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Microbiol Immunol 2008 52 118 127 doi 10 1111 j 1348 0421 2008 00011 x EDITOR COMMUNICATED PAPER Co infection of respiratory bacterium with severe acute respiratory syndrome coronavirus induces an exacerbated pneumonia in mice Yasushi Ami Noriyo Nagata Kazuya Shirato Rie Watanabe Naoko Iwata Keiko Nakagaki Shuetsu Fukushi Masayuki Saijo Shigeru Morikawa and Fumihiro Taguchi National Institute of Infectious Diseases Murayama Branch 4 7 1 Gakuen Musashi Murayama Tokyo 208 0011 Japan Correspondence Fumihiro Taguchi Department of Virology III Division of Respiratory Virus Diseases National Institute of Infectious Diseases 4 7 1 Gakuen Musashi Murayama Tokyo 208 0011 Japan Tel 81 42 561 0771 ext 533 email ftaguchi nih go jp Received 4 December 2007 accepted 6 December 2007 List of Abbreviations ACE2 angiotensin converting enzyme 2 ARDS acute respiratory distress syndrome BAL bronchoalveolar lavage BSL 3 biosafety level three DMEM Dulbecco s modified minimal essential medium FCS fetal calf serum FGF fibroblast growth factor Fr 1 Frankfurt 1 Fr mo Fr 1 passaged 10 times through mice GM CSF granulocyte macrophage colony stimulating factor IFN interferon IL interleukin i n intranasally IP IFN inducible protein KC keratinocyte derived cytokine LPS lipopolysaccharide MCP monocyte chemotactic protein MHV murine coronavirus mouse hepatitis virus MIG monokine induced by IFN PBS phosphate buffered saline p i post infection Pp Pasteurella pneumotropica SARS severe acute respiratory syndrome SARS CoV severe acute respiratory syndrome coronavirus TNF tumor necrosis factor VEGF vascular endothelial growth factor Key words coronavirus elastase mouse SARS ABSTRACT SARS CoV grows in a variety of tissues that express its receptor although the mechanism for high replication in the lungs and severe respiratory illness is not well understood We recently showed that elastase enhances SARS CoV infection in cultured cells which suggests that SARS develop ment may be due to elastase mediated enhanced SARS CoV infection in the lungs To explore this possibility we examined whether co infection of mice with SARS CoV and Pp a low pathogenic bacterium which elicits elastaseproductioninthelungs inducesexacerbationofpneumonia Mice co infected with SARS CoV and Pp developed severe respiratory disease withextensiveweightloss resultingina33 90 mortalityrate Micewith exacerbated pneumonia showed enhanced virus infection in the lungs and histopathological lesions similar to those found in human SARS cases In tranasal administration of LPS another elastase inducer showed an effect similar to that of Pp infection Thus this study shows that exacerbated pneumonia in mice results from co infection with SARS CoV and a respi ratory bacterium that induces elastase production in the lungs suggesting apossibleroleforelastaseintheexacerbationofpneumonia SARS CoV is responsible for a life threatening disease that affected nearly 800 individuals in 2002 2003 1 3 The virus genome sequence of about 30 kilobases was identified within the month after its isolation 4 5 Its receptor ACE2 was also discovered within several months of identification of the causative agent 6 The 118 c 2008 The Societies and Blackwell Publishing Asia Pty Ltd Mouse SARS by SARS CoV and Pp Infection advancement of SARS CoV research has been overwhelm ing This virus has become one of the most studied among the coronaviruses In spite of these extensive studies the pathogenesis and mechanisms of development of this se vere respiratory disease have not yet been fully elucidated Although a number of animal species have been found to permit SARS CoV replication 7 12 there has been only limited success in developing histopathology simi lar to that of human SARS in animal models Recently transgenic mice expressing human ACE2 were reported to develop pneumonia after SARS CoV infection The pathogenesis of SARS CoV infection in those mice was however slightly different from that of human SARS 13 14 Human SARS patients generally die from pulmonary failure whereas infection of the central nervous system is the major factor responsible for a fatal outcome in trans genic mice 13 In another transgenic model high repli cation of SARS CoV in the brain likely contributed to the deaths 14 More recently Roberts et al 15 reported that a mouse adapted SARS CoV causes fatal respiratory disease in young mice which reproduced many aspects of human SARS Respiratory agents such as human metapneumovirus or chlamydia have been isolated from SARS patients 1 16 and were initially suspected to be the causative agents ofSARS However SARS CoVwasfinallyidentifiedas the agent of SARS since it fulfilled Koch s postulate 7 Nevertheless when animals were infected with SARS CoV alone most failed to develop SARS like severe pneumonia 12 These results may imply that the respiratory agents foundinsomeSARScasescouldworkincombinationwith SARS CoV in order to induce a severe form of pneumonia In