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JOURNAL OF VIROLOGY Apr 2011 p 4025 4030 Vol 85 No 8 0022 538X 11 12 00 doi 10 1128 JVI 02292 10 Copyright 2011 American Society for Microbiology All Rights Reserved Epithelial Cells Lining Salivary Gland Ducts Are Early Target Cells of Severe Acute Respiratory Syndrome Coronavirus Infection in the Upper Respiratory Tracts of Rhesus Macaques H17188 Li Liu 1 Qiang Wei 2 Xavier Alvarez 3 Haibo Wang 1 Yanhua Du 1 Hua Zhu 2 Hong Jiang 2 Jingying Zhou 1 Pokman Lam 1 Linqi Zhang 4 Andrew Lackner 3 Chuan Qin 2 and Zhiwei Chen 1 5 AIDS Institute Li Ka Shing Faculty of Medicine The University of Hong Kong Hong Kong SAR People s Republic of China 1 Institute of Laboratory Animal Science Chinese Academy of Medical Sciences CAMS and Peking Union Medical College PUMC No 5 Panjiayuan Nanli Chaoyang District Beijing People s Republic of China 2 Division of Comparative Pathology Tulane National Primate Research Center Covington Louisiana 3 Comprehensive AIDS Research Center Tsinghua University Beijing People s Republic of China 4 and Department of Microbiology and Research Center for Infection and Immunology Li Ka Shing Faculty of Medicine The University of Hong Kong Hong Kong SAR People s Republic of China 5 Received 3 November 2010 Accepted 26 January 2011 The shedding of severe acute respiratory syndrome coronavirus SARS CoV into saliva droplets plays a critical role in viral transmission The source of high viral loads in saliva however remains elusive Here we investigate the early target cells of infection in the entire array of respiratory tissues in Chinese macaques after intranasal inoculations with a single cycle pseudotyped virus and a pathogenic SARS CoV We found that angiotensin converting enzyme 2 positive ACE2 H11545 cells were widely distributed in the upper respiratory tract and ACE2 H11545 epithelial cells lining salivary gland ducts were the early target cells productively infected Our findings also have implications for SARS CoV early diagnosis and prevention Severe acute respiratory syndrome SARS is a recently emerged human infectious disease caused by a zoonotic coro navirus SARS CoV that has a mortality rate of approxi mately 10 11 20 24 30 Although it is known that SARS CoV is transmitted via saliva droplets the source of high viral loads up to 6 38 H11003 10 8 copies ml in patients saliva remains elusive especially during the acute phase of viral replication 41 We hypothesized that viral replication in the upper res piratory tract may contribute to the rapid viral shedding into saliva droplets To address this hypothesis it is necessary to investigate SARS CoV infection at the site of viral entry in cluding the identification of early target cells Our current understandings of the target cells for SARS CoV are largely based on autopsy specimens of patients who died of SARS Multiple cell types in the lower respiratory tract were found to be infected including type I alveolar epithelium macrophages and putative CD34 H11001 Oct 4 H11001 stem progenitor cells in human lungs 2 7 10 13 28 29 These results while very informative do not address the question of which cell types support the initial seeding of infection in the upper respiratory tract Since the SARS outbreak in humans has subsided and because many of the current scientific questions are difficult to address in humans several animal models have been developed Animal models that have been used to study SARS CoV infection include mice hamsters ferrets cats and nonhuman primates cynomolgus macaques rhesus macaques common marmosets and African green monkeys 1 12 14 22 26 27 32 33 35 Although none of these animal models reproduce lethal SARS most support SARS CoV infection to some ex tent and therefore have contributed greatly to attempts to develop vaccines and therapeutics 1 9 14 19 43 We have recently demonstrated that rhesus angiotensin converting en zyme 2 ACE2 the primary receptor of SARS CoV supports viral entry as efficiently as does its human homologue 3 23 Moreover Chinese origin rhesus macaques Macaca mulatta experimentally infected with a pathogenic SARS CoV devel oped lung damage similar to that seen in humans with SARS 31 The Chinese rhesus macaque model therefore offers us an opportunity to study the early target cells of SARS CoV in the respiratory tract To allow careful examination of the initial targets of infec tion we developed a single cycle reporter virus pseudotyped with a functional Opt9 or a nonfunctional SH9004 422 463 spike S glycoprotein of SARS CoV 5 45 As previously described by others and us the commonly used pseudoviruses were generated by cotransfection of HEK293T cells with hu man immunodeficiency virus HIV Luc H11001 Env H11002 Vpr H11002 back bone and S glycoprotein expression plasmids 18 25 45 46 These pseudoviruses however are not feasible for monkey experiments due to host factors e g Trim 5H9251 which restrict HIV based pseudovirus in simian cells 38 We have there fore