key: cord-1027816-rrhh2alf authors: Ng, Lisa FP; Hibberd, Martin L; Ooi, Eng-Eong; Tang, Kin-Fai; Neo, Soek-Ying; Tan, Jenny; Krishna Murthy, Karuturi R; Vega, Vinsensius B; Chia, Jer-Ming; Liu, Edison T; Ren, Ee-Chee title: A human in vitro model system for investigating genome-wide host responses to SARS coronavirus infection date: 2004-09-09 journal: BMC Infect Dis DOI: 10.1186/1471-2334-4-34 sha: 45d2d838cb3d5ae2dabd7bb7c82329b398d1c65f doc_id: 1027816 cord_uid: rrhh2alf BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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] [2] [3] with genome sequence revealing no strong homology to existing known coronaviruses [4] [5] [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 [7] . 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) [7] . Some coronaviruses like HCoV have restricted tissue tropism, including macrophages [8] , 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 [2] . 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 [9] , together with perhaps limited amounts of IL8 and other chemokines [10] that may depend on which cell type is infected [11] . In experimental systems the immediate innate immune response has been shown to be directed by the monocytemacrophage-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. 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. At each time point (4 hours, 8 hours, 12 hours, 24 hours), 5 × 10 7 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. 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. We obtained PBMCs from 6 healthy volunteers by Ficoll-Hypaque separation of whole blood. Of the 6 donor PBMCs tested, all were able to support SARS-CoV replication when infected with multiplicity of infection (MOI) of 0.1. The first sampling, taken from cells infected for 4 hours, showed an average copy number of 32 × 10 3 (Fig. 1A ) and represents the initial inoculum level. Over the course of the next 8 days, there was a steady rise in viral load, reaching as high as 480 × 10 3 copies per well in one donor, which could only be explained by active replication of the SARS-CoV intracellularly. This work is supported by recent in vivo evidence suggesting that SARS-CoV may have infected and replicated within PBMCs of SARS patients [15] and cells from humans and animals [16] . An indication of the PBMCs lineage involvement was provided by repeating the experiment using the monocyte-macrophage cell line THP-1 [17] , in which viral replication was similar to the primary cell culture over the first 4 days (Fig. 1B) . In the primary cultures, the nonadherent cell fraction which comprises mainly lymphocytes and granulocytes showed dramatically less viral replication in our assay as did all cells infected at MOI of 0.01 (Fig. 1A) . 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 × 10 3 copies per well which is 4 times more than that attained by donor e, with 120 × 10 3 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 [18] . 