the course of studying the cell entry mechanismt for SARS CoV we found that some proteases produced in the host animals such as trypsin and elastase enhanced SARS CoV infection in cultured cells 17 These in vitro observations hinted at the highly pathogenic feature of this virus in the lungs where elastase is predominantly pro duced as a result of inflammation In the present study we examined whether or not SARS CoV infection is enhanced by weak inflammation in the lungs induced by infection with low pathogenic bacteria which induce elastase Our results show that both low virulent Pp infection and ad ministration of LPS derived from Escherichiacoli induced elastase in the lungs and enhanced the replication of SARS CoV resulting in exacerbation of the respiratory disease caused by SARS CoV infection and a high mortality rate These results indicate that co infection of SARS CoV with low virulent microorganisms induces exacerbated pneu monia and suggest the possibility that elastase is involved in the pathogenesis of exacerbated pneumonia caused by SARS CoV infection MATERIALS AND METHODS Virus and virus titration Fr 1 1 18 kindly provided by Dr John Ziebuhr as well as Fr mo was propagated and plaque assayed with VeroE6 cells as previously described 17 Compared with the orig inal virus Fr 1 used in our laboratory has two amino acid changes at position 641 His to Tyr of S protein and ORF1a 429 Ala to Ser 18 which are presumed to have arisen during passage through VeroE6 cells Passage of Fr 1 through mice to obtain Fr mo was done as follows Mice were inoculated i n with 20 lofFr 1 and their lungs were washed with PBS pH 7 2 contain ing 0 1 bovine serum albumin Sigma St Louis MO USA 20 IU of penicillin G Sigma 20 lofstrepto mycin Sigma and 1 g of amphotericin B Gibco Grand Island NY USA per ml on day three after infection The resultant bronchoalveolar wash was inoculated into mice by the i n route and passaged a total of 10 times Finally the lungs of the infected mice were homogenized and the homogenate was spun at 2000 rpm for 10 minutes at 4 C The supernatant was inoculated onto VeroE6 cells and culture fluid from infected cells was used to infect the mice Fr mo has amino acid mutations in the S protein at positions 480 Asp to Glu as well as 641 His to Tyr the latter is identical to the mutation found in Fr 1 Fr mo has two additional mutations in the ORF1a 3534 Phe to Leu and ORF1ab 5172 Thr to Ile though the mutation at ORF1a 429 found in Fr 1 was not present For titration of virus the lungs were aseptically isolated at intervals after inoculation and 10 homogenates were prepared by using a grinder and silica sand in chilled PBS containing 200 g ml of kanamycin Meiji seika Tokyo Japan The homogenates were centrifuged at 2000 rpm for 10 minutes at 4 C The supernatants were isolated and kept at 80 C until virus titration VeroE6 cells were grown and maintained in DMEM Nissui Tokyo Japan contain ing 5 FCS Sigma and virus infectivity was determined by plaque assay as described previously 17 Mice Six week old BALB c male mice were purchased from SLC Hamamatsu Japan or Charles River Japan CRJ Tokyo Japan Those mice are serologically confirmed to be free from infections with pathogenic microorganisms includ ing Pp Mice were anesthetized with intraperitoneal ad ministration of xylazine and ketamine and inoculated i n with 20 lofPp MaM strain 19 suspended in PBS In some experiments mice were administered i n with 20 l of LPS O55 B5 Sigma dissolved in PBS at a concentration of 1 mg ml Those mice were inoculated i n with 20 lof c 2008 The Societies and Blackwell Publishing Asia Pty Ltd 119 Y Ami et al SRAS CoV one day after Pp infection or LPS administra tion and kept in globe box isolators in a BSL 3 labora tory in our institute during the experimental period Mice were killed at selected intervals after SARS CoV infection and lungs and other organs were aseptically collected for virus titration histopathological examination detection of virus specific antigens and measurement of elastase ac tivity Mice were maintained in keeping with the animal experiment guidelines of our institute Histopathology and immunohistochemistry Mice inoculated with Pp and or SARS CoV were killed at selected intervals after infection Tissues from those mice were fixed in 10 neutral buffered formalin pH 7 4 and subjected to routine pathological examination For detection of virus antigens streptavidin biotin complex methods DAKO Japan were used with rabbit hyperim mune serum produced in our institute against SARS CoV according to the manufacturer s instructions Elastase activity Elastase activity was examined using the synthetic sub strate Suc Ala Ala Pro Val pNA which is highly specific for neutrophil elastase in lung tissue as described by Yoshimura et al 20 Todetermineelastaseactivity we employed 10 lung homogenates prepared as described above or BAL For the collection of BAL one ml of chilled saline containing 0 38 citric acid was injected into the lungs