developed SIVmac Luc H11001 Env H11002 Vpr H11002 as the backbone vec Corresponding author Mailing address for Chuan Qin Institute of Laboratory Animal Science 5 Panjiayuan Nanli Chaoyang District Beijing 100021 People s Republic of China Phone 86 10 67776529 Fax 86 10 67710812 E mail qinchuan Mailing address for Zhiwei Chen AIDS Institute Li Ka Shing Faculty of Medicine The University of Hong Kong 21 Sassoon Road Pokfulam Hong Kong SAR People s Republic of China Phone 852 28199831 Fax 852 28177805 E mail zchenai hku hk The first two authors contributed equally to this work H17188 Published ahead of print on 2 February 2011 4025 on April 13 2015 by NYU MEDICAL CENTER LIBRARY http jvi asm org Downloaded from tor which we constructed by placing the luciferase gene in place of the viral nef gene Fig 1A to generate the functional pseudovirus simian immunodeficiency virus SIV OPT9 4 6 The nonfunctional envelope SH9004 422 463 was included to generate a control pseudovirus SIV SH9004 422 463 45 Pseu doviral stocks were quantified by SIV p27 enzyme linked im munosorbent assay ELISA and tested for their infectivity in HEK293T human ACE2 huACE2 cells or HEK293T cells transfected with Chinese macaque ACE2 cmACE2 3 45 As shown in Fig 1B SIV OPT9 pseudovirus infected effi ciently both HEK293T huACE2 cells and HEK293T cmACE2 cells As expected the control SIV SH9004 422 463 failed to infect either cell type Fig 1B Moreover cells incubated with SIV OPT9 showed strong cytoplasmic expression of luciferase around 48 h postinfection Fig 1C whereas no positive sig nals were found in cells incubated with SIV SH9004 422 463 or in uninfected cells Fig 1C These results confirmed that the detection of reporter luciferase expression is a specific indica tor of SIV OPT9 infection Since luciferase expression peaks around 48 h postinfection both in vitro and in vivo we chose this time point for our monkey studies 17 To identify the early target cells of SARS CoV in vivo seven Chinese macaques were included in this experiment and all were inoculated via the intranasal route three with pseudovi rus SIV OPT9 two with SIV SH9004 422 463 and two with cell culture medium A high dose of each pseudovirus approxi mately 400 ng p27 ml which contains about 10 9 RNA copies of virus was administered to each animal Animals were sacri ficed 48 h after inoculation and tissue specimens were col lected for analysis from throughout the respiratory tract in cluding the nasal cavity nasopharynx oropharynx epiglottis larynx trachea left bronchus right bronchus and lungs left and right cranial middle and caudal lobes and right accessory lobe The animal protocols were approved by our institutional animal welfare committee It is known that ACE2 is the functional receptor of SARS CoV and plays a crucial role in SARS lung pathogenesis 21 23 Although ACE2 mRNA and protein have been found in FIG 1 Construction and characterization of single cycle pseudoviruses SIV OPT9 and SIV SH9004 422 463 A Genomic organizations of SIVmac Luc H11001 Env H11002 Vpr H11002 OPT9 and SH9004 422 4630 are shown Pseudoviruses SIV OPT9 and SIV SH9004 422 463 were generated by cotransfection of HEK293T cells with SIVmac Luc H11001 Env H11002 Vpr H11002 and a full length SARS CoV Spike gene OPT9 or SH9004 422 463 45 LTR long terminal repeat CMV cytomegalovirus promoter B Entry assay of SIV OPT9 and SIV SH9004 422 463 pseudotyped viruses Five nanograms measured by p27 of each pseudovirus was used to infect transfected HEK293 huACE2 and HEK293 cmACE2 cells Luciferase activity was measured 48 h postinfection using a commercial kit Promega Madison WI Triplicates were tested in each experiment The average values and standard error bars are presented The experiment was repeated three times with similar results being obtained each time C Immunohistological detection of luciferase expression in pseudovirus infected cells Both HEK293T huACE2 and HEK293T cmACE2 cells infected with SIV OPT9 show a strong cytoplasmic expression of luciferase panels 1 and 4 whereas no positive signals were found in cells incubated with SIV SH9004 422 463 panels 2 and 5 and in uninfected cells panels 3 and 6 4026 NOTES J VIROL on April 13 2015 by NYU MEDICAL CENTER LIBRARY http jvi asm org Downloaded from multiple human organs the protein distribution throughout the respiratory tract has not been comprehensively investigated 15 16 To address this question and to identify the possible route of viral infection we investigated the localization and distribution of ACE2 H11001 cells in the entire respiratory tract of Chinese rhesus macaques By immunohistochemical IHC analysis ACE2 H11001 cells were found to be abundantly present throughout the respiratory tract Fig 2 panels 1 and 2 Sim ilar to the human situation numerous ACE2 positive cells were observed in the epithelial cells lining the respiratory tract and in the lamina propria as well as the cells morphologically compatible with salivary gland duct epithelium Control rabbit antibodies