【病毒外文文献】2004 Tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome_ an _em_in-situ__e

Journal of Pathology J Pathol 2004 202 157 163 Published online in Wiley InterScience DOI 10 1002 path 1510 Original Paper Tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome an in situ hybridization study of fatal cases KF To 1 Joanna HM Tong 1 Paul KS Chan 2 Florence WL Au 1 Stephen SC Chim 3 KC Allen Chan 3 Jo LK Cheung 2 Esther YM Liu 2 Gary MK Tse 1 Anthony WI Lo 1 YM Dennis Lo 3 and HK Ng 1 1 Department of Anatomical and Cellular Pathology The Chinese University of Hong Kong Hong Kong SAR China 2 Department of Microbiology Prince of Wales Hospital The Chinese University of Hong Kong Hong Kong SAR China 3 Department of Chemical Pathology The Chinese University of Hong Kong Hong Kong SAR China Correspondence to Professor KF To Department of Anatomical and Cellular Pathology The Chinese University of Hong Kong Prince of Wales Hospital 30 32 Ngan Shing Street Shatin NT Hong Kong SAR China E mail kfto cuhk edu hk Received 21 July 2003 Revised 6 September 2003 Accepted 28 October 2003 Abstract Severe acute respiratory syndrome SARS is a new human infectious disease with significant morbidity and mortality The disease has been shown to be associated with a new coronavirus SARS CoV The clinical and epidemiological aspects of SARS have been described Moreover the viral genome of SARS CoV has been fully sequenced However much of the biological behaviour of the virus is not known and data on the tissue and cellular tropism of SARS CoV are limited In this study six fatal cases of SARS were investigated for the tissue and cellular tropism of SARS CoV using an in situ hybridization ISH technique Among all the tissues studied positive signals were seen in pneumocytes in the lungs and surface enterocytes in the small bowel Infected pneumocytes were further confirmed by immunofluorescence fluorescence in situ hybridization FISH analysis These results provide important information concerning the tissue tropism of SARS CoV which is distinct from previously identified human coronaviruses and suggest the possible involvement of novel receptors in this infection Whereas the lung pathology was dominated by diffuse alveolar damage the gut was relatively intact These findings indicated that tissue responses to SARS CoV infection are distinct in different organs Copyright 2004 John Wiley coronavirus viral tropism Introduction Severe acute respiratory syndrome SARS is an emerging human disease with significant morbid ity and mortality The worldwide outbreak affected more than 8000 patients from over 30 countries with an associated cumulative mortality of 9 2 1 A new coronavirus SARS CoV has been aetiologi cally linked to SARS 2 4 The supporting evi dence includes serological studies detection of the viral genome by polymerase chain reaction PCR based molecular methods and viral isolation from respiratory specimens 2 5 Although a highly sensi tive diagnostic test is not available and SARS CoV is not detected in every clinically defined case 5 monkey inoculation experiments reproduce a sim ilar disease in the primate to that in the human and further strengthen the aetiological role of SARS CoV 6 Much has been documented on the clini cal and epidemiological characteristics of SARS Epi demiological modelling shows that SARS CoV is highly contagious However much of the biologi cal behaviour of the virus is not known and data on the tissue and cellular tropism of SARS CoV are limited The known human coronaviruses types 229E and OC43 generally cause the common cold These viruses have only rarely been associated with more severe respiratory diseases such as pneumonia in neonates the elderly or immunocompromised patients 7 9 However SARS is a lower respiratory tract disease Upper respiratory tract symptoms are uncom mon and mild 10 Moreover the genomic sequence of SARS CoV is distinct from other coronaviruses including the two known human pathogenic coro naviruses 229E and OC43 11 12 On the basis of ultrastructural findings SARS CoV appears to infect lung pneumocytes 13 14 However extra pulmonary manifestations including diarrhoea lym phopaenia impaired liver function and elevation of non cardiac creatine kinase are common and suggest that the virus may have tropism for non pulmonary tissues 10 Thus the tissue and cellular tropism of SARS CoV is likely to be distinct from that of the known human coronaviruses At present com prehensive documentation of the various cell types and organs infected by SARS CoV is lacking In this report we investigate the tissue and cellular tropism of SARS CoV in fatal SARS cases by in situ hybridization Copyright 2004 John Wiley RNase A was pretreated by boiling at 70 C for 10 min to remove possible DNase contaminants for 2 h at 37 C before the hybridization and 2 omission of the probes in the hybridization mixture In addition tissue sec tions form three autopsy cases of non SARS patients collected during the SARS outbreak in