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] . To further elucidate the molecular processes of SARS-CoV infection, PBMCs from 3 healthy individuals were infected separately in vitro with SARS-CoV (0.1 MOI) and harvested at 4 hours, 8 hours, 12 hours and 24 hours time intervals post-infection. As controls, uninfected aliquots of the same PBMCs were also harvested at the corresponding time points. Total RNA extracted from the PBMCs of the 3 individuals were pooled, labeled and hybridized to human oligonucleotide arrays consisting of ~19,200 gene features. Reciprocal dye swap replicate hybridizations were performed to minimize technical noise. Analysis of variance in expression levels for each gene across all the time points indicated the ~1200 genes which showed the largest variability ( Fig. 2A and 2B) . In order to focus the analysis, we queried the entire data set for genes related to the immune response by keyword searches on their gene ontology descriptions with the aim of describing the specific host-pathogen interaction. In common with other studies of respiratory pathogens [9] [10] [11] [12] [13] , our data points towards two critical time points in the response, with the first 12 hours representing a primary pro-inflammatory cytokine profile while longer periods represent the consequence of this activity and a secondary cascade of responses [9] [10] [11] [12] [13] . We observed that within the first 12 hours of SARS-CoV infection, evidence of this monocyte-macrophage activation was seen, indicated by enhanced expression of CD14, TLR9 plus NFKβ1 and GATA signaling ( Fig. 2C and Table 1 ). In addition, the MRC2 endocytotic receptor was upregulated as was the complement pathway (C1q, C6) . Taken together, these data suggest an early activation of the innate immunity pathway. This activation was accompanied by an unusual cytokine transcriptional profile ( Fig. 2C and Table 1 ). While IL1β (up regulated for the first 12 hours) would be expected following macrophage activation [21] , TNFα, IFNγ and IL6 were noted by their surprisingly low level of expression. This is in spite of the presence of elevated IL19 which is thought to enhance their up regulation [22] . In some clinical investigation, concentrations of TNF and IL 6 measured during active disease were found to be relatively low [23, 24] , reflecting our findings. This paper did not report on IFN levels, however, we found them to be low (Supplementary figure [see Additional file 1]). This is of particular interest as IFNs have been shown to have significant anti-SARS-CoV effects [25] . Such effects suggest that alteration of the IFN response and perhaps other immune modulators might provide opportunity for novel treatment and management regimes for SARS patients to be developed. 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 [26] . 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] [28] [29] [30] [31] [32] . Specific trafficking of these cells to the lung may account for the localized nature of the response [33] . 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. It is interesting to note that the TLR9 was highly expressed in comparison to other TLR receptors, implying some degree of TLR specificity for the virus (Fig. 3A ). TLR9 is known to respond to CpG signaling motifs (GTCGTT) [37] [38] [39] and one possibility is that the virus is activating directly through this mechanism. In support of this, we found that the SARS-CoV viral sequence contains the highest number (7 copies) of such specific signaling motifs compared to other coronaviruses and significantly more than several other viruses involved in respiratory diseases (Fig. 3B) . It is conceivable that TLR9 may be aiding host recognition of the virus via the CpG groups and contributing to the initiation of the innate host inflammatory response. An alternative explanation is that TLR9 is being stimulated by mechanisms unrelated to CpG recognition. 