did not result in any positive signals in consecutive tissue sections Fig 2 panel 3 The finding of ACE2 H11001 cells in salivary gland duct epithelium has not been described previ ously probably due to the lack of examination of the relevant tissues 15 These findings suggest that target cells for SARS CoV may be more widespread in the respiratory tract than previously recognized particularly early in infection 15 To determine early target cells of SARS CoV we used a triple immunofluorescence staining technique to detect the luciferase protein Luc H11001 and various cell type specific mark ers Infected cells were identified in all three macaques 3 3 who received SIV OPT9 In contrast positive cells were not identified in any tissues of the four control macaques 4 4 who received either SIV SH9004 422 463 or placebo Luciferase sig FIG 2 Detection of ACE2 H11001 cells by immunohistochemistry at multiple levels of the respiratory tracts of rhesus macaques Positive labeling for ACE2 was found at all levels of the respiratory tract For each level examined a low magnification overview is shown in panel 1 with a higher magnification of the boxed area in panel 1 shown in panel 2 Panel 3 shows the same tissue immunolabeled with control rabbit serum The magnifications of each image are as follows H1100340 A1 H11003100 A3 B1 B3 C1 C3 D1 D3 E1 and E3 H11003200 A2 F1 F3 G1 and G3 and H11003400 B2 C2 D2 E2 F2 and G2 In nasopharynx A and oropharynx B ACE2 H11001 cells were identified in lymphatic nodules black arrow A1 vascular endothelium yellow arrow A1 the epithelium of salivary gland ducts yellow arrow A2 some muscle cells yellow arrow B1 epithelium lining yellow arrows B2 lamina propria black arrows B2 and basal layer of the epidermis red arrow B2 In epiglottis C ACE2 H11001 cells were found in lamina propria black arrow panel 1 the epithelial lining yellow arrow panel 2 and capillaries black arrow panel 2 In larynx D ACE2 H11001 cells were observed in glands red arrow panel 1 the epithelial lining yellow arrow panel 2 and lamina propria black arrows panel 2 In trachea E numerous ACE2 H11001 cells were found in the epithelial lining yellow arrow panel 2 and lamina propria black arrow panel 2 In bronchus F many ACE2 H11001 cells were identified in lamina propria black arrows panel 1 and in the epithelial lining yellow arrows panel 2 In lungs G ACE2 H11001 cells were observed on type I pneumocytes yellow arrow panel 2 and type II pneumocytes black arrow panel 2 VOL 85 2011 NOTES 4027 on April 13 2015 by NYU MEDICAL CENTER LIBRARY http jvi asm org Downloaded from nals were not detected in ACE2 H11001 cells in epithelial layers of the nasal cavity nasopharynx epiglottis larynx and trachea However two of three SIV OPT9 infected macaques dis played many infected cells in the epithelial layers of the sali vary gland ducts in laryngopharynx Fig 3A Positive cells were not found in other tissue sections examined By testing various cell surface markers these positive cells appeared to be Luc H11001 ACE2 H11001 cytokeratin H11001 Fig 3A Infected cells appeared to have weaker ACE2 staining Fig 3A panel 2b which was probably due to infection induced partial ACE2 downregula tion 21 To confirm this finding in situ hybridization ISH was performed on all tissue sections to detect SIV genome with digoxigenin 11 dUTP labeled RNA probe as previously de scribed 42 Consistent with the results of the immunofluo rescence microscopy data labeled cells were found almost exclusively in the epithelium of the salivary gland ducts in the laryngopharynx Fig 3C We did not detect positive cells in the laryngopharynx of the third monkey who received SIV OPT9 however the tissues collected were lacking salivary gland ducts Interestingly this animal displayed cytoplasmic staining of luciferase in sections of 3 lobes of the lungs right cranial lobe middle lobe and left caudal lobe We found that most of the Luc H11001 cells also were ACE2 H11001 in the lungs with colocalization of cytokeratin on some of the cells Fig 3B suggesting that the pulmonary epithelium is also a major early target of the virus This finding is consistent with previous studies on human fatal cases 13 28 29 39 40 44 Our results are significant for understanding early events of SARS CoV infection However the use of SIV based pseu dovirus can be a limitation of the study To address this issue another four Chinese macaques were challenged with a live pathogenic SARS CoV as previously described 5 Tissue sec tions of laryngopharynx trachea left bronchus right bronchus and lung were stained with a commercial rabbit polyclonal antibody against SARS CoV N protein 48 h post viral chal lenge The reason that we chose 48 h was mainly based on the natural course of SARS CoV infection in Chinese macaques We and others previously showed that it takes 48 h to easily detect SARS CoV proteins 31 which is consistent with other animal model studies 8 34 36 37 Moreover this time point was chosen to match the 48 h time