our hospital were included as negative controls All three of these cases were negative for viral isolation and serology for SARS Tissues sections of various organs includ ing lung intestine heart liver kidney spleen lymph nodes bone marrow and muscles were studied Immunofluorescene fluorescence in situ hybridization FISH studies Cytokeratin AE1 AE3 Dako 1 50 was used as an epithelial marker and CD68 Dako 1 1000 was used as a macrophage marker Double immunofluo rescence FISH was performed as for ISH with the additional immunofluorescence performed by incuba tion of primary antibodies overnight before detec tion of signals with the appropriate fluorescent sec ondary antibodies ISH signals were detected using anti digoxigenin rhodamine Roche The secondary antibody for immunofluorescence was generally anti mouse fluorescein thiocyanate FITC Nuclei were counterstained by 4 6 diamidino 2 phenylindole DAPI in anti fade mountant Vectorshield Vec tor Laboratories A hundred infected cells FISH positive in the alveolar spaces were counted in each immunofluorescence FISH experiment Epiflu orescence microscopy was performed on a Zeiss Axo plan 2 microscope equipped with the appropriate sets Copyright 2004 John Wiley 202 157 163 Tropisms of coronavirus SARS CoV an ISH study 159 of excitation and emission filters Images were cap tured by a cooled charge coupled device as grey scale images Results In Vero cell cultures infected by SARS CoV ISH and FISH using the RT PCR product of the membrane protein gene M revealed strong signals in the cyto plasm Figure 1A This FISH signal is specific for infected cells Similar cytoplasmic signals and aut ofluorescence were not detected in uninfected Vero cell cultures Figure 1B These results correlated with electron microscopic findings in which viral particles were mainly observed in the cytoplasm of infected cells 2 4 13 We next performed ISH for SARS CoV on all avail able tissue sections obtained from autopsies of fatal cases of SARS and the control non SARS cases The tissue sections from various organs of non SARS patients were all negative for ISH indicat ing its specificity Positive ISH signals were detected in those samples only when SARS CoV was iso lated by virological cell culture in SARS patients Lung sections from three cases and small intesti nal sections from four cases were all positive for viral culture These samples were all positive for ISH Table 1 Other tissue samples including the heart liver spleen kidney lymph nodes bone mar row and muscles were all culture negative and they were all found to be ISH negative Table 1 The tra chea samples were also negative for ISH The results of FISH in all cases were the same as those of ISH Hence the lung and the intestine were the only two organs where SARS CoV was explicitly identi fied On close examination of the lung positive cyto plasmic signals were seen in pneumocytes lining the alveolar septa Figure 2A and within the alveo lar spaces Figure 2B No signals were detected in Figure 1 Fluorescence in situ hybridization FISH of SARS CoV in infected A and non infected B Vero cells The Vero cells are indicated by arrows The composite picture is shown in i Images captured under the red green and blue filters are shown as grey scale images in ii iii and iv respectively FISH signals of SARS CoV were detected in the cytoplasm of infected but not in uninfected Vero cells ii No bleaching of filters or autofluorescence was detected iii The nuclei were counterstained with DAPI iv Figure 2 In situ hybridization ISH of SARS CoV in the lung A Infected pneumocytes arrow with positive cytoplasmic signals are seen lining the alveolar septa The other cellular components in the alveolar septa and histiocytes arrow head are negative 400 B Scattered infected cells show a diffuse cytoplasmic distribution of the viral genome in infected cells indicated by arrows 400 The infected cells as suggested by the immunofluorescence FISH study Figure 3 were pneumocytes C Control non SARS lung shows no specific ISH signals 400 Copyright 2004 John Wiley 202 157 163 160 KF To et al Table 1 Viral isolation and ISH results in autopsy samples of fatal cases of SARS Tissue Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Lung Small intestine NA Heart Liver Spleen Kidney Bone marrow NA NA NA NA Lymph node NA NA NA NA indicates positive by viral isolation positive by ISH indicates negative by viral isolation negative by ISH NA indicates not available for viral isolation negative by ISH the non SARS lung control Figure 2C The viral genome as revealed by ISH either was present dif fusely throughout the cytoplasm of the infected cells or formed aggregates within the cytoplasm In the lungs of fatal cases multinucleate cells are one of the characteristic features of SARS Detectable sig nals however were not seen in these cells Detached cells in the alveoli might be detached pneumocytes or macrophages The nature of the viral RNA positive detached cells in the alveolar spaces was ascer