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] [41] [42] , further study of the regulatory mechanisms for chemokine-cytokine production will likely improve their accuracy and perhaps allow development of new treatment protocols. Figure 3 Expression of TLR9 in response to SARS-CoV infection. A. Expression range (log 2 ) for TLR9, TLR2 and TLR4. The expression range for TLR9 was greater than expected (* represents a P-value for TLR9 of 0.016). B. Comparison of the CpG motif (GTCGTT) copy number in coronaviruses and other viruses linked to respiratory diseases. Accession numbers are as follows: SARS coronavirus SIN2500 -AY283794, Human coronavirus 229E -NC_002645, Murine hepatitis virus -NC_001846, Avian infectious bronchitis virus -NC_001451, Bovine coronavirus -NC_003045, Human rhinovirus B -NC_001490, Human parainfluenza virus 1 -NC_003461, Human respiratory syncytial virus -NC_001781, Human metapneumovirus -NC_004148. 3314 1089116 ; 1089116; Hs.188691 1091254 N4BP3 80828 1081054 KIAA0924; 1081054; Hs 351428 1086300 FLJ13941; 1086300; Hs.187617 1078744 LGALS3BP 6874 1085972 FLJ20280; 1085972; Hs.270134 1076739 GPX5 2157 1082995 DKFZP564D166; 1082995; Hs.4996 1093600 SH3GLP3; 1093600; 1078099 ; 1078099; Hs 26232 1091782 EBF 32425 1083484 RALY 76111 1076985 FLJ22615; 1076985; Hs.266746 1077149 ITGAV 295726 1093408 ; 1093408; Hs 247748 1076310 ; 1076310; 1077045 PCLO 1079053 TH; 1079053; Hs 6191 1083121 ; 1083121; Hs.82503 1076580 FLJ20729 42768 1077644 TFAM 1081068 ; 1081068; Hs.306618 1086001 ; 1086001; Hs.306864 1082405 LOC149414; 1082405 40337 1083559 MATR3; 1083559; 1083183 FLJ12960 SCAM-1; 1076449 169849 1093618 ; 1093618; Hs.306691 1087594 SERPINI1 13277 1080493 FLJ13881; 1080493; Hs.115412 1077088 PYCS 1077296 TES; 1077296; Hs.165986 1076355 BIRC1 75511 1085653 LOC63928; 1085653; Hs.178589 1076550 RA-GEF-2; 1076550; Hs 24587 1079722 FLJ21924; 1079722; Hs.143509 1076439 TROAP 171955 1077540 LCT 99291 1091655 ; 1091655; Hs.352044 1080463 NEUROG1 158333 1075708 ; 1075708; Hs.308467 1076501 LOC57401; 1076501; Hs.287378 1093645 FLNA 51743 1078951 PDZ-GEF1; 1078951; Hs.154545 1085256 ; 1085256; Hs 1086102 TNFRSF13B; 1086102; Hs 115365 1083796 MLL; 1083796; 1083494 MGC27165 25882 1084348 DIS3 1076377 FLJ10979; 1076377; Hs 72782 1077941 GIF; 1077941; Hs.110014 1092416 ; 1092416; Hs 107139 1094058 ; 1094058; Hs.58220 1076255 SPRY3 159223 1086116 ; 1086116; Hs.287566 1084545 HDS; 1084545; Hs 53531 1085869 ; 1085869; Hs.289078 1082111 49881 1085693 MGC27165; 1085693; Hs.146360 1077447 GSK3A 123116 1077874 ; 1077874; Hs.10268 1077643 NFIL3 2605 1091694 SNN 74376 1092577 KIAA1387; 1092577; Hs.301434 1088628 NME6 1089815 DKFZp434H2226; 1089815; Hs.274457 1076937 FLJ22028; 1076937; Hs.192570 1078080 KIAA1243; 1078080; Hs.151076 1077336 SAE1 279868 1075821 ; 1075821; 1082542 FLJ20276; 1082542; Hs.270502 1076289 TM7SF2 103283 1077457 ; 1077457; Hs.131887 1087742 SEC22L2 225977 1078253 FAP; 1078253; Hs.418 1089805 MEST 79284 1089597 ; 1089597; Hs.82932 1080586 SMT3H2 183857 1093777 UGDH 28309 1084342 KIAA0916; 1084342; Hs.151411 1082372 ; 1082372; Hs 6462 1083459 FLJ20635; 1083459; Hs 23076 1084139 FTHFD 272789 1086145 FLJ20241; 1086145; Hs.181780 1093794 MFN1 283110 1087615 ; 1087615; Hs.306787 1079458 LOC284018; 