point used with the pseu dotyped virus to show that the results were not an artifact of the construct Like SIV OPT9 infected animals epithelial cells of the salivary gland ducts and lungs were infected by SARS CoV in three 3 4 Rh0505 Rh0506 and Rh0507 and two 2 4 Rh0506 and Rh0508 of the four macaques respectively Fig 4A and B The tissue sections collected from the fourth monkey were lacking salivary gland ducts Moreover a few sporadic positive signals were found in the epithelial layers of the trachea and bronchus in one of four infected animals 1 4 Rh0505 Fig 4C which was in contrast to a large number of ACE2 H11001 cells identified at these locations Fig 2E and F This finding suggested that these sites were not yet significantly infected by SARS CoV probably due to the resistance of ep ithelial and mucus barriers at the time of examination Impor tantly SARS CoV was readily detected in oral swabs but not in the blood of each of these infected macaques 4 4 on day 2 after intranasal challenges Fig 4D These data are consistent FIG 3 ACE2 H11001 epithelial cells lining salivary gland ducts are early target cells of SARS CoV infection A and B Triple immunofluorescence labeling of luciferase green as indicated by yellow arrows in A1 and B1 ACE2 red in A1 A2b A3b B2a and B3 cytokeratin AE1 AE3 red in A2a A3a and B2c and cell nuclei blue on frozen sections of the laryngopharynx A and lung B respectively The magnifications of each image are as follows H1100340 A1 and B1 and H11003400 A2a A2b A3a A3b and A3c The numbered insets correspond to the enlarged images in panels A and B The triple labels involved a fluorescein isothiocyanate conjugated goat antiluciferase antibody Rockland Immunochemicals Inc Gilbertsville PA a rabbit anti human ACE2 antibody or control rabbit serum and a mouse anti human cytokeratin AE1 AE3 or control mouse serum DakoCytomation Inc Carpinteria CA The secondary antibodies used to detect ACE2 or AE1 AE3 included an Alexa 568 conjugated goat anti rabbit IgG antibody or an Alexa 647 conjugated goat anti mouse IgG antibody Molecular Probes Inc Eugene OR Triple label deconvolved images of the same tissue sections were collected using a DeltaVision deconvolution microscope Applied Precision Mercer Island WA Luciferase labeling was detected with fluorescein isothiocyanate filters as depicted in green A and B ACE2 was detected with the Alexa Fluor 568 filter as depicted in red A2b A3b B2a and B3 Cytokeratin AE1 AE3 was detected with the Cy5 filter as depicted in red A2a A3a and B2c Hoechst stained nuclei were detected with a 4H11032 6 diamidino 2 phenylindole filter as depicted in blue A and B Sections from control animals or stained with control serum show no staining of luciferase labeling B3 C ISH for the detection of SIV OPT9 infected cells panel 1 Numerous positive cells with blue black labeling are present in the ductal epithelium magnification H11003200 indicated by red arrows using a method previously described 42 Control sections show no staining panel 2 4028 NOTES J VIROL on April 13 2015 by NYU MEDICAL CENTER LIBRARY http jvi asm org Downloaded from with a previous study in humans where a larger amount of SARS CoV RNA was found in saliva from all specimens of 17 SARS patients including four patients before the development of lung lesions 41 The finding of viral loads in oral swabs from 4 4 animals in comparison to infected lungs in 2 4 ma caques would likely suggest a critical role of viral production in upper respiratory sites including salivary gland ducts early in infection Our data therefore suggest that ACE2 H11001 cytokeratin H11001 cells lining salivary gland ducts are early target cells of SARS CoV and a likely source of the virions found in patients saliva droplets especially early in infection Our findings however do not exclude the possibility that saliva virions might also come from other sources e g respiratory secretions espe cially later in infection It remains to be further studied how many virions produced in trachea bronchus lungs or other tissues would contribute to the saliva viral load Moreover a serial time point study would be necessary to further investi gate the impact of upper respiratory tract infection on SARS CoV transmission and pathogenesis In conclusion we report here the distribution of ACE2 H11001 cells in the respiratory tract of Chinese rhesus macaques which mimics the human situation and extends those observations to more proximal tissues of the respiratory tract than have been examined in humans Moreover we have developed a safe pseudoviral system to study the early target cells of SARS CoV in monkeys We demonstrated for the first time that ACE2 H11001 cytokeratin H11001 epithelial cells of salivary gland ducts in the upper respiratory tract are early targets of SARS CoV infection in addition to other cells such as ACE2 H11001 cytokeratin H11001 pneumo cytes in lungs This finding was confirmed by data generated using a live pathogenic SARS CoV in Chinese rhesus m