (32), diagnostics (5, 28, 43, 52, 54), clinical features and progression in relation to viral load (22, 37), treatment (7, 9, 46), infection control (44), and immunization (55). However little is known about the pathogenesis at the cellular level, despite the identification of ACE2 and L-SIGN as the receptors for binding of SARS-CoV to host cells (30, 23).
One important limitation to studies at the cellular level is the absence of a relevant cell line which can be lytically infected by SARS-CoV. Initially, only embryonal monkey kidney cell lines such as Vero or FRhk-4 and their derivatives could be readily infected. Subsequent investigation showed that human colon adenocarcinoma cell lines Caco-2, CL-14, and LoVo could also be infected (3, 10). However, inflammation or cell death is not manifested on endoscopic examination of the intestines of SARS patients (29). Examination of intestinal tissue biopsy by electron microscopy revealed abundant intracellular viral particles, and there was negligible inflammatory cells or cellular apoptosis on light microscopy (29). In fact, a high TCID50 in the culture supernatant of persistently infected LoVo cells can be achieved without any cytopathic effects (3). Recently a gene expression analysis showed that SARS-CoV upregulated antiapoptotic genes and several CXC chemokines while downregulating proapoptotic genes, IL-18, and macrophage migration inhibitory factor (10) at 24 h postinoculation of Caco-2 cell lines. These patterns of gene expression appeared to explain the severe watery diarrhea without clinical or endoscopic signs of necrosis or inflammation in SARS patients (29). However, no data on early gene expression before the onset of cytolysis were reported. Similarly, no comparative study of the host cell transcriptional profile and those for other, less virulent coronaviruses was ever reported.
No pneumocyte cell line has yet been found to support lytic or nonlytic infection by SARS-CoV. However, the human hepatoma cell line Huh7 was found to be susceptible to SARS-CoV. This is not completely unexpected, because the mouse hepatitis virus, a group 2 coronavirus, is known to infect Huh7 cells (25). In this study HCoV-229E was found to produce good lytic infection within 48 h postinoculation. A high multiplicity of infection of 100 TCID50s per cell was used to ensure the reproducibility of gene expression, as previously reported (41). Since the expression of a high number of genes was expected to change significantly when virus-induced cytopathology was impending for a rapidly lytic viral infection, we tried to study the difference in the expression profile at the relatively early stages of the infection of 2 and 4 h postinoculation, as reported for herpes simplex virus type 1 (17). This time frame is biologically relevant because proliferation of the Golgi complex and related vesicles and swelling of trans-Golgi sacs were observed in infected cells within the first hour of infection. Extracellular virus particles were present in 5% and 30% of the cell populations at 5 and 6 h postinoculation, respectively (35). This would also facilitate the analysis, since a lower number of altered genes were involved. In the comparative transcriptomic study, far more genes (n = 136) were upregulated by SARS-CoV than by HCoV-229E. In contrast to the reported findings of increased antiapoptoptic/inflammatory gene expression and decreased proapoptotic/inflammatory gene expression in enterocyte cell lines (10), far more proapoptotic and proinflammatory genes were expressed in Huh7 cells infected by SARS-CoV but not HCoV-229E. For instance, expression of BCL2 was induced by SARS-CoV in enterocytes, yet we observed upregulation of its antagonists, including BAX and BCL2L11, in Huh7 cells. Moreover, much higher expression of other proapoptotic proteins, including CASP7, CARD10, PMAIP1, and GADD45B, was also induced by SARS-CoV than by HCoV-229E. Furthermore, there was marked perturbation of genes involved in cell cycle regulation, including induction of the CDKN2B, gene which can mediate growth arrest at the G1 phase. The induction of proinflammatory cytokines by SARS-CoV was even more prominent when compared to HCoV-229E. The induction of IL-8 may be of pathogenic importance, as its level has been positively correlated with disease severity in pulmonary infection by respiratory syncytial virus. Thus, the observed significantly higher level of IL-8 induced by SARS-CoV compared with HCoV-229E in Huh7 cells may recapitulate the host response to these viruses by pneumocytes. The induction of various chemokines of the CXC or CCL family may mediate the chemotaxis of lymphocytes and neutrophils. These alterations in gene expression are in keeping with the histological changes of SARS hepatitis, in which cellular apoptosis, marked accumulation of cells in mitosis with ballooning degeneration of hepatocytes, and moderate lymphocytic infiltration were found in liver tissue biopsy samples (4). Recently, a vaccine study in ferrets immunized with the modified vaccinia virus Ankara carrying the spike protein showed that the ferrets developed hepatitis after being challenged with wild SARS-CoV. This finding tends to suggest that the Huh7 cell line model may have some relevance to the pathogenesis of SARS (50), but our present findings still may not be directly applicable to the pneumocytes involved in SARS.
