A human in vitro model system for investigating genome-wide host responses to SARS coronavirus infection
- Lisa FP Ng†1,
- Martin L Hibberd†1, 2,
- Eng-Eong Ooi3,
- Kin-Fai Tang3,
- Soek-Ying Neo1,
- Jenny Tan1,
- Karuturi R Krishna Murthy1,
- Vinsensius B Vega1,
- Jer-Ming Chia1,
- Edison T Liu1 and
- Ee-Chee Ren1, 2Email author
© Ng et al; licensee BioMed Central Ltd. 2004
Received: 11 June 2004
Accepted: 09 September 2004
Published: 09 September 2004
The molecular basis of severe acute respiratory syndrome (SARS) coronavirus (CoV) induced pathology is still largely unclear. Many SARS patients suffer respiratory distress brought on by interstitial infiltration and frequently show peripheral blood lymphopenia and occasional leucopenia. One possible cause of this could be interstitial inflammation, following a localized host response. In this study, we therefore examine the immune response of SARS-CoV in human peripheral blood mononuclear cells (PBMCs) over the first 24 hours.
PBMCs from normal healthy donors were inoculated in vitro with SARS-CoV and the viral replication kinetics was studied by real-time quantitative assays. SARS-CoV specific gene expression changes were examined by high-density oligonucleotide array analysis.
We observed that SARS-CoV was capable of infecting and replicating in PBMCs and the kinetics of viral replication was variable among the donors. SARS-CoV antibody binding assays indicated that SARS specific antibodies inhibited SARS-CoV viral replication. Array data showed monocyte-macrophage cell activation, coagulation pathway upregulation and cytokine production together with lung trafficking chemokines such as IL8 and IL17, possibly activated through the TLR9 signaling pathway; that mimicked clinical features of the disease.
The identification of human blood mononuclear cells as a direct target of SARS-CoV in the model system described here provides a new insight into disease pathology and a tool for investigating the host response and mechanisms of pathogenesis.
The causative agent for SARS has been identified as a novel coronavirus [1–3] with genome sequence revealing no strong homology to existing known coronaviruses [4–6]. Coronaviruses belong to the family of enveloped viruses called Coronaviridae, and have the largest known single-stranded viral RNA genomes (27 to 32 kb). Coronaviruses, have both "early" and "late" phases of gene expression. Regulatory proteins are synthesized as "early" non-structural proteins, while the structural proteins are synthesized as "late" proteins. "Late" structural proteins are usually required in greater amounts thus, there is a necessity to regulate the expression of the viral genes quantitatively. After the viral entry via endocytosis or through specific receptors, the 5'-end of the viral genome is translated directly giving rise to twenty-three viral proteins, including the RNA dependent RNA polymerase (RdRp), and other functional products involved in transcription, replication, viral assembly and cell death. Coronaviruses can be classified into species and three major antigenic groups based on, serology, natural hosts, monoclonal antibody recognition and nucleotide sequencing . Most coronaviruses have restricted host ranges as they infect only one host species or, at most, a few related species, they are an important group of animal pathogens. Group one (I) includes human coronavirus 229E (HCoV), porcine transmissible gastro-enteritis virus (TGEV) and feline enteric coronavirus (FECoV). Group two (II) includes bovine coronavirus (BCoV), murine hepatitis virus (MHV), and HCoV-OC43; and Group three (III) includes avian infectious bronchitis virus (IBV) . Some coronaviruses like HCoV have restricted tissue tropism, including macrophages , although most strains that infect humans cause only mild respiratory infections.
However, SARS has rapidly caused a world-wide problem. The earliest known cases of SARS was reported in Guandong Province, China in November 2002, becoming more widespread by March 2003, when it was introduced to Canada, Singapore, Taiwan and Vietnam via Hong Kong. The largest number of infected patients has been in China with a worldwide incidence totalling more than 8,400 by July 2003. Infection by the virus induces high morbidity and mortality, the latter being estimated at 15% by the World Health Organisation. SARS is characterized by high fever, non-productive cough or dyspnea and in many cases may progress to generalized, interstitial infiltrates in the lung, thus needing intubation and mechanical ventilation . The characteristic compression of alveolar sacs seen in atypical pneumonia is largely due to fluid build up outside the alveoli. One possible cause of this could be interstitial inflammation, following a localised host response. To date, the details of the host response to SARS-CoV infection is still largely unknown and consequently the most appropriate treatment regime remains to be established. Typically a pro-inflammatory cytokine profile (up regulated TNFα, Il1, IL6 and IFNs) is seen in viral infections such as influenza , together with perhaps limited amounts of IL8 and other chemokines  that may depend on which cell type is infected . In experimental systems the immediate innate immune response has been shown to be directed by the monocyte-macrophage-dendritic lineage to a range of different organisms [12, 13] and consists of a core set of pathways common to all, together with pathogen specific pathways. This data points to critical time points in the response, with the first 12 hours representing primary events while longer periods the consequence of this activity and a secondary (perhaps larger) cascade of responses.