tained by immunofluorescence FISH with cytoker atin AE1 AE3 Figures 3A and 3B and macrophage CD68 markers Figure 3D Among 100 infected cells in alveolar spaces counted in each immunoflu orescence FISH experiment all were cytokeratin positive and none were CD68 positive Other cellular components in the lung including bronchial epithelial cells endothelial cells fibroblasts and lymphocytes were all negative No probe controls with immunoflu orescence for cytokeratin or macrophage markers Figures 3C and 3E respectively indicated that the FISH signals seen were specific Background fluores cence was limited to red blood cells and some connec tive tissue components The cytoplasm of the detached pneumocytes and macrophages demonstrated minimal background signals In the small intestine positive cytoplasmic signals of SARS CoV ISH were detected in the surface ente rocytes but not in the other cells Figures 4Ai and 4Aii The signals appeared concentrated at the api cal regions of the enterocytes No detectable ISH signals were seen in non SARS small intestinal con trol Figures 4Bi and 4Bii FISH showed the same findings as ISH in the small intestinal sections Double immunofluorescence FISH with cytokeratin AE1 AE3 further confirmed that the SARS CoV positive cells were enterocytes Figure 5A arrows The no probe control Figure 5B again indicated that autofluorescence signals were minimal in the detached surface enterocytes arrows It is important to note that the tissue responses in the intestines were quite different from those in the lungs Apart from autolytic changes no gross or histological abnormality was apparent in the small intestine of any of these fatal cases of SARS In particular inflammatory processes were not detected Discussion We have studied the distribution of SARS CoV in fatal cases of SARS using in situ hybridization Among all of the tissues available only the lungs and the small intestines were positive Cell types infected were limited to pneumocytes and enterocytes in these two organs respectively In this study the results of the ISH hybridization study were concordant with viral isolation results in our autopsy series Despite anti viral treatment in most of these cases persistent infection was detected in the lungs and small intestines in these fatal cases The identification of SARS CoV RNA in the pneu mocytes is consistent with the reported ultrastruc tural findings 13 14 As SARS is a lower respi ratory tract disease one may expect to see SARS CoV infecting pneumocytes This is in contrast to the other two known pathogenic coronaviruses types 229E and OC43 These two viruses produce the com mon cold and are expected to affect the upper res piratory tract However apart from trachea samples other upper respiratory tract tissues were not avail able from these SARS victims in our series for further study Macrophages are commonly seen in lung tissues from SARS patients and it is reasonable to suggest that they might play an important role in the patho genesis of pulmonary injury However it is not known whether these macrophages are infected by the SARS CoV Our study clearly demonstrates that at least in fatal cases persistent SARS CoV infection is demon strable in pneumocytes but not in macrophages or other cell types in lung tissues This result is con sistent with the report of the haematological manifes tations in SARS patients in which direct infection of SARS CoV in haemato lymphoid tissues has not been documented 17 The presence of viral RNA in the surface entero cytes is intriguing Data from recent outbreaks indicate that concurrent gastrointestinal symptoms especially diarrhoea are common 10 18 19 Human coronavirus or coronavirus like particles CVLPs have been sug gested to be the aetiological agent of diarrhoea in susceptible subjects including patients with acquired immunodeficiency syndrome and in children from Copyright 2004 John Wiley 202 157 163 Tropisms of coronavirus SARS CoV an ISH study 161 Figure 3 Immunofluorescence FISH with cytokeratin AE1 AE3 and macrophage CD68 markers in lung tissue The panels show i the composite picture ii FISH signals of SARS CoV in the red channel iii immunofluorescence in the green channel and iv counterstaining of the nuclei in the blue channel Panels ii iv are shown as grey scale captured images A Low power view of the lung showing a SARS CoV infected pneumocyte double arrow among other uninfected pneumocytes arrows 400 Pneumocytes were highlighted by immunofluorescence using a cytokeratin marker AE1 AE3 B High power view of A showing clearly the cytoplasmic signals red and in ii in the infected pneumocytes double arrows 1000 Among 100 infected cells double arrow counted in each positive case all were pneumocytes C High power view of the no probe control in the same lung specimen Arrows indicate some of the detached pneumocytes that stained positive for cytokeratin with minimal cytoplasmic signals when viewed under the red filter The oval background red signals represent