The upregulation of genes involved in procoagulation and platelet activation was interesting. TFPI2 inhibits thrombin generation by binding and inactivation of the tissue factor-factor VIIa complex. Upregulation of the gene probably represents an inhibitory response to restrain the activation of the coagulation pathway during acute inflammation. In contrast, TFPI2 also inhibits both free and matrix/cell-associated plasmin, thus favoring fibrin deposition, and may have positive role in matrix turnover (11). Upregulation of the PAI1 gene accompanied by a dramatic increase in the protein level results in an antifibrinolytic response, favoring fibrin deposition during the acute inflammatory phase of the disease. It is important to remember that mouse hepatitis virus can activate the immune coagulation system by the fgl2 gene, encoding a prothrombinase (12). This enzyme can induce macrophage procoagulation activity which results in fibrin deposition on the endothelia of intrahepatic veins and hepatic sinusoids. The end result could be confluent hepatocellular necrosis. The low number of liver biopsies done with these patients may account for the lack of reports on these full-blown changes related to vascular damage. However, systemic vasculitis, including edema, localized fibrinoid necrosis, and infiltration by monocytes, lymphocytes, and plasma cells into vessel walls in various tissues, was reported (13). Thrombosis was found in small veins. Interestingly, marked upregulation of a proapoptotic gene, PHLDA1, was observed in SARS-CoV infection of Huh7. A previous study has shown that overexpression of this gene in vascular endothelial cells would lead to decreased cell adhesion and induce detachment-mediated apoptosis. This gene, if similarly induced in vascular endothelial cells infected by SARS-CoV, may contribute to the vascular damage induced by SARS-CoV infection (21).
The clinical manifestations and histological changes involving the pneumocytes, enterocytes, and vascular endothelium were not surprising because ACE2, the host cell receptor for viral entry, was present in all these cell types (19). Similarly, abnormal urinalysis associated with increased viral load in urine is expected, since SARS-CoV could be cultured from or antigen expression could be detected in kidney cell lines and kidney tissues of patients (22). However, neither ACE2 nor L-SIGN could be detected by immunohistochemical staining in normal human hepatocytes or Huh7 (1, 42). This could be explained by an alternative cellular receptor for viral entry or just because the amount of receptor required for cell entry is so low that the cells may appear negative on immunohistochemical staining. Our previous study has shown that a high viral load in serum or stool is associated with hepatic dysfunction (22). The viral load in serum reflects the dynamic of the viral replication from any organs and the clearance mechanism of the host, whereas a high viral load in the stool may indirectly reflect the degree of portal venous viremia. Thus, the lack of cell death and inflammation in the intestine may still have importance in the pathogenesis of SARS in other body sites.
In conclusion, SARS-CoV produces more severe disturbance of host cell gene expression in a human epithelial cell line of liver origin than does HCoV-229E at the early stage of infection. There are marked alterations in gene expression related to apoptosis, inflammation, and procoagulation. These findings are consistent with the histological changes of SARS, especially in the liver and blood vessels. Besides antivirals (6, 24), other modalities of treatment, such as antiapoptotic agents (34), immunomodulators against inflammation (7), and modifiers of coagulation, should be considered in future research. It is important to remember that many patients continued to deteriorate despite a decreasing viral load 2 to 3 weeks after the onset of SARS.
ACKNOWLEDGMENTS
We thank William Mak, Carol Chan, and Tommy Tang, Genome Research Centre, The University of Hong Kong, for their excellent technical assistance. Special thanks to all the staff members of the University and Hospital Departments of Microbiology at Queen Mary Hospital.
We acknowledge research funding from the Research Fund for the Control of Infectious Diseases of the Health, Welfare and Food Bureau of the Hong Kong SAR government, Infectious Diseases Fund (William Benter), Infectious Diseases Fund (Teresa Lim), and Tung Wah Group of Hospitals Hospitals’ Fund for Research in Infectious Diseases.