We postulated that the pulmonary damage in SARS may not be a direct effect of the virus on the alveoli, but represents a secondary effect of cytokines or other factors proximal to but not from the lung tissue mediated by the host either as the primary or secondary response [2, 14]. In this study, we have addressed this question by developing a human in vitro model system that will in the future allow detailed investigations of the host response to be made.
Cell culture and virus infection
PBMCs were obtained by Ficoll-Hypaque separation of whole blood. 2 × 105 PBMCs were seeded into each well of a 24-well culture plate, 0.5 ml of complete RPMI-1640 (Life Technologies-Invitrogen, USA) added to each and cultured overnight at 37°C (5% CO2). A seed stock of SARS-CoV (strain SIN 2774) passaged in Vero E6 cells was used for infection. Vero E6 culture supernatants were added to each well in 50 μl volume at a concentration of 0.1 or 0.01 MOI (based on plaque forming units) and a control plate (media only). Each culture was set up in duplicate. After 4 hours of incubation, one set of the duplicate wells of the control plate and a 4 hours incubation plate were harvested while the rest received 0.5 ml media top-up and incubated for a further 2, 4, 6 and 8 days.
Cell harvest and RNA isolation
Harvesting was performed by gently flushing the wells with a Pasteur pipette to removed non-adherent cells, followed by a rinse of 1 ml RPMI. The rinsed fraction was pooled with the first harvest aliquot and spun at 1500 rpm. The cell pellet was washed twice with 2 ml RPMI to remove virus in the supernatant. 1.5 ml Trizol (Invitrogen, USA) was then added to the adherent cell fraction as well as the non-adherent cell fraction to lyse cells and stabilize the RNA. Extraction of total RNA was then performed following manufacturer's protocol and the resultant RNA dissolved in 40 μl water.
Real-time quantitative polymerase chain reaction (PCR)
The amount of SARS-CoV in each cell fraction was measured by real-time quantitative PCR assay. 2 μl of RNA was reverse transcribed and amplified in 20 μl using 0.9 μM each of forward (5'-GGTTGGGAT TATCCAAAATGTGA-3') and reverse (5'-AGAACAAGAGAGGCCATTATCCTAAG-3') primers, and 0.25 μM of TaqMan® MGB probe: 5'-(6-FAM)AGAGCCATGCCTAACAT(NFQ)-3') in a one step PCR using master mix from Applied Biosystems (USA) according to manufacturers' recommendations. Reactions were performed using an ABI PRISM 7900 sequence detection system (48°C for 30 min, followed by 95°C for 10 min and 40 cycles of 95°C for 15 sec and 60°C for 1 min) and quantitation achieved using standard curves generated from in vitro transcribed RNA.
High density oligonucleotide array hybridization
At each time point (4 hours, 8 hours, 12 hours, 24 hours), 5 × 107 cells of mock-infected and infected cells were harvested and lysed using Trizol (Invitrogen, USA). Total RNA was isolated according to the manufacturer's recommendation. Quality of the total RNA was judged from the ratio between 28S and 18S RNA after agarose gel electrophoresis. 20 μg of total RNA was labeled with Cy-3 or Cy-5 using the Superscript II reverse-transcription kit (Invitrogen, USA) and hybridization was carried out overnight (16 hours) at 42°C on high-density oligonucleotide arrays (~19,200 gene features, Compugen) using universal human reference (Stratagene, USA) as a reference. Hybridized arrays were scanned at 5 μm resolution on a GenePix 4000A scanner (Axon Instruments) with variable photo-multiplier tube voltage to obtain maximal signal intensities, and the resulting images were analyzed via GenePix Pro v4.0 (Axon Instruments) as described in the manual.
Microarray data analysis
Raw data were analyzed on GenePix analysis software version 4.0 (Axon Instruments) and uploaded to a relational database. The logarithmic expression ratio for a spot on each array was normalized by subtracting the median logarithmic ratio for the same array. Data were filtered to exclude spots with a size of less than 25 μm and any poor quality or missing spots. Since the correlation of the overall data from reciprocal labeling was good, values obtained from reciprocal labeling experiments were averaged. In addition, the data were distilled to the set of gene features that were present at all 4 time points in both the viral infected samples and the negative controls. The results were represented as the logarithmic ratio of gene expression between the viral infected samples and their corresponding negative controls at the various time points. Application of these filters resulted in the inclusion of ~12,900 of the total ~19,200 gene features in subsequent analyses.