1079458; Hs.90790 1090936 B3GNT3 155986 1089698 TNFRSF10B; 1089698; Hs.51233 1075706 ACAS2 170263 1089946 OS4; 1089946; Hs.180669 1083199 COL5A1 336224 1083785 LOX; 1083785; Hs.102267 1093300 MPP1 1861 1085954 PAEP 16621 1091686 SDNSF 147472 1091603 PSK 92002 1076431 RORB 1497 1086203 ARHGAP11A; 1086203; Hs.172652 1075842 CCL25 274313 1094150 POSH 251673 1079799 TNXB; 1079799; 1092438 FLJ10846 158299 1077899 COMP 1077783 ZFR; 1077783; Hs.173518 1078509 ; 1078509; Hs.306888 1077087 SPUVE 1086297 BDNF 56023 1076915 DKFZP564C196; 1076915; Hs.127384 1085336 GPLD1 278422 1093966 ; 1093966; Hs.218008 1077814 107127 1076391 KIAA1613; 1076391; Hs.287380 1094373 MPP5 306219 1084530 ; 1084530; Hs 112842 1088497 ; 1088497; Hs.306804 1082951 INSR 89695 1087772 ; 1087772 1082079 CMT2; 1082079; Hs.124 1078217 ; 1078217; Hs 112933 1086130 ; 1086130; Hs 348198 1079025 FLJ13291; 1079025; Hs.334848 1085844 RU2 152009 1080453 NMB 2582 1081996 FLJ10631; 1081996; Hs.238944 1083485 BAG5 5443 1086352 AKAP12; 1086352; Hs.788 1087404 NASP 241573 1081129 FLJ12735; 1081129; Hs 1083481 C20orf36; 1083481; Hs.184628 1077493 MS4A5 178066 1080443 ; 1080443; Hs.350531 1084296 LLGL2 77196 1093143 FLJ10747; 1093143; Hs.189782 1077730 THRB 23740 1084379 DNTT 91161 1076452 CLECSF12; 1076452; Hs.161786 1087935 PRKAG2 259842 1090948 HGF; 1090948; Hs 63304 1094113 OMP; 1094113; Hs.248153 1092594 LIM; 1092594; Hs.154103 1081703 CACNA1A 105584 1079183 FLJ20244; 1079183; Hs.158947 1081228 HS6ST1 74070 1094004 ; 1094004; 1092488 FLJ10375; 1092488; Hs.319088 1083800 IGF2R 76473 1082305 JRK; 1082305; Hs.142296 1083650 APOB; 1083650; Hs.585 1086066 ; 1086066; Hs 166085 1094568 ; 1094568; Hs.306746 1089644 KIAA0173; 1089644; Hs.169910 1086177 ANGPT2 274184 1076920 ; 1076920; Hs.306342 1093530 KIAA1007; 1093530; Hs.279949 1093737 MAN2B1 279854 1091622 ; 1091622; Hs.301472 1087651 CALB3; 1087651; Hs.639 1085400 S100A13 14331 1091219 ; 1091219; Hs.4892 1087709 FLJ10620 99445 1081204 FLJ10572; 1081204; Hs.220998 1085004 ; 1085004; Hs.114293 1092598 FLJ21080 169745 1078960 ; 1078960; Hs.293930 1080954 HSPC063 278947 1088682 EED; 1088682; Hs.151461 1082738 EFNA1 1624 1090676 ; 1090676; Hs.22545 1081939 SORL1 54433 1077487 KIAA1320; 1077487; Hs.117414 1085754 DKK4 1084185 TTK; 1084185; Hs.169840 1090099 MUC5B 1079397 UTY; 1079397; Hs.115277 1077516 ; 1077516; Hs.293916 1078417 ; 1078417; Hs 30213 1090079 FLJ10486; 1090079; Hs.173946 1076051 IL1RL1; 1076051; Hs 145477 1093779 ; 1093779; Hs.302052 1077732 PRDM11 283097 1093696 FRAT2 140720 1088385 SPS; 1088385; Hs.124027 1083853 PKP1 6335 1090113 ; 1090113; 1084038 FLJ13322; 1084038; Hs.110298 1083798 FLJ12525; 1083798; Hs 1093412 DKFZp761A132; 1093412; Hs.301372 1093598 KIAA0794; 1093598; Hs.127287 1083428 H6PD 83551 1089979 KIAA1404; 1089979; Hs 306216 1089559 MGEA5; 1089559; 1093216 ; 1093216; Hs.246472 1090206 TPM2 96149 1094419 ; 1094419; Hs 183889 1090026 CPSF5 1085916 C20orf51; 1085916; Hs.187773 1086027 KIAA0415; 1086027; Hs.229950 1094210 APM2 142989 1084199 ; 1084199; Hs.123060 1083897 GNB5 1090937 KEL; 1090937; Hs.157 1083560 KIAA0276; 1083560; Hs.240112 1085200 FLJ00012 102929 1092291 KIAA0140; 1092291; Hs.156016 1093344 C4BPB 145078 1086425 ; 1086425; Hs.152939 1077428 SDHC 155223 1092858 ; 1092858 272927 1079867 ; 1079867; Hs.274547 1085065 ; 1085065; Hs.288218 1075544 DXS9879E 18212 1077050 KIAA1324; 1077050; Hs.104696 1075441 SLC17A7 