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Division of Haematology
Division of Anatomical Pathology, Department of Pathology, The University of Hong Kong, Hong Kong
ABSTRACT
The pathogenesis of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) at the cellular level is unclear. No human cell line was previously known to be susceptible to both SARS-CoV and other human coronaviruses. Huh7 cells were found to be susceptible to both SARS-CoV, associated with SARS, and human coronavirus 229E (HCoV-229E), usually associated with the common cold. Highly lytic and productive rates of infections within 48 h of inoculation were reproducible with both viruses. The early transcriptional profiles of host cell response to both types of infection at 2 and 4 h postinoculation were determined by using the Affymetrix HG-U133A microarray (about 22,000 genes). Much more perturbation of cellular gene transcription was observed after infection by SARS-CoV than after infection by HCoV-229E. Besides the upregulation of genes associated with apoptosis, which was exactly opposite to the previously reported effect of SARS-CoV in a colonic carcinoma cell line, genes related to inflammation, stress response, and procoagulation were also upregulated. These findings were confirmed by semiquantitative reverse transcription-PCR, reverse transcription-quantitative PCR for mRNA of genes, and immunoassays for some encoded proteins. These transcriptomal changes are compatible with the histological changes of pulmonary vasculitis and microvascular thrombosis in addition to the diffuse alveolar damage involving the pneumocytes.
INTRODUCTION
Severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) is the etiological agent of SARS (26, 39). The disease is associated with significant mortality and morbidity (38). Such aggressive clinical behavior is very different from that of other known human coronaviruses, such as the group 1 coronaviruses 229E and NL63 or the group 2 coronavirus OC43. Although these viruses are generally associated with mild upper respiratory tract infection such as the common cold (47-49), these human coronaviruses can also cause pneumonia when the very young, elderly, or immunosuppressed hosts are affected (14, 15, 40). Animal models of SARS were established by infecting cynomolgus monkeys, ferrets, domestic cats, or mice (16, 27, 31, 33, 55), but there are questions about their reproducibility of the pathology mimicking human disease. Although the cellular receptors for attachment of SARS-CoV were found to be ACE2 (30) and, recently, L-SIGN (23), pathogenesis at the cellular level is largely unknown.
SARS-CoV is an enveloped, positive-sense, single-stranded RNA virus which can grow in embryonal monkey cell lines, including Vero E6 and fetal rhesus monkey kidney (FRhk-4) cells (26, 39). It can be subcultured onto other Vero cells and colonic carcinoma cell lines such as Caco-2 or LoVo (3, 10). Unlike other human coronaviruses, SARS-CoV proliferates rapidly and causes obvious cytopathic effects in Vero E6 cells within 48 h of inoculation (35). There are no other human cell lines known to be susceptible to infection by both SARS-CoV and other human coronaviruses. Recently there have been reports of a human hepatoma cell line, Huh7, which can be infected by pseudotyped lentiviral particles carrying the spike protein of SARS-CoV and wild-type replicative SARS-CoV (6, 20, 45). We report in this study the susceptibility of the cell line Huh7 to infection by both SARS-CoV and human coronavirus 229E (HCoV-229E). A comparative gene transcriptional profile at an early stage of infection of Huh7 cells by these two viruses was performed to elucidate their difference and its importance in the pathogenesis of disease.
MATERIALS AND METHODS
Cell lines and virus. The human hepatoma cell line Huh7 (courtesy of David Ho, Aaron Diamond AIDS Research Center) was used throughout this study. The cells were incubated at 37°C in minimal essential medium supplemented with 10% fetal calf serum and 100 IU/ml of penicillin and 100 μg/ml of streptomycin. Our prototype virus (SARS-CoV HKU-39849) was isolated from the lung tissue biopsy of the brother-in-law of the index patient who traveled from Guangzhou and started a superspreading event in the Hong Kong Special Administrative Region leading to the pandemic (37). The HCoV-229E strain (ATCC VR-740) was used in this study. The SARS-CoV and HCoV-229E strains used in our experiments had undergone three passages in the FRhk-4 cell and MRC-5 cell lines, respectively, and were stored at –70°C. Viral titers were determined as median tissue culture infective dose (TCID50) per ml in confluent Huh7 cells in 96-well microtiter plates, which standardized the viral inoculum and measured the relative susceptibility of the Huh7 cell line to these two viruses. The relative susceptibilities of the Vero 1008, Vero 76, Vero, and Huh7 cell lines to SARS-CoV and HCoV-229E were also tested by TCID50. One hundred TCID50 was confirmed by plaque assays to be equivalent to 85 PFU. All work with infectious virus was performed inside a type II biosafety cabinet in a biosafety containment level III facility, and the personnel wore powered air-purifying respirators (HEPA Airmate; 3 M, Saint Paul, Minn.).