of dead mice via the trachea using a canula com bined with a syringe and then the saline was recovered After removing cells containing in the BAL by spinning at 10000 rpm for five minutes supernatants were used to determine elastase activity The lung homogenates di luted 10 fold with PBS finally 1 lung homogenates or BAL were incubated in 0 1 M Tris HCl buffer pH 8 0 containing 0 5 M NaCl and 1 mM substrate for 24 hours at 37 C pNA released from the substrate was measured spectrophotometrically at 405 nm and shown as elastase activity as described previously 20 Analysis of cytokines and chemokines Cytokines and chemokines were assayed mostly as pre viously reported 9 Lung homogenates prepared as de scribed above were diluted 10 times with a lysis buffer Viruses included in the materials were completely inac tivated by ultraviolet irradiation for 10 minutes Using a Mouse cytokine 20 plex antibody bead kit Bioscience International Inc Camarillo CA USA as previously re ported 9 these materials were examined with Luminex 200 Luminex Co Austin TX USA for the following cytokines and chemokines FGF basic GM CSF IFN IL 10 IL 12 IL 13 IL 17 IL 1 IL 1 IL 2 IL 4 IL 5 IL 6 IL 10 KC MCP 1 MIG IP 10 TNF VEGF RESULTS Induction of elastase in mouse lungs by Pp infection and its effect on respiratory disease caused by SARS CoV infection It has been shown that SARS CoV replication is enhanced in the presence of proteases such as trypsin or elastase 17 The possible molecular mechanism of this enhance ment is the potentiation of SARS CoV infection from the cell surface which is more efficient than the infection via an endosomal pathway which takes place in the absence of proteases 17 Elastase a major protease produced in lung inflammation could enhance SARS CoV replication in the lungs which would in turn result in severe damage to this organ We first examined whether infection with Pp induced elastase in the lungs Pp is of low virulence and as a rule causes only mild never severe respiratory disease in adult mice 21 Six week old BALB c mice were in fected i n with 2 0 10 7 c f u of Pp and elastase activity in both lung homogenates and BAL was monitored At one day p i significant amounts of elastase were detected in the BAL and lungs of Pp infected mice while elastase activity was undetectable in samples from uninfected mice Fig 1a We also examined the effect of LPS administra tion on elastase production in the lungs since LPS has been used as an elastase inducer 22 Mice administered i n with 20 l of LPS 1 mg ml dissolved in PBS were shown to produce elastase in the lungs one to two days af ter administration similar to infection with Pp Fig 1b These results show that Pp and LPS are appropriate agents for inducing elastase in the lungs and BAL Next we examined the effect of Pp on SARS CoV in fection We used two different SARS CoVs one was Fr 1 isolated from diseased humans 1 18 and the other a mouse adapted virus called Fr mo which was passaged 10 times through the mice and finally grown in VeroE6 cells as described in Materials and Methods Fr mo has four amino acid changes in the spike ORF1a and ORF1b com pared with those of Fr 1 reported previously 18 Mice were infected with 1 3 10 7 c f u of Pp and one day later were co infected with 1 1 10 6 and 0 8 10 6 p f u of Fr 1 and Fr mo respectively by the i n route As shown in Figures 2a and b mice inoculated with Pp alone showed a transientlossofbodyweightandruffledhairfromoneto four days pi Mice infected with Fr 1 or Fr mo alone rarely showed weight loss and ruffled hair When mice were in fected with both Pp and Fr 1 the clinical symptoms were similar to those of mice inoculated with Pp alone and included transient loss of body weight and ruffled hair 120 c 2008 The Societies and Blackwell Publishing Asia Pty Ltd Mouse SARS by SARS CoV and Pp Infection 0 10 20 30 No 1 day 2 day 1 day 2 day Pp LPS 0 10 20 30 40 Lung BAL Elastase activity nm ol m l a b Fig 1 Elastase production in the lungs of mice after Pp infection or LPS administration Mice inoculated i n with 1 5 10 7 cfu of Pp suspended in 20 lPBS or mock infected mice were killed one day after infection and elastase activity in BAL and lung homogenates were measured a Mice were inoculated i n with 2 3 10 7 cfu of Pp or administered with 20 l of LPS 1 mg ml dissolved in PBS and elastase activity in lung homogenates was determined one and two days after inoculation Mock infected mice No were also examined for elastase activity b Mean values with standard deviation of three to five samples in each group are shown a b c Body w e igh ts 60 80 100 120 Time days after virus inoculation Body w e igh t 40 60 80 100 120 60 80 100 120 Fig 2 Body weights of mice infected with SARS CoV Mice were in fected i n with Pp 1 3 10 7 cfu and 1 day later with SARS CoV ei ther 1 1 10 6 of p f u Fr 1 a or 0 8 10 6 p f u of Fr mo b and were weighed daily after Pp infection Mice were administered i n with 20 l of LPS 1 mg ml infected with 0 8 10 6 p f u of Fr mo 1 