key: cord-0901428-l3a3polk
authors: Chan, Paul K.S.; To, Ka‐Fai; Lo, Anthony W.I.; Cheung, Jo L.K.; Chu, Ida; Au, Florence W.L.; Tong, Joanna H.M.; Tam, John S.; Sung, Joseph J.Y.; Ng, Ho‐Keung
title: Persistent infection of SARS coronavirus in colonic cells in vitro
date: 2004-07-13
journal: J Med Virol
DOI: 10.1002/jmv.20138
sha: 9b1a57ac566a5ac1a8e97824347865e473343335
doc_id: 901428
cord_uid: l3a3polk

Severe acute respiratory syndrome coronavirus (SARS‐CoV) can produce gastrointestinal symptoms. The intestinal tract is the only extrapulmonary site where viable viruses have been detected. This study examined seven established human intestinal cell lines, DLD‐1, HCT‐116, HT‐29, LoVo, LS‐180, SW‐480 and SW‐620, for their permissiveness to SARS‐CoV infection. The results showed that only LoVo cells were permissive to SARS‐CoV infection as evident by positive findings from indirect immunofluorescence staining for intracellular viral antigens, in situ hybridization for intracellular viral RNA, and electron microscopy for intracellular viral particles. In contrast to Vero cells, SARS‐CoV did not produce cytopathic effects on LoVo cells. However, LoVo cells were found to be highly permissive for productive infection with a high viral titre (>3 × 10(7) viral copies/ml) produced in culture supernatant following a few days of incubation. SARS‐CoV established a stable persistent chronic infection that could be maintained after multiple passages. Being a cell line of human origin, LoVo cells could be a useful in vitro model for studying the biology and persistent infection of SARS‐CoV. Our results on the expression of angiotensin‐converting enzyme 2 (ACE2), a recently identified cellular receptor for SARS‐CoV, in these cell lines indicated that it might not be the sole determinant for cells to be susceptible to SARS‐CoV infection. J. Med. Virol. 74:1–7, 2004. © 2004 Wiley‐Liss, Inc.

Severe acute respiratory syndrome (SARS) is a newly emerged human disease caused by a novel coronavirus (SARS-CoV) Ksiazek et al., 2003; Peiris et al., 2003a] . SARS was first identified from Southern China in November 2002. Up to July 2003 when the global outbreak came to a halt, a total of 8,098 cases had been reported to the World Health Organization [WHO, 2003] . SARS in adult is characterized by an acute onset of fever with subsequent progression to pneumonia Lee et al., 2003; Poutanen et al., 2003] , whereas infection in children usually presents as a mild upper respiratory tract illness [Hon et al., 2003] . The causative agent, SARS-associated coronavirus (SARS-CoV) is phylogenetically distinct from previous known human and animal coronaviruses [Marra et al., 2003; Rota et al., 2003] . SARS-CoV does not grow in cell lines used commonly for isolating respiratory viruses, such as Hep2, HeLa, Madin-Darby Canine kidney (MDCK) and LLC-MK2 ], but produce readily lytic infection in African Green monkey (Vero) cells Ng et al., 2003a,b] . While SARS-CoV is notorious for producing severe respiratory tract disease, a substantial proportion of patients had concurrent gastrointestinal symptoms Leung et al., 2003; Peiris et al., 2003b] . SARS-CoV was found in intestinal biopsy samples taken during the early phase of the illness . Studies on fatal cases also demonstrated that the gastrointestinal tract was the only extra-pulmonary site where viable viruses could be found .

The pathogenesis of SARS-associated diseases remained elusive. Although viable viruses could be found in lung and intestinal tissues, the pathology of the lung was different from those of the gastrointestinal tract Tse et al., 2004] . While the SARS-CoVinfected lung was dominated by a diffuse alveolar damage picture with giant cell and atypical pneumocyte formations, the gastrointestinal tract appeared intact and syncytium-forming giant cells were not seen .

At present, a human cell-derived culture model for SARS-CoV is not available. Here, we report our findings on searching for human intestinal cell lines that may represent a more appropriate model for studying the biology of SARS-CoV.

Seven human colorectal adenocarcinoma cell lines were investigated for their permissiveness to SARS-CoV infection. These cell lines were originally obtained from American Type Culture Collection (ATCC, Manassas, VA) and maintained with the recommended medium ( Table I) . The CUHK-W1 strain of SARS-CoV (GenBank accession no. AY278554) was grown in Vero cells and the third passage at a concentration of 5 Â 10 6 50% tissue culture infective doses (TCID 50 )/ml was kept at À708C for experiments.

