【病毒外文文獻】2006 Murine encephalitis caused by HCoV-OC43, a human coronavirus with broad species specificity, is partly immune-media

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is University Drosten et al 2003 Fouchier et al 2003 Since then two additional coronaviruses HCoV NL63 and HCoV HKU1 have termed HCoV OC43 NV HCoV OC43 NV was then propagated in tissue culture cells generating a tissue culture adapted ence with pas Virology 347 2006 410 been identified these agents cause upper and lower respiratory Keywords Coronavirus Rodent model Encephalitis Introduction Until recently human coronaviruses were mostly associated with mild upper respiratory tract infections the common cold and occasionally with outbreaks of gastroenteritis Vabret et al 2003 However with the recognition that the Severe Acute Respiratory Syndrome SARS was caused by a coronavirus it became apparent that coronaviruses could also cause more significant disease in the human population tract diseases that are much less severe than SARS van der Hoek et al 2004 Woo et al 2005 HCoV OC43 and HCoV 229E are the etiological agents for many coronavirus induced upper respiratory tract infections HCoV OC43 harvested from a patient with an upper respira tory tract infection was originally isolated after passage in human embryonic tracheal organ cultures this virus caused neurological disease after only one passage in suckling mice and encephalitis within 2 4 passages McIntosh et al 1967 Abstract The human coronavirus HCoV OC43 causes a significant fraction of upper respiratory tract infections Most coronaviruses show a strong species specificity although the SARS Coronavirus crossed species from palm civet cats to infect humans Similarly HCoV OC43 likely a member of the same coronavirus group as SARS CoV readily crossed the species barrier as evidenced by its rapid adaptation to the murine brain McIntosh K Becker W B Chanock R M 1967 Growth in suckling mouse brain of IBV like viruses from patients with upper respiratory tract disease Proc Natl Acad Sci U S A 58 2268 73 Herein we investigated two consequences of this plasticity in species tropism First we showed that HCoV OC43 was able to infect cells from a large number of mammalian species Second we showed that virus that was passed exclusively in suckling mouse brains was highly virulent and caused a uniformly fatal encephalitis in adult mice The surface glycoprotein is a major virulence factor in most coronavirus infections We identified three changes in the HCoV OC43 surface glycoprotein that correlated with enhanced neurovirulence in mice these were located in the domain of the protein responsible for binding to host cells These data suggest that some coronaviruses including HCoV OC43 and SARS CoV readily adapt to growth in cells from heterologous species This adaptability has facilitated the isolation of HCoV OC43 viral variants with markedly differing abilities to infect animals and tissue culture cells D 2005 Elsevier Inc All rights reserved Received 19 September 2005 returned to author for revision 26 October 2005 accepted 30 November 2005 Available online 18 January 2006 Murine encephalitis caused by HCoV broad species specificity Noah Butler a Lecia Pewe b Kathryn a Interdisciplinary Program in Immunology b Department of Pediatrics University c Medical Scientist Training Program University d Department of Microbiology University 0042 6822 see front matter D 2005 Elsevier Inc All rights reserved doi 10 1016 j virol 2005 11 044 Corresponding author Department of Pediatrics 2042 Medical Laborato ries University of Iowa Iowa City IA 52242 USA E mail address stanley perlman uiowa edu S Perlman OC43 a human coronavirus with partly immune mediated Trandem c Stanley Perlman a b d of Iowa Iowa City IA 52242 USA of Iowa Iowa City IA 52242 USA of Iowa Iowa City IA 52242 USA of Iowa Iowa City IA 52242 USA 421 variant termed HCoV OC43 TC HCoV OC43 showed increasing neurovirul sage through the murine brain McIntosh et al 1967 however most recent studies have used CNS adapted viruses that were further propagated at least for a few passages in studies several groups showed that the adaptation of the SARS CoV to humans during the 2003 epidemic included several mutations in the S protein These mutations were shown to enhance binding to human angiotensin converting enzyme 2 ACE 2 the host cell receptor for SARS CoV Kan et al 2005 Li et al 2005 Therefore to investigate the role of the S protein in HCoV OC43 pathogenesis we sequenced the S genes of HCoV OC43 TC and HCoV OC43 NV and compared the results to published sequences of the S genes of several other HCoV