Monitoring of virus-induced cytopathic effect, antigen detection, and semiquantitative and quantitative RT-PCR. Huh7 cells and culture supernatants infected with either SARS-CoV or HCoV-229E at a multiplicity of infection of 100 TCID50 per cell were collected at 2, 4, 12, and 24 h postinfection. A washing step was performed 1 h postinoculation. The percentages of cells developing cytopathic effects were counted by inverted light microscopy at 24 and 48 h. The rate of viral replication was measured by reverse transcription-quantitative PCR (RT-qPCR) on the culture filtrate. The amount of coronavirus antigen expression in infected cells was measured by indirect immunofluorescence with convalescent-phase sera of patients with SARS-CoV or HCoV-229E infection, as reported previously (2, 8). Briefly, harvested cells were prepared and fixed in ice-cold acetone for 10 min. Convalescent-phase serum at a dilution of 1 in 100 was used to react with the infected cells harvested at various time points. After 30 min incubation, the cells were washed twice in phosphate-buffered saline for 5 min each, and then goat anti-human fluorescein isothiocyanate conjugate (INOVA Diagnostics, Inc., San Diego, CA) was added and the cells were further incubated for 30 min at 37°C. The cells were washed again as described above, and the percentage of positive cells was manually estimated under UV microscopy.
Reverse transcription-PCR (RT-PCR) for SARS-CoV and HCoV-229E was done directly on culture filtrate according to our previous protocol (2). Briefly, total RNA extracted from culture filtrate with the QIAamp virus RNA minikit (Qiagen) as instructed by the manufacturer was reverse transcribed with random hexamers. cDNA was amplified with SARS-CoV primers (forward, 5'-TACACACCTCAGCGTTG-3'; reverse, 5'-CACGAACGTGACGAAT-3') and HCoV-229E primers (forward, 5'-GGTACTCCTAAGCCTTCTTCG-3'; reverse, 5'-GACTATCAAACAGCATAGCAGC-3'). Using real-time RT-qPCR assays, cDNA was amplified in SYBR Green I fluorescence reactions (Roche, Mannheim, Germany). For the RT-qPCR of SARS-CoV, a 20-μl reaction mixtures containing 2 μl cDNA, 3.5 mmol/liter magnesium chloride, and 0.25 μmol/liter of the same forward and reverse primers as in the reaction mixtures were thermal cycled with a Light Cycler (Roche) (95°C for 10 min, followed by 50 cycles of 95°C for 10 s, 57°C for 5 s, and 72°C for 9 s with ramp rates of 20°C/s). For the RT-qPCR of HCoV-229E, the conditions were similar to those described above except that 45 cycles of 95°C for 10 s, 65°C for 3 s, and 72°C for 12 s were used. Plasmids with the target sequences were used to generate the standard curve. At the end of the assay, the PCR products of SARS-CoV and HCoV-229E (182 bp and 295 bp, respectively) were subjected to a melting curve analysis (65 to 95°C, 0.1°C/s) to determine the specificity of the assay. All assays were performed in replicates.