autofluorescence from red blood cells D Macrophages were seen among the floating cells and were recognized by their CD68 immunoreactivity arrows Among 100 infected cells double arrow counted in each positive case none were macrophages 400 E High power view of the no probe control in the same lung specimen Arrows indicate macrophages in the alveolar space No autofluorescence signal is seen in these cells developing countries 20 21 However enteric tissue tropism of SARS CoV has not been documented Our results indicate that the surface enterocytes are infected by the SARS CoV but in contrast to the lungs no evidence of tissue destruction or inflammatory pro cesses was seen in association with viral infection in the intestines The mechanism underlying diarrhoea in SARS remains unclear However our observations suggest that the underlying pathology might well be in the small intestine and that active viral replica tion within intestinal cells rather than an inflammatory lesion might be responsible for the pathophysiology of the SARS related diarrhoea Nevertheless our study indicates that tissue responses to SARS CoV infection are distinct in different organs Although brain tissues and samples during the early phase of SARS were not available to assess com prehensively the temporal tissue tropism and neu rotropism of the virus our study does not support persistent viral infection in other organs in these fatal cases Thus it is possible that the extra pulmonary manifestations may be related to systemic effects Copyright 2004 John Wiley 202 157 163 162 KF To et al Figure 4 In situ hybridization ISH of SARS CoV in the small intestine The panels show i a low power view 40 and ii a high power view 400 A Despite the autolytic changes positive signals were seen and mainly detected in the surface enterocytes arrows High power view showing the positive cytoplasmic signals detected in the partially detached surface enterocytes The positive signals appear polarized towards the apical part of the enterocytes B Control non SARS intestine section reveals no specific ISH signals High power view shows no cytoplasmic signals in the detached surface enterocytes Figure5 Immunofluorescence FISH with cytokeratin AE1 AE3 in the small intestine The panels show i the composite picture ii FISH signals of SARS CoV in the red channel iii immunofluorescence in the green channel and iv counterstaining of the nuclei in the blue channel Panels ii iv are shown as grey scale captured images A High power view of the small intestine showing SARS CoV infected surface enterocytes arrows that have become detached 1000 Surface enterocytes are highlighted by immunofluorescence using the cytokeratin marker AE1 AE3 B High power view of the no probe control showing minimal autofluorescence signals in the detached enterocytes arrows of the abnormal inflammatory or immune reactions towards SARS CoV Multiple factors contribute to tissues and cellular tropism On the basis of genomic data SARS CoV is distinct from other coronaviruses including the two known human pathogenic coronaviruses 229E and OC43 11 12 Emerging data suggest that differences in the spike protein of the virus may be related to tissue tropism of bovine coronavirus 22 The host cell surface proteins are also important The membrane protein aminopeptidase N CD13 acts as a receptor for human coronavirus 229E and mediates its tissue Copyright 2004 John Wiley 202 157 163 Tropisms of coronavirus SARS CoV an ISH study 163 tropism 23 CD13 is expressed on various polarized epithelial cells including surface enterocytes but not pneumocytes It is also interesting to note that the macrophages in the lungs in these cases also expressed CD13 KFT unpublished data However even in areas with virus positive pneumocytes none of the adjacent macrophages were infected The surface receptor for SARS CoV is currently unknown but our data suggest that CD13 is probably not the receptor for SARS CoV In conclusion we have demonstrated SARS CoV tropism towards lung pneumocytes and intestinal sur face enterocytes in fatal cases of SARS The results contribute to our understanding of the pathology of this new human infectious disease The distinct tis sues and cellular tropism highlight the novel biology of SARS CoV which may require a distinct set of receptor proteins for its tropism Further studies of these receptors may have an important impact on the development of drugs for the treatment of this new human disease References 1 World Health Organization Severe acute respiratory syndrome SARS Multi countries outbreak update 73 http www who int csr don 2003 06 04 en Access date 4 June 2003 2 Peiris JS Lai ST Poon LL et al Coronavirus as a possible cause of severe acute respiratory syndrome Lancet 2003 361 1319 1325 3 Drosten C Gunther S Preiser W et al Identification of a novel coronavirus in patients with severe acute respiratory syndrome N Engl J Med 2003 348 1967 1976 4 Ksiazek TG Erdman D Goldsmith CS et al A novel coronavirus associated with