OC43 isolates Results Intranasal inoculation of HCoV OC43 NV but not HCoV OC43 TC is uniformly fatal to 5 to 8 week old C57BL 6 B6 mice In confirmation of our preliminary results intranasal inoculation of HCoV OC43 NV resulted in 100 mortality in mice ranging from 5 to 8 weeks old Fig 1A Mice developed signs of acute encephalitis including hunched posture lethargy and wasting by days 6 7 Mortality was associated with a 30 35 loss of body mass Fig 1B Severe clinical encephalitis was associated with widespread mononuclear cell infiltration including perivascular cuffing and with loss of CNS architec 347 2006 410 421 411 tissue culture cells For example Talbot and co workers showed using the mouse adapted virus after passage in tissue culture cells termed HCoV OC43 QUE herein that mice infected intranasally with 10 4 10 5 TCID 50 developed enceph alitis if inoculated 8 days but not 21 days postnatally Jacomy and Talbot 2003 HCoV OC43 QUE was passaged 5 6 times in tissue culture prior to use in mice personal communication Dr Pierre Talbot INRS Institut Armand Frappier Laval Quebec and consequently may be less virulent than virus isolated directly from infected suckling mouse brains Consistent with this possibility we observed in preliminary experiments that virus directly harvested from suckling mouse brains caused a lethal infection after intranasal inoculation of 5 to 8 week old mice Mice died 9 11 days after inoculation a time when the adaptive immune response to another coronavirus mouse hepatitis virus MHV is maximal Bergmann et al 1999 To begin to understand these differences in virulence we initiated a more complete study of the disease caused by the neurovirulent strain of HCoV OC43 This ability of HCoV OC43 to cross species barriers to infect mice and to gain virulence in the new host contrasts with the strict species specificity exhibited by most coronaviruses For example the group I coronavirus HCoV 229E does not readily infect mice even transgenic mice expressing human aminopeptidase N the virus receptor for HCoV 229E Went worth et al 2005 However the ability of the group II coronavirus HCoV OC43 to adapt easily to replication within the murine brain suggests that it may be more lax in its species specificity than other coronaviruses In that sense it resembles another group II coronavirus SARS CoV which likely crossed the species barrier from animals such as palm civet cats to infect humans CSMEC 2004 Guan et al 2003 Song et al 2005 Unlike other coronaviruses HCoV OC43 and the closely related bovine coronavirus BCoV appear to bind to cells via N acetyl neuraminic acid Schultze and Herrler 1992 Vlasak et al 1988 although there are data to suggest that HCoV OC43 can also employ MHC class I antigen as a host cell receptor Collins 1993 This use of a sugar moiety for entry would also be consistent with the ability to infect a broader range of species than most coronaviruses This possibility was investigated by infecting tissue culture cells from several different animal species with HCoV OC43 using both the mouse adapted and the tissue culture adapted strains The ability to rapidly gain virulence after passage in the murine brain is likely to occur via selection of mutations in the S protein that optimize binding and entry to target cells Passage of neurovirulent variants of MHV in tissue culture selects for viruses that are attenuated in vivo but enhanced for replication in vitro These changes map to the surface S glycoprotein Gallagher and Buchmeier 2001 Tsai et al 2003 In addition infection of rats with uncloned stocks of MHV resulted in the selection of virulent strains of virus again virulence correlated with changes in the S protein Taguchi et al 1985 Also replacement of the S gene in the N Butler et al Virology moderately virulent A59 strain of MHV with the gene encoding the S protein of the virulent JHM strain resulted in a gain in virulence in mice Phillips et al 1999 In other ture data not shown In contrast intranasal inoculation of 5 week old C57BL 6 mice with HCoV OC43 TC did not cause any clinical disease including any weight loss Fig 1 Consistent with the uniformly lethal outcome observed in Fig 1 HCoV OC43 NV is uniformly lethal to wild type C57BL 6 mice 5 week old mice were inoculated intranasally with 30 LD 50 HCoV OC43 NV or 10 6 TCID 50 HCoV OC43 TC as described in Materials and methods Mice were monitored daily for survival A and weight loss B Indicates P 0 0002 mice infected with HCoV OC43 NV we detected high titers of virus in the CNS of infected mice HCoV OC43 NV grew poorly in tissue culture cells and we could only reliably titer