Microarray analysis. Human genome-wide gene expression was examined with the GeneChip system HG-U133A microarray (Affymetrix Inc., Santa Clara, CA), which is composed of more than 22,000 oligonucleotide probe sets interrogating approximately 18,400 unique transcripts, including 14,500 well-characterized human genes. Quality control, GeneChip hybridization, and data acquisition and analysis were performed at the Genome Research Centre, The University of Hong Kong, according to the standard protocols available from Affymetrix. In brief, total RNAs of the infected or uninfected cell lines at different time points were extracted using the RNeasy minikit (Qiagen, Valencia, CA). Double-stranded cDNA was synthesized from 10 μg of total RNA with the GeneChipT7-Oligo (dT) Promoter Primer Kit (Affymetrix, Inc.) and the SuperScript Choice System (Invitrogen). Biotin-labeled cRNA was then synthesized by in vitro transcription using the BioArray High Yield RNA Transcript Labeling Kit (Affymetrix, Inc). After fragmentation, 15 μg of labeled cRNA was hybridized to the oligonucleotide microarray. The chips were washed and stained using the GeneChip Fluidics Station 400 (Affymetrix) and then scanned with the GeneChip Scanner 3000 (Affymetrix). Data analysis was performed using the Microarray Suite Expression Analysis software (version 5.1; Affymetrix). For comparison across different arrays, the data for each array were normalized by a global scaling strategy, using a scaling target intensity of 500. By using the Affymetrix-defined comparison mathematical algorithms, a fold change in expression between each of the infected samples in comparison to the uninfected mock control was calculated, log2 transformed, and further classified as not changed, increased (signal log ratio change P value of <0.005), decreased (signal log ratio change P value of >0.995), or marginally increased or decreased. To classify a gene as significantly upregulated or downregulated after infection at a specific time point, two additional criteria were used: (i) the fold change must be greater than or equal to 2 (signal log ratio of 1 if upregulated or 1 if downregulated) to be classified as increased or decreased, and (ii) genes that were classified as upregulated must be flagged as present in the infected samples, while genes that were classified as downregulated must be flagged as present in the uninfected control sample. All gene chip procedures were performed in replicates.
Gene expression analysis by semiquantitative PCR, RT-qPCR, and immunoassays. Genes with significant transcriptional changes known to be associated with biological significance were selected for further analysis by semiquantitative PCR, RT-qPCR, and immunoassays. RT-qPCR was performed according to our previous protocol (56). The extracted RNA was pretreated with DNase. Primers which specifically amplified nine genes related to apoptosis, inflammation, and coagulation were designed (Table 1). First-strand cDNA was synthesized from the total RNA by reverse transcription with random hexamers. Semiqualitative comparison was performed using simple gel electrophoresis and ethidium bromide staining (10). Quantitative PCR was performed using the SYBR Green I fluorescence reactions in a Light Cycler as described above. Detailed PCR conditions are available upon request. Serial dilutions of a reference cDNA derived from the SARS-CoV-infected sample were used to generate the standard curve. Melting curve analysis was performed for each primer pair at the end of the reaction to confirm the specificity of the assay. The housekeeping porphobilinogen deaminase gene was used for standardization of the initial RNA content of a sample. Experiments were performed in duplicate, and the result for an individual sample was expressed as the mean expression level of a specific gene/porphobilinogen deaminase gene relative to the reference cDNA. The relative expression between each infected sample and the uninfected control was then calculated and expressed as fold change.
Three sets of immunoassays (human interleukin-8 [IL-8] from BD Biosciences and PAI1 [serpine 1] and TFPI2 from Diagnostica Stago) were performed according to our previous protocols (5, 28) and the manufacturers' instructions. Briefly, for IL-8 assay, 100 μl culture supernatant or standard was added to IL-8 monoclonal antibody-coated wells and incubated for 2 h at room temperature. After washing five times, 100 μl a biotinylated anti-human IL-8 monoclonal antibody was added to each well. After five washings, 100 μl of tetramethyl benzidine substrate was added, followed by incubation for 30 min at room temperature. The reaction was stopped by adding 50 μl stop solution. The absorbance was read at 450 nm within 30 min. The experimental conditions and procedure were similar for the immunoassays for PAI1 and TFP1. All experiments were done in replicates to ensure accuracy and reproducibility.
Statistical analysis. The fold change of the target gene expressions and the difference in the concentrations of expressed proteins between SARS-CoV and HCoV-229E at different postinoculation time points were compared by Student's t test. A P value of <0.05 was considered significant. A statistical package (SPSS 10.0; SPSS Hong Kong, Hong Kong) was used for all analyses.
RESULTS
Susceptibility of the Huh7 cell line to SARS-CoV and HCoV-229E. With a multiplicity of infection of 100, a cytopathic effect was visible in Huh7 cells at 24 and progressed to about 50% cell death at 48 h with both viruses. SARS-CoV but not HCoV-229E grew well in the Vero cell line. The viruses produced comparable TCID50 of around 107 per ml in the culture supernatant of Huh7 cells at 48 h (Table 2). In terms of viral load, a 1-log-unit increase of viral genome copy was noted at 12 h postinoculation with both viruses, which was followed by a peak at 24 h (Table 3). Antigen expression could be observed by indirect immunofluorescence in over 50% of the cells at 24 h postinoculation for both viruses.