Cell lines at 60-70% confluence in 25-cm 2 flasks were inoculated with 300 ml of virus suspension to provide a multiplicity of infection (MOI) of 10. Inoculated cell cultures were incubated at 378C. A mock infection was performed in parallel for each cell line. The cell monolayers were examined daily for cytopathic effects. Cells were harvested after seven days of incubation for virus detection.

Intracellular viral antigens were detected by indirect immunofluorescence staining based on a convalescent serum collected from a SARS patient as previously described [Chan et al., 2004a] . Briefly, cells were spotted onto a 12-well Teflon-coated glass microscope slides, and fixed with 100% pre-chilled acetone. A 25-ml aliquot of the 1:160 diluted convalescent serum was placed on the coated wells, and incubated at 378C for 30 min in a moist chamber. After washing, a fluorescein isothiocyanate (FITC)-conjugated rabbit anti-human IgG antibody (Dako, Glostrup, Denmark) was added at a dilution of 1:40, and incubated for 30 min at 378C. Epifluorescence microscopy was performed on a Zeiss Axoplan 2 microscope equipped with the appropriate sets of excitation and emission filters. Grey scale images were captured by a cooled charge-coupled device.

Intracellular viral RNA was detected by in situ hybridization based on a digoxigenin-labeled DNA probe targeting the putative membrane protein encoding region of SARS-CoV as previously described . Briefly, formalin-fixed cell block sections were treated with microwave heating in 0.01M sodium citrate buffer, and then fixed in 4% paraformaldehyde. Hybridization was carried out at high stringency with DNA probe at a concentration of 2 ng/ml in 15 ml of hybridization mix (50% formamide, 10% dextran sulphate in 2Â Saline Sodium Citrate [SSC]) at 428C for 16 hr under 20 Â 20 mm coverslips in a moist chamber. Excess probe was removed by washing in 2Â SSC for 5 min at room temperature. This was followed by three times high stringency washes in 0.1Â SSC for 10 min at 618C. Immunological detection was conducted with anti-digoxigenin-rhodamine (Roche, Indianapolis, IN) following the manufacturer's instruction, and counterstained with 4,6 diamindino-2-phenylindol (DAPI) in an anti-fade mountant (Vectorshield, Vector Laboratory, Burlingame, CA). Cell block preparations from SARS-CoV-infected and non-infected Vero cell culture were used as positive and negative controls, respectively. The signal specificity was assessed by digestion of the sections with RNase A prior to hybridization, and by omission of the probes in the hybridization mixture. 

Cell pellets harvested after 7 days of incubation were fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer (pH 7.2), and processed for examination by transmission electron microscopy.

Further experiments were undertaken on LoVo cells as they were the only cells found to be permissive to SARS-CoV. Cell culture flasks containing LoVo cells at 60-70% confluence were inoculated with SARS-CoV at 10, 1 and 0.1 MOI, respectively. The concentration of virus in cell culture supernatant was monitored by real time reverse transcription polymerase chain reaction (RT-PCR). Aliquots of 140 ml cell culture supernatant were collected daily for RNA extraction using the Qiagen RNA Mini Kit (Qiagen, Hilden, Germany). One-step real time quantitative RT-PCR was carried out using primers Pol-1 5 0 -AGC TCG CGT CTC AGT TTC AAG-3 0 and Pol-2 5 0 -TGC CAG AAG CTG CAT GCA-3 0 targeting the orf1ab polyprotein of SARS-CoV (nt 14442-14511 of strain CUHK-W1, GenBank accession no. AY278554); and TaqMan MGB probe 5 0 -(FAM)-GAT CAG CAG CAT ACA ACT-(MGBNFQ)-3 0 . The RT-PCR reactions were set up using the TaqMan 1 EZ RNA PCR reagent kit, (Applied Biosystems, Foster City, CA) following the manufacturer's protocol. Samples, reaction controls and standards were tested in duplicates. The thermal cycling parameters were optimized for the Sequence Detector 7700 (Applied Biosystems). The conditions were 508C for 2 min for the activity of uracil N-glycosylase (UNG), followed by reverse transcription at 608C for 30 min, inactivation of UNG at 958C for 5 min, then 40 cycles of 948C for 20 sec and 608C for 1 min.