169111 1083260 FLJ20200; 1083260; Hs.165803 1075745 ABHD2 70337 1083924 ; 1083924; Hs.158530 1081706 TFF1 84232 1090519 LOC51249; 1090519; Hs.184456 1085098 CRTAP 100602 1088905 AIM1; 1088905; 1077195 FLJ13621; 1077195; Hs.287583 1077793 IL19 29191 1091367 ; 1091367; Hs MR-1 10114 1092754 ; 1092754; Hs.75452 1079301 TFAP2A 1093207 SORD; 1093207; Hs 12056 1081715 ; 1081715; Hs.306751 1086553 SSFA2 153863 1080008 ; 1080008; Hs.186874 1091405 NRXN1 63795 1081660 HIST1H4L; 1081660; Hs 169857 1077253 NPY 1019 1084603 ; 1084603; Hs.7709 1076845 PRC1 283099 1093913 FLJ20255; 1093913; Hs.274247 1081795 DCTN1 76751 1080217 ; 1080217; Hs.335835 1092439 NEU1 118721 1086498 CIDEB 91973 1086559 FLJ10726; 1086559; Hs.268561 1092695 CENPE 16244 1079550 APTX 1079097 CCNF 173987 1080924 ESDN 123642 1086504 GNPI 108557 1079478 DLST 296348 1076333 SSA2; 1076333; Hs.554 1087037 HSPC150 8768 1082766 ; 1082766; Hs.272227 1086159 CDKL5 85137 1078618 ; 1078618; Hs.306767 1082210 FLJ22170; 1082210; Hs.288573 1079284 DNAJA3 1094593 CEACAM8; 1094593; Hs.41 1076338 THY1 36451 1081299 KIAA1061; 1081299; Hs.123420 1093157 IMPG1 236204 1083511 ; 1083511; Hs 159456 1076587 ; 1076587; Hs.168232 1084839 KIAA1238; 1084839; Hs.236463 1077790 ATP2B2 1075659 DKFZP434I092; 1075659; Hs.120021 1077220 58382 1092243 FLJ10547; 1092243; Hs 1093846 FRSB 142245 1081658 ; 1081658; Hs.288965 1087110 C14orf101; 1087110; Hs 75789 1080945 FLJ10210; 1080945; Hs.183639 1082751 KIAA0595; 1082751; Hs 11713 1075539 OSBP 274361 1093888 FAH 75615 1093105 KRT18; 1093105; 1091009 FLJ23323 1089521 DKFZp547D155; 1089521; 1090567 ; 1090567; Hs.306409 1090952 PPARGC1 1079527 13CDNA73; 1079527; Hs.181304 1085602 GPR66 173081 1093504 ATRN 1077650 FLJ20499; 1077650; Hs.122275 1076841 AP2A2 6066 1077963 ; 1077963; Hs.323370 1086617 ; 1086617; Hs.306614 1080452 DKFZp434O0515 104019 1077033 KIAA0179; 1077033; Hs 159360 1094006 AHSG 29385 1086755 MONDOA 4188 1077294 SERPINA3; 1077294; Hs.234726 1086793 CMG1 1086648 ; 1086648; Hs.7886 1085215 117852 1083409 ; 1083409; 1086167 ; 1086167; Hs.172627 1090578 NDUFB2 1078254 HSCBCIP1; 1078254; Hs.283783 1087774 ; 1087774; Hs.272203 1090147 IRA1 77813 1080921 ; 1080921; 1084261 FLJ22169; 1084261; Hs.323363 1078830 H2AFZ 79133 1086065 LOC88745; 1086065; Hs.283636 1080273 ; 1080273; Hs 112986 1082236 MIPEP 11747 1085920 FLJ11457; 1085920; Hs.126707 1092046 BTG4 55498 1090452 ; 1090452; Hs 61828 1089935 KIAA1649; 1089935; Hs 27495 1080747 LRBA 154149 1088407 ; 1088407; Hs 100016 1089590 PARG 155595 1075488 KIAA0057; 1075488; Hs.153954 1078878 SLC25A10; 1078878; Hs.285829 1086660 LOC148894; 1086660; Hs 11806 1082458 ; 1082458; Hs 1082491 FLJ22595; 1082491; Hs 211582 1090423 FLJ12998; 1090423; Hs.343627 1080636 PCSK4 46884 1079288 FLJ11042; 1079288; Hs.274208 1091520 ; 1091520; 1089661 LOC152559; 1089661; Hs.26860 1091078 FH 123090 1082436 FLJ23598; 1082436; Hs 249212 1092047 GMNN 278627 1083102 VIP 129445 1084095 TMOD 79914 1082947 ; 1082947; Hs.46801 1090696 MAPRE3 172740 1092150 PXN; 1092150; Hs.102497 1086099 VCAM1 17211 1093120 ; 1093120; Hs.38034 1078306 SNCAIP 1077359 KIAA0419; 1077359; Hs.279912 1093496 MSH6 151428 1092523 ; 1092523; Hs.226440 1091498 LEPREL2 171921 1084345 HEMGN 7911 1092494 ; 1092494; Hs.12385 1082997 MAPK4 180034 1084430 HAKAI 292767 1084745 FLNC 58414 1076664 ; 1076664; Hs.288660 1084601 AVP 1084752 TGFBR1; 1084752; Hs.220 1080199 ARHE 184227 1081324 