day later and weighed daily after LPS administration c Mean body weights are shown as a percentage compared with the mean weights of all mice measured just before Pp or LPS inoculation Mice were inoculated with SARS CoV alone Pp or LPS alone triangle orPp or LPS SARS CoV square Two of six mice infected withPp Fr mo died by day five p i and the body weight from days five to seven p i showed a significant difference when compared with those of Pp infected mice P 0 001 P 0 005 P 0 01 by Student s t test b Three of six mice treated with LPS and infected with Fr mo died on day four and body weights were signif icantly lower P 0 001 on days three to four in comparison to those of mice treated with LPS alone c Fig 2a In contrast mice co infected withPp Fr mo had severe weight loss and most of them did not recover during the observation period eight days p i Fig 2b Those mice showed clinical symptoms such as ruffled hair from one day after Pp infection and hunched pos ture from three to four days after Fr mo infection These symptoms continued during the observation period On days four to eight p i these mice lost 30 or greater c 2008 The Societies and Blackwell Publishing Asia Pty Ltd 121 Y Ami et al a b c Vi rus tit er i n t h e lu ng lo g 10 p f u 0 2 4 6 8 Time days after SARS CoV infection Time days after SARS CoV infection 2 d 4 d4 d 2 d 10 4 10 2 Virus titer inoculated Fig 3 Virus titers in the lungs of mice infected with Pp and or SARS CoV Mice infected solid line or mock infected broken line with Pp and one day later with Fr 1 a orFr mo b as described in the legend to Fig 2 were killed on days two four six and eight and virus titers in the lungs were determined by a plaque assay Significant difference was shown P 0 001 P 0 01 by Student s t test a and b Mice were infected with Pp 2 0 10 7 cfu black column or mock infected shaded column and one day later further infected with 1 10 4 10 4 or 1 10 2 10 2 p f u of Fr mo Virus titers in the lungs were examined on days two 2 d and four 4 d after Fr mo infection c Significant differ ence was shown P 0 001 P 0 006 by Student s t test c Virus titers are indicated in p f u in log 10 50 mg tissue weight Mean values of the titers with standard deviation are shown Groups a and b consisted of four to five mice each and group c three mice of their body weight and more than 33 of those mice died after exhibiting severe respiratory disease Thus mice co infected with Pp Fr mo developed severe respiratory disease suggesting the possibility that elastase produced by Pp infection exacerbated infection by SARS CoV adapted to mice We also infected mice that had been administered LPS with Fr mo Fig 2c These mice developed weight loss and clinical symptoms similar to those displayed by mice inoculated withPp Fr mo One half of the infected mice died by day four p i but the remaining mice grad ually recovered Fig 2c This finding also supported the possibility that elastase is involved in the exacerbation of respiratory disease caused by SARS CoV infection Effect of Pp infection on virus growth in the lungs We then examined the virus titers of mice infected with SARS CoV alone and those doubly infected with Pp and SARS CoV Those mice were infected under conditions identical to those shown in Figure 2 When examined on days two and four p i Fr mo had grown at a rate 10 to 50 fold higher in the lungs of mice than had Fr 1 Fig 3a and b showing that Fr mo has a higher potential to grow in mice On day two p i virus titers in the lungs were about 100 fold higher in mice infected with Pp and Fr 1 than in mice infected with Fr 1 alone However by the fourth day p i there was not a significant difference between the two groups Fig 3a On day two p i the virus titers of mice infected with Pp Fr mo were slightly but significantly higher than those of mice infected with Fr mo alone The difference in virus titers between mice infected with Pp Fr mo and those with Fr mo alone was also evident at four days p i Fig 3b These results show that infection with Pp greatly enhanced the infection of Fr 1 but had a less remarkable effect in the infection of Fr mo in the early phase of viral infection More significant enhancement of Fr mo infection was observed when mice were infected with low titers of virus When mice were infected with 1 10 4 or 1 10 2 p f u the virus titers were about 10 fold higher in mice co infected with Pp Fr mo than in those infected with Fr mo alone on both two and four days p i Fig 3c Enhancement of Fr mo infection in the lungs was also observed in mice that had received LPS as compared with mice without LPS administration data not shown These results collectively suggest that the elastase induced by Pp infection or LPS administration enhanced the infection produced by mouse adapted Fr mo in mouse lungs The elastase activities of those mice infected with Fr 1 Pp and Fr 1 Fr mo Pp Fr mo or Pp alone were ex amined on days two and four after SARS CoV infection that is three and five days after Pp infe