Effects on gene expression of host cells determined by microarray. Two hundred twenty-four genes were significantly altered within 4 h postinoculation in the transcriptomal expression analysis. Only 21 genes were perturbed by HCoV-229E per se, whereas 164 genes were altered by SARS-CoV infection only, and the remaining 39 genes were altered by both coronaviruses. Out of the 164 genes with altered expression in SARS-CoV, 38 were upregulated and only one was downregulated at both 2 and 4 h postinoculation. Forty-three were upregulated and 16 were downregulated at 2 h postinoculation. Forty-nine were upregulated and 17 were downregulated at 4 h postinoculation. In contrast, for HCoV-229E infection, only one gene was upregulated and no gene was downregulated at both 2 and 4 h postinoculation. No gene was upregulated and two were downregulated at 2 h postinoculation. Fourteen genes were upregulated and four were downregulated at 4 h postinoculation. When multiple transcripts of the same gene were eliminated and analyzed, genes related to apoptosis (n = 23), inflammatory or immune response (n = 34), and coagulation (n = 5) were identified, in addition to the expected genes related to the stress response and metabolism and other unknown genes (Table 4). Of the 23 apoptotic genes affected, 13 of them are proapoptotic and 11 of them are upregulated in SARS-CoV infection, compared with only 3 in HCoV-229E infection (Table 5). For inflammation and immune response, 32 genes are upregulated in SARS-CoV, compared with only 3 in HCoV-229E. These include the genes for NFKB1A; NFKB2; IL-8; transforming growth factor ?2; chemokines CXCL1, -2, -3, -5, -6, and -10; ICAM1; and tumor necrosis factor alpha-induced proteins. Quite unexpectedly, genes of the procoagulation pathway were also affected by SARS-CoV infection, with upregulation of PLSCR1 (phospholipid scramblase 1), EGR1 (early growth response 1 gene), PAI1/SERPINE1 (plasminogen activator inhibitor 1), and THBS1 (thrombospondin 1). In terms of stress response, seven genes were upregulated in SARS-CoV infection, compared with only one in HCoV-229E infection. Overall there were far more changes in expression of genes related to the cell cycle, transcription, and metabolism and those with miscellaneous and unknown functions in SARS-CoV infection. When the Pathway Assist software (Ariadne Genomics Inc.) was used for linking the altered genes in cellular pathways for SARS-CoV, there were clear clusterings of altered genes related to apoptosis, inflammation, and coagulation (Fig. 1).
Confirmation of cellular gene and protein expression by semiquantitative PCR, RT-qPCR, and immunoassays. Since previous studies have documented the alterations of the apoptotic and inflammatory pathways in relation to SARS-CoV infection, only key genes related to inflammation and apoptosis were chosen to confirm the results of microarray analysis by semiquantitative RT-PCR and RT-qPCR. For the coagulation pathway, both procoagulation and anticoagulation genes were chosen. A similar trend of upregulation of gene expression was found by RT-qPCR, with SARS-CoV showing a 1.4- to 10.8-fold increase compared with HCoV-229E for coagulation (TFPI2, PAI1, and THBS1), inflammation (IL-8 and NFKB2), transcription (JUNB), and apoptotic (PHLDA1, CARD10, and BAX) pathways (Fig. 2 and 3). IL-8 was increased in both SARS-CoV and HCoV-229E infection, but the increase was much higher in the case of SARS-CoV. The enzyme immunoassay showed that SARS-CoV induced higher concentrations of PAI1 and IL-8 than HCoV-229E at 2, 4, 12, and 24 h postinoculation. Both SARS-CoV and HCoV-229E induced similar TFPI2 expression at 4, 12, and 24 h postinoculation, but at 2 h postinoculation SARS-CoV induced a lower concentration of this predominately anticoagulation protein (Fig. 4).
DISCUSSION
Of the four coronaviruses known to infect human, HCoV-229E, HCoV-OC43, and NL63 are generally associated with mild upper respiratory tract infection such as the common cold in immunocompetent hosts (47-49). In contrast, SARS-CoV causes respiratory failure in over 60% of affected persons, with a mortality rate of 15% (37, 38). Besides pneumonia, SARS is also clinically manifested as watery diarrhea without enterocolitis (48.6%) (8), hepatitis without liver failure (49.4%) (22), lymphopenia (75%) (37), impaired coagulation (63%) (51), and occasionally pulmonary vasculitis and thrombosis in the lungs of those who died (36, 13). Much is already known about many aspects of SARS, including the virology (18, 26, 39, 53), genomics (32), diagnostics (5, 28, 43, 52, 54), clinical features and progression in relation to viral load (22, 37), treatment (7, 9, 46), infection control (44), and immunization (55). However little is known about the pathogenesis at the cellular level, despite the identification of ACE2 and L-SIGN as the receptors for binding of SARS-CoV to host cells (30, 23).