DKFZp762E1312; 1081324; Hs.104859 1094494 TERT 115256 1079966 ; 1079966; 1080018 FLJ22374 8769 1079860 C4orf7; 1079860; 1075863 ; 1075863; Hs.288741 1084255 WNT8B 137595 1076992 KIAA1524; 1076992; Hs.151343 1086595 HSPC228 267288 1079830 ; 1079830; Hs.27801 1093948 DIM1 5074 1093952 NS; 1093952; Hs.279923 1078457 C14orf136; 1078457; Hs.164866 1083224 ; 1083224; Hs 105940 1084401 HUMAUANTIG; 1084401; Hs.75528 1086503 ZNF268 183291 1081114 ; 1081114; Hs.144871 1082735 FBXW2 5320 1084860 HIST1H4E; 1084860; Hs.240135 1080149 CTNND2 98017 1084890 FLJ20986; 1084890; Hs.324507 1094296 EEG1 301667 1084716 ; 1084716; Hs.333421 1094316 ; 1094316; Hs.287451 1076895 KIF14 3104 1094248 FLJ23360; 1094248; Hs.161279 1092069 HEPH 31720 1084648 NGFR 1827 1091894 KANK 159140 1087429 FLJ10283; 1087429; Hs.284216 1078146 PTTG1 1093594 NT5C; 1093594; 1080458 ; 1080458; Hs.10862 1080450 APOB; 1080450; Hs 301526 1093188 NMA 82002 1084998 DFFA 152316 1093096 CRKL 37078 1081448 BLu; 1081448; Hs.167380 1081854 ID3 168289 1080263 IFIT2 95577 1079412 C20orf177; 1079412; Hs.286184 1079599 TM4SF3 84072 1082949 COL4A3; 1082949; Hs.530 1090239 HRC 1086475 C20orf67; 1086475; Hs.272814 1083950 ; 1083950; 1093493 ; 1093493; Hs 25846 1085805 ; 1085805; Hs.242038 1091003 PRKAR1A 155499 1075874 LOC147991; 1075874; Hs.29808 1075590 MGC10500; 1075590; Hs.271599 1088702 GPR75 300622 1077517 ; 1077517; Hs.257696 1076050 RARB 171495 1075439 LOC83693; 1075439; Hs.171937 1091423 HSMCR30 122744 1093746 TACSTD1; 1093746; Hs.692 1091298 MMD 79889 1088691 FLJ22655; 1088691; Hs.115497 1079761 FLJ23091; 1079761; Hs.250746 1090251 TNFRSF11B; 1090251; Hs.81791 1093290 HBG1 266959 1077897 ; 1077897; Hs.105664 1094103 STRAIT11499; 1094103; Hs.236556 1083014 NEK9 309763 1093745 RDX; 1093745; Hs.263671 1090650 BK65A6.2; 1090650; Hs Rab11-FIP3; 1093995; Hs.119004 1076916 10711 1090870 BID; 1090870; Hs.315689 1076010 PRKWNK1 15744 1078662 KIAA0406; 1078662; Hs 288809 1089718 FLJ22789; 1089718; 1086410 PRO2831 250568 1078940 HBZ; 1078940; Hs.272003 1084390 M6PR 1077059 ; 1077059; Hs 1089552 ETBR-LP-2; 1089552; Hs.132049 1081555 NOTCH2; 1081555; 1081662 FLJ20695 91747 1081764 FLJ23416; 1081764; Hs.306900 1081605 NAGLU 155462 1083540 LOC220963; 1083540; Hs 1094242 DNAH9; 1094242; 1077159 NMES1; 1077159; 1085057 ; 1085057; Hs.222024 1083387 PCBD 6088 1083595 DKFZP434F122; 1083595; Hs.159352 1080171 SFRS2IP 155596 1094415 SIT 248140 1093529 ; 1093529; Hs.306574 1090292 SERPINI2; 1090292; Hs.158308 1079189 CAT 28907 1082665 ; 1082665; Hs.272249 1088235 FNTA 3363 1078346 KIAA0545; 1078346; Hs.129943 1078603 SLC6A5 136557 1084047 KIAA1695; 1084047; Hs.288841 1094201 CSNK1G3 251394 1085516 ; 1085516; 1093813 ; 1093813; Hs SARS study group: Coronavirus as a possible caused of severe acute respiratory syndrome SARS Working Group: A novel coronavirus associated with severe acute respiratory syndrome Identification of a novel coronavirus in patients with severe acute respiratory syndrome Characterization of a novel coronavirus associated with severe acute respiratory syndrome Comparative full-length genome sequence analysis of 14 SARS coronavirus isolates and common mutations associated with putative origins of infection Coronaviridae and their replication Macrophage infiltration, but not apoptosis, is correlated with immune-mediated demyelination following murine infection with a neutropic coronavirus Induction