The infected LoVo cell cultures were split at a ratio of 1:4 after every 7 days of incubation, and continued for a total of seven passages. The proportion of infected cells obtained at the end of each passage was assessed by immunofluorescence staining, and with the concentration of virus in culture supernatant assessed by real time RT-PCR.

The expression of angiotensin-converting enzyme 2 (ACE2) in these cell lines was examined as described previously [Harmer et al., 2002] . Briefly, total RNA was extracted using Qiagen RNeasy kit (Qiagen) according to the manufacturer's instructions. cDNA was synthesized from 2 mg of total RNA using Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA) with random primers. The expression level of ACE2 mRNA was determined by real time RT-PCR using the Taq-Man 1 EZ RNA PCR reagent kit (Applied Biosystems). Variations in input RNA amounts were compensated by measuring the RNA level of a housekeeping gene, betaactin. The sequences of the primers and probes were as follows: ACE2 forward primer: 5 0 -CAT TGG AGC AAG TGT TGG ATC TT-3 0 ; ACE2 reverse primer: 5 0 -GAG CTA ATG CAT GCC ATT CTC A-3 0 ; ACE2 probe: 5 0 -(FAM)-CTT GCA GCT ACA CCA GTT CCC AGG CA (TAMRA)-3 0 ; beta-actin forward primer: 5 0 -CTG GCA CCC AGC ACA ATG-3 0 ; beta-actin reverse primer: 5 0 -GCC GAT CCA CAC GGA GTA CT-3 0 ; beta-actin probe: 5 0 -(VIC)-TCA AGA TCA TTG CTC CTC CTG AGC GC-(TAMRA)-3 0 . The PCR and thermal cycling conditions were as described previously [Harmer et al., 2002] . The relative expression of ACE2 of different cell lines were analyzed using the comparative threshold cycle (C T ) method as detailed in the ABI PRISM 7700 Sequence Detection System User Bulletin Number 2. Briefly, the DC T for each sample was calculated by subtracting the average C T of beta-actin from the average C T of ACE2. The cell line with the lowest ACE2 expression level was used as a reference for calculating DDC T , which was obtained by subtracting DC T(reference) from DC T(sample) . The relative quantitation was then calculated by 2 ÀDDC T . The mRNA quantity for the reference cell line was expressed as 1.0, and all other quantities were expressed as a number of fold differences relative to the reference cell line.

Seven established cell lines of human intestinal origin, DLD-1, HCT-116, HT-29, LoVo, LS-180, SW-480 and SW-620, were investigated for their permissiveness to SARS-CoV infection. None of them showed cytopathic effects during the 7 days of incubation. Fluorescence in situ hybridization (FISH) and indirect immunofluorescence staining revealed the presence of SARS-CoV in LoVo cells, whereas all other cell lines were virus-free after 7 days of incubation. Viral genome was detected in the cytoplasm of infected LoVo cells by FISH using a probe specific to the putative M protein encoding region. For the FISH, positive signals appeared as red fluorescence as shown in Figure 1 . Perinuclear accentuation of positive signals was also observed. The nuclei of infected and non-infected LoVo cells appeared blue due to counter stain (Fig. 1) . Indirect immunofluorescence staining also indicated the presence of viral antigens in the cytoplasm of infected LoVo cells. SARS-CoV antigens were appeared as fluorescence green, whereas the background appeared red due to counter stain (Fig. 2) .

While no morphological changes of infected LoVo cells could be observed with light microscopy at magnification of 400Â, ultrastructural changes resulting from viral infection were found when examined by electron microscopy. The infected cells contained cytoplasmic vesicles, consistent with dilated endoplasmic reticulum and Golgi apparatus, which were packed with numerous spherical viral cores at different stages of maturation as shown in Figure 3A ,B. These cores contained both viral proteins and the genome of SARS-CoV. In addition, mature virus particles of sizes ranging from 80 to 120 nm were found on the outer surface of the plasma membrane ( Fig. 3C) . Thus, FISH, indirect immunofluorescence and ultrastructural studies confirmed the permissiveness of LoVo cells to SARS-CoV infection.

The concentration of viruses in the cell culture supernatants collected over the first week after virus inoculation is shown in Figure 4 . The production of virus increased exponentially after 2-3 days of virus inoculation, and appeared in a dose-dependent manner. The infected LoVo cells sustained a persistent infection for all the passages that we had performed. At the end of each passage, about 50-60% of cells were infected as revealed by immunofluorescence staining, and a virus concentration in the range of 2-9 Â 10 7 copies/ml was detected from the cell culture supernatant.