One important limitation to studies at the cellular level is the absence of a relevant cell line which can be lytically infected by SARS-CoV. Initially, only embryonal monkey kidney cell lines such as Vero or FRhk-4 and their derivatives could be readily infected. Subsequent investigation showed that human colon adenocarcinoma cell lines Caco-2, CL-14, and LoVo could also be infected (3, 10). However, inflammation or cell death is not manifested on endoscopic examination of the intestines of SARS patients (29). Examination of intestinal tissue biopsy by electron microscopy revealed abundant intracellular viral particles, and there was negligible inflammatory cells or cellular apoptosis on light microscopy (29). In fact, a high TCID50 in the culture supernatant of persistently infected LoVo cells can be achieved without any cytopathic effects (3). Recently a gene expression analysis showed that SARS-CoV upregulated antiapoptotic genes and several CXC chemokines while downregulating proapoptotic genes, IL-18, and macrophage migration inhibitory factor (10) at 24 h postinoculation of Caco-2 cell lines. These patterns of gene expression appeared to explain the severe watery diarrhea without clinical or endoscopic signs of necrosis or inflammation in SARS patients (29). However, no data on early gene expression before the onset of cytolysis were reported. Similarly, no comparative study of the host cell transcriptional profile and those for other, less virulent coronaviruses was ever reported.
No pneumocyte cell line has yet been found to support lytic or nonlytic infection by SARS-CoV. However, the human hepatoma cell line Huh7 was found to be susceptible to SARS-CoV. This is not completely unexpected, because the mouse hepatitis virus, a group 2 coronavirus, is known to infect Huh7 cells (25). In this study HCoV-229E was found to produce good lytic infection within 48 h postinoculation. A high multiplicity of infection of 100 TCID50s per cell was used to ensure the reproducibility of gene expression, as previously reported (41). Since the expression of a high number of genes was expected to change significantly when virus-induced cytopathology was impending for a rapidly lytic viral infection, we tried to study the difference in the expression profile at the relatively early stages of the infection of 2 and 4 h postinoculation, as reported for herpes simplex virus type 1 (17). This time frame is biologically relevant because proliferation of the Golgi complex and related vesicles and swelling of trans-Golgi sacs were observed in infected cells within the first hour of infection. Extracellular virus particles were present in 5% and 30% of the cell populations at 5 and 6 h postinoculation, respectively (35). This would also facilitate the analysis, since a lower number of altered genes were involved. In the comparative transcriptomic study, far more genes (n = 136) were upregulated by SARS-CoV than by HCoV-229E. In contrast to the reported findings of increased antiapoptoptic/inflammatory gene expression and decreased proapoptotic/inflammatory gene expression in enterocyte cell lines (10), far more proapoptotic and proinflammatory genes were expressed in Huh7 cells infected by SARS-CoV but not HCoV-229E. For instance, expression of BCL2 was induced by SARS-CoV in enterocytes, yet we observed upregulation of its antagonists, including BAX and BCL2L11, in Huh7 cells. Moreover, much higher expression of other proapoptotic proteins, including CASP7, CARD10, PMAIP1, and GADD45B, was also induced by SARS-CoV than by HCoV-229E. Furthermore, there was marked perturbation of genes involved in cell cycle regulation, including induction of the CDKN2B, gene which can mediate growth arrest at the G1 phase. The induction of proinflammatory cytokines by SARS-CoV was even more prominent when compared to HCoV-229E. The induction of IL-8 may be of pathogenic importance, as its level has been positively correlated with disease severity in pulmonary infection by respiratory syncytial virus. Thus, the observed significantly higher level of IL-8 induced by SARS-CoV compared with HCoV-229E in Huh7 cells may recapitulate the host response to these viruses by pneumocytes. The induction of various chemokines of the CXC or CCL family may mediate the chemotaxis of lymphocytes and neutrophils. These alterations in gene expression are in keeping with the histological changes of SARS hepatitis, in which cellular apoptosis, marked accumulation of cells in mitosis with ballooning degeneration of hepatocytes, and moderate lymphocytic infiltration were found in liver tissue biopsy samples (4). Recently, a vaccine study in ferrets immunized with the modified vaccinia virus Ankara carrying the spike protein showed that the ferrets developed hepatitis after being challenged with wild SARS-CoV. This finding tends to suggest that the Huh7 cell line model may have some relevance to the pathogenesis of SARS (50), but our present findings still may not be directly applicable to the pneumocytes involved in SARS.