To discover patterns of gene expression, the values associated with each gene feature f were translated so that their means were zero. Similar genes, whose translated gene-features exhibited same induction-repression pattern, were grouped together. Genes g i , g j were said to be similar if they satisfied the following condition:
where g it and g jt denote the translated values of gene features g i , g j at time t respectively; and N is the number of time points for which the expression of a gene was observed. Similar genes, based on the above criteria, are grouped together. Within each group, the genes were ordered in the descending order of their expression range (defined as the difference between the maximum and minimum ratios of gene expression). This algorithm is a special case of the Friendly Neighbor algorithm currently under development. The final plots were generated using the original expression ratios while preserving the clustering and ordering discovered by the above algorithm. To determine whether a gene observed to be responsive could appear merely by chance, 100,000 expression profiles were generated by randomly sampling the expression ratios from the entire dataset with replacement. The P value of a gene is the fraction of the random profiles whose logarithmic expression range is as good as, or better than that of the selected gene.
Results and Discussion
Kinetics of SARS-CoV replication
The kinetics of viral replication was variable among the 6 donors (Fig. 1C). There was a lag phase of 2 days in the case of donors a, c and e; and 4 days for donors b, d and f before any significant increase could be detected. The viral replication generally peaked at either day 4 or day 6. The exception was donor b, in which the virus seemed to replicate at a much slower pace compared to the other 5 donor samples. Equally interesting was the different levels of virus attained. Donor d seems to stand out from the rest, reaching a peak of 480 × 103 copies per well which is 4 times more than that attained by donor e, with 120 × 103 copies per well. Such variation strongly suggests that there is an underlying host-pathogen interaction influencing the kinetics of SARS-CoV replication efficiency. These in vitro observations may reflect the wide range of patient outcomes after SARS-CoV infection .
Antibody blocking experiments were also performed in which SARS-CoV was pre-incubated with convalescent patient sera for 30 minutes before introduction to the PBMCs and after a 4 day incubation period, the adherent cell fraction was harvested and assayed for SARS-CoV viral titer. Results clearly showed that even at high dilution, convalescent sera inhibited SARS viral replication (data not shown), presumably by blocking viral entry. This supports other reports indicating that SARS-CoV is not endocytosed through antibody mediated mechanisms and confirms a protective role for antibodies elicited either by the infection or through immunization [19, 20].
SARS-CoV specific gene expression changes
Representation of selected immune-related genes upregulated during the first 12 hours post-infection.
Unique Gene ID.
Expression range (log2)
Immune Response Genes:
Monocyte to macrophage
Blood Coagulation Genes:
Apoptosis, caspase activator
Signaling, scavenger activity
Apoptosis, ion channel activity
Intracellular receptor signaling
Defense response, maintenance
Extracellular defense response
Cellular defense response
Extracellular defense response
Extracellular defense response
Extracellular defense response
JAK-STAT cascade, recptor
Invasive growth response
A number of CC chemokines (CCL4, CCL20, CCL22, CCL25, CCL27) and their receptors (CCR4 and CCR7) were highly expressed in response to the infection (Fig. 2C and Table 1), indicating a rapid mobilization and increased trafficking, in particular of the monocyte-macrophage lineage very early on in the infection . CXC chemokines (CXCL9, CXCL12) were also highly expressed suggesting significant increase in neutrophil homing as well. These are likely to be lung directed as IL8 and IL17 were also highly expressed [27–32]. Specific trafficking of these cells to the lung may account for the localized nature of the response .
Surprisingly, a number of blood coagulation genes were highly expressed early during our in vitro infection (Fig. 2C and Table 1), in particular TBXAS, which has been implicated in vasoconstriction, platelet aggregation, membrane lysis and increased permeability [34, 35]; fibrin (FGB and FGG) and the coagulation pathway directly (SERPINs D1 and A3 together with Factors 10, 3 and 2). This gives a pro-coagulation profile, which mimics the clinical-pathological observations: at autopsy, many SARS patients have unusually disseminated small vessel thromboses in the lungs without evidence of disseminated intravascular coagulation [1, 36]. Again, these expression profiles provide an experimental framework to explore an important aspect of SARS pathobiology and treatment.
The emerging picture from this study implicates a central role for the immune response in the pathobiology of a SARS infection. While detailed in vivo studies of the host response are now required, the in vitro model described here will allow responses to specific modulators (such as therapeutics) to be investigated. In future developments of the model, it will be interesting to compare the host response to different SARS-CoV isolates with inactivated preparations of the virus. In other diseases, in vitro models have revealed a number of key processes relevant to the clinical diseases [9, 12, 13] and it is likely that the responses identified here will prove to be equally important. Although some clinical parameters have now been used as prognostic markers [40–42], further study of the regulatory mechanisms for chemokine-cytokine production will likely improve their accuracy and perhaps allow development of new treatment protocols.
This study was supported by grants from the Agency for Science, Technology and Research (A*STAR), Singapore. Lisa F. P. Ng is also supported by a postdoctoral scholarship from the Singapore Millennium Foundation, Singapore Technologies. We thank Lora V. Agathe, Patricia Tay Pei-Wen, Khoo Chen-Ai and Li Pin (Genome Institute of Singapore); Renita Danabalan (National Environment Agency) for technical assistance and Dr Ling Ai-Ee (Department of Pathology, Singapore General Hospital) for the SARS-CoV isolate.
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