of proinflammatory cytokines in human macrophages by influenza A (H5N1) viruses: a mechanism for the unusual severity of human diseases? Lancet Inflammatory responses in influenza A virus infection Release of macrophage migration inhibitory factor and CXCL8/interleukin-8 from lung epithelial cells rendered necrotic by influenza A virus infection Human macrophage activation programs induced by bacterial pathogens The plasticity of dendritic cell responses to pathogens and their components Lung pathogen of fatal severe acute respiratory syndrom SARS-coronavirus replicates in mononuclear cells of peripheral blood (PBMCs) from SARS patients Discovery of novel human and animal cells infected by the severe acute respiratory syndrome coronavirus by replication-specific multiplex reverse transcription-PCR Bacterial activity of a monocytic cell line (THP-1) against common respiratory tract bacterial pathogens is depressed after infection with respiratory syncytial virus Severe acute respiratory syndrome without respiratory symptoms or abnormal chest radiograph findings An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus Antibody responses against SARS-coronavirus and its nucleocapsid in SARS patients Alternative activation of macrophages IL-19 induces production of IL-6 and TNF-alpha and results in cell apoptosis through TNF-alpha Beijing Group of National Research projects for SARS: Dynamic changes in blood cytokine levels as clinical indicators in severe acute respiratory syndrome Tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in-situ hybridization study of fatal cases Treatment of SARS with human interferons Role of chemokines in the biology of natural killer cells Endogenous IL-17 as a mediator of neutrophil recruitment caused by endotoxin exposure in mouse airways Potential role of IL-8, platelet-activating factor and TNF-alpha in the sequestration of neutrophils in the lung: effects on neutrophil deformability, adhesion receptor expression, and chemotaxis G-CSF and IL-8 but not GM-CSF correlate with severity of pulmonary neutrophilia in acute respiratory distress syndrome MRP8/MRO14, CD11b and HLA-DR expression of alveolar macrophages in pneumonia Induction of interleukin 8 (IL-8) production by pseudomonas nitrite reductase in human alveolar macrophages and epithelial cells Interleukin-8 induces lymphocyte chemotaxis into the pleural space. Role of pleural macrophages Role of thromboxane, prostaglandins and leukotrienes in endotoxic and septic shock Thromoxane synthesis is increased by upregulation of cytosolic phospholipase A2 and cyclooxygenase-2 in peripheral polymorphonuclear leukocytes during bacterial infection in childhood Lung pathology of severe acute respiratory syndrome (SARS): a study of 8 autopsy cases from Singapore Recognition of pathogen-associated molecular patterns by TLR family Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides A Toll-like receptor recognizes bacterial DNA Prognostic factors for severe acute respiratory syndrome: a clinical analysis of 165 cases Pattern of disease in patients with severe acute respiratory syndrome Validation of a novel severe acute respiratory syndrome scoring system 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. Order is arranged from the highest to lowest value based on the expression range (log 2 ). The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/4/34/prepub