The upregulation of genes involved in procoagulation and platelet activation was interesting. TFPI2 inhibits thrombin generation by binding and inactivation of the tissue factor-factor VIIa complex. Upregulation of the gene probably represents an inhibitory response to restrain the activation of the coagulation pathway during acute inflammation. In contrast, TFPI2 also inhibits both free and matrix/cell-associated plasmin, thus favoring fibrin deposition, and may have positive role in matrix turnover (11). Upregulation of the PAI1 gene accompanied by a dramatic increase in the protein level results in an antifibrinolytic response, favoring fibrin deposition during the acute inflammatory phase of the disease. It is important to remember that mouse hepatitis virus can activate the immune coagulation system by the fgl2 gene, encoding a prothrombinase (12). This enzyme can induce macrophage procoagulation activity which results in fibrin deposition on the endothelia of intrahepatic veins and hepatic sinusoids. The end result could be confluent hepatocellular necrosis. The low number of liver biopsies done with these patients may account for the lack of reports on these full-blown changes related to vascular damage. However, systemic vasculitis, including edema, localized fibrinoid necrosis, and infiltration by monocytes, lymphocytes, and plasma cells into vessel walls in various tissues, was reported (13). Thrombosis was found in small veins. Interestingly, marked upregulation of a proapoptotic gene, PHLDA1, was observed in SARS-CoV infection of Huh7. A previous study has shown that overexpression of this gene in vascular endothelial cells would lead to decreased cell adhesion and induce detachment-mediated apoptosis. This gene, if similarly induced in vascular endothelial cells infected by SARS-CoV, may contribute to the vascular damage induced by SARS-CoV infection (21).
The clinical manifestations and histological changes involving the pneumocytes, enterocytes, and vascular endothelium were not surprising because ACE2, the host cell receptor for viral entry, was present in all these cell types (19). Similarly, abnormal urinalysis associated with increased viral load in urine is expected, since SARS-CoV could be cultured from or antigen expression could be detected in kidney cell lines and kidney tissues of patients (22). However, neither ACE2 nor L-SIGN could be detected by immunohistochemical staining in normal human hepatocytes or Huh7 (1, 42). This could be explained by an alternative cellular receptor for viral entry or just because the amount of receptor required for cell entry is so low that the cells may appear negative on immunohistochemical staining. Our previous study has shown that a high viral load in serum or stool is associated with hepatic dysfunction (22). The viral load in serum reflects the dynamic of the viral replication from any organs and the clearance mechanism of the host, whereas a high viral load in the stool may indirectly reflect the degree of portal venous viremia. Thus, the lack of cell death and inflammation in the intestine may still have importance in the pathogenesis of SARS in other body sites.
In conclusion, SARS-CoV produces more severe disturbance of host cell gene expression in a human epithelial cell line of liver origin than does HCoV-229E at the early stage of infection. There are marked alterations in gene expression related to apoptosis, inflammation, and procoagulation. These findings are consistent with the histological changes of SARS, especially in the liver and blood vessels. Besides antivirals (6, 24), other modalities of treatment, such as antiapoptotic agents (34), immunomodulators against inflammation (7), and modifiers of coagulation, should be considered in future research. It is important to remember that many patients continued to deteriorate despite a decreasing viral load 2 to 3 weeks after the onset of SARS.
ACKNOWLEDGMENTS
We thank William Mak, Carol Chan, and Tommy Tang, Genome Research Centre, The University of Hong Kong, for their excellent technical assistance. Special thanks to all the staff members of the University and Hospital Departments of Microbiology at Queen Mary Hospital.
We acknowledge research funding from the Research Fund for the Control of Infectious Diseases of the Health, Welfare and Food Bureau of the Hong Kong SAR government, Infectious Diseases Fund (William Benter), Infectious Diseases Fund (Teresa Lim), and Tung Wah Group of Hospitals Hospitals’ Fund for Research in Infectious Diseases.
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