In view of the current evidence of ACE2 as the cellular receptor for SARS-CoV, we investigated the relationship between permissiveness to SARS-CoV infection and ACE2 expression in these colorectal adenocarcinoma cell lines. Table II shows the results of relative expressional level of ACE2 using real time RT-PCR. Surprisingly, apart from LoVo cells, ACE2 was also found to be expressed in other cell lines that were not permissive to SARS-CoV infection. Remarkably, the cell lines LS-180 and SW620 expressing high levels of ACE2 mRNA were found to be non-permissive to SARS-CoV infection.

Our results showed that the human colorectal adenocarcinoma-derived LoVo cell line was susceptible to SARS-CoV infection. SARS-CoV produced a persistent infection in LoVo cells, and the infection could be maintained after multiple passages. SARS-CoV did not produce observable cytopathic effects on LoVo cells at the microscopic level. This is in stark contrast to Vero cells, where SARS-CoV produced a lytic infection with characteristic refractile rounding cytopathic effects [Ng et al., 2003a,b] . The fact that SARS-CoV did not produce lytic infection in this intestinal cell line is reminiscence of what we have observed from the endoscopic intestinal biopsies obtained from a SARS patient , and in fatal cases of SARS . Although viral particles inside the ileal and colonic epithelial cells were revealed by electron microscopy, the SARS-CoV-infected ileal and colonic mucosa appeared normal at microscopic level and no viral inclusion bodies could be detected. Multinucleated pneumocytes were observed in SARS-CoV-infected lungs in human and monkey models Kuiken et al., 2003; To et al., 2004] , and formation of multinucleated syncytia was also observed in human fibroblast 293T cells rendered permissive by ACE2 transfection . However, multinucleated syncytium was not observed in SARS-CoV-infected LoVo cells. This observation accords the histopathological findings in intestinal biopsies and autopsy specimens obtained from SARS patients To et al., 2004] .

The current finding of persistent infection in colonic cells in vitro is in line with the clinical observation of prolonged shedding of SARS-CoV in stool samples. It has been reported that shedding of SARS-CoV in stool for 3-4 weeks after the onset of illness was common, and prolonged shedding for more than 2 months occurred in some patients [Chan et al., 2004b] . The use of a non-lytic productive cycle by the virus in cultured intestinal cell lines may provide a biological basis for these clinical observations.

Our data from the previous outbreak showed that only 30% of RT-PCR-positive nasophayngeal/tracheal aspirate samples, and <1% of RT-PCR-positive stool samples had viruses isolated by Vero E6 cells [Chan et al., 2004b] . The reasons for the poor sensitivity of Vero E6 cells in detecting SARS-CoV are not entirely clear. On one hand, cell culture-based assay requires intact and functional viral particles while nucleic acid-based method requires only a partial viral genome. On the other hand, species and tissue type differences may play a significant role in the overall sensitivity of the cell culture-based method. Our results show that LoVo cells can serve as an alternative human cell line for virus isolation. It is worthwhile to investigate whether LoVo cells could provide a more sensitive system for isolating SARS-CoV from clinical samples. LoVo cells, being an immortalized adenocarcinoma cell line, also have the advantages of fast doubling time and requiring relatively simple culture media to maintain. In addition, LoVo cells being originated from human, appears to recapture, at least partially, the biological consequences of the intestinal epithelium following infection with SARS CoV. The LoVo cells could potentially be a useful model for the in vitro study of the pathogenesis of SARS-CoV-related diseases.

Recently, ACE2 has been shown to be the cellular receptor for SARS-CoV in Vero E6 cells Wong et al., 2004] . Apart from the cardiovascular system and kidney, intestinal tissues were also shown to express high levels of ACE2 [Harmer et al., 2002] . Our results indicated that ACE2 was expressed in all the seven adenocarcinoma cell lines investigated. The ACE2 expression level of LoVo cells was intermediate as compared to others (Table II) . However, only the LoVo cells were found to be permissive to SARS-CoV infection. These results indicate that ACE2, by itself, is insufficient to explain the infectivity of SARS-CoV in intestinal cells. Hence, other cellular factors may play a role in completing the full life cycle of SARS-CoV. The possible factors that determine the susceptibility of different cell types to SARS-CoV infection deserve further investigations.

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