infectious virus using suckling mice as described in Materials and methods Virus titers increased from days 5 to 7 Fig 2 To confirm these results we also measured viral loads using a real time RT PCR assay As shown in Figs 2A and B there was a strong positive correlation between recovery of infectious virus and the detection of OC43 nucleocapsid RNA in the brains of mice Also and in agreement with the results of Jacomy et al Jacomy and Talbot 2003 HCoV OC43 infected other organs to a small extent such as the lungs and intestines when viral RNAwas assayed by real time RT PCR Fig 2C Notably the results also suggested that virus was in the process of clearance at the time of death since virus titers RNA levels declined between days 7 and 9 p i Fig 2C In mice infected intranasally with neurovirulent strains of MHV virus enters the CNS via the olfactory nerves with subsequent transneuronal retrograde dissemination to distant connections of the olfactory bulb Perlman et al 1989 1990 Using in situ hybridization and immunohistochemistry to track HCoV OC43 RNA and antigen respectively we detected mice for N Butler et al Virology 347 2006 410 421412 Fig 2 Viral titers and viral RNA burdens in the brains of HCoV OC43 NV infected LD 50 HCoV OC43 NV A and B Whole brains were isolated at the indicated time described in Materials and methods Titers from 3 representative mice are shown and viral RNA burden C HCoV OC43 RNA titers increase until day 7 p i then decline for virus burden with real time RT PCR Data in panel C are expressed as mean T SEM Cord versus Lung and Intestine Indicates P 0 05 Wild type and RAG1 C0 C0 C57BL 6 mice were infected intranasally with 30 points and titers of infectious virus and virus RNA burden were determined as each time point demonstrating correlation between recovery of infectious virus by day 9 Whole RNAwas isolated from the indicated tissue and assayed for 4 6 mice per time point Note the different scales in Brain and Spinal overlap between the pathways of entry and spread used by MHV and those used by HCoV OC43 after intranasal inoculation HCoV OC43 was detected in the olfactory bulb and the olfactory nucleus at 3 days p i but was cleared from these structures by day 5 p i At later time points days 7 and 9 p i HCoV OC43 RNA and antigen were detected primarily in the brainstem and the spinal cord Fig 3A data not shown Unlike MHV which is known to infect oligodendrocytes and is present throughout the white matter in the infected CNS HCoV OC43 was not detected in the white matter at any time point examined shown at day 9 p i in Figs 3A B HCoV OC43 NV infection of mouse CNS is restricted to neurons The ability of HCoV OC43 NV to cause lethal encephalitis in adult animals contrasted with that reported for HCoV OC43 QUE Jacomy et al reported that HCoV OC43 QUE infection was primarily restricted to neurons in vivo Jacomy and Talbot 2003 Thus one possible explanation for the enhanced neurovirulence of HCoV OC43 NV relative to HCoV OC43 QUE is an expanded cell tropism To more directly assess this possibility we used combined in situ hybridization and immunohistochemistry to identify cell types harboring viral RNA and antigen We found that viral product was restricted to cells with morphologies consistent with neurons Figs 3C D Moreover in some animals evaluated at late times postinfection were infected by HCoV OC43 NV in vivo we found no evidence of colocalization of HCoV OC43 RNA and the astrocyte specific marker GFAP antigen Figs 3F H We also performed a series of colocalization experiments for viral RNA and F4 80 a macrophage microglia specific cell marker but could not identify F4 80 cells that were positive for viral product To confirm these results we sorted F4 80 CD45 hi macrophages and F4 80 CD45 int microglia cells from the CNS of infected mice at 7 days postinfection collected them onto glass slides and stained them for virus antigen Neither macrophages nor microglia stained positive for viral antigen while infected tissue culture cells processed in parallel stained positively data not shown These data suggest that unlike MHV HCoV OC43 does not infect astrocytes or macrophages microglia Based on the morphology of the infected cells as well as the lack of virus staining in the white matter or in GFAP or F4 80 cells we conclude that the predominant if not sole targets for HCoV OC43 NV are neurons Host adaptive immune response contributes to HCoV OC43 induced morbidity and mortality Viral RNA burden in the CNS was diminishing at the time of death Fig 2C consistent with a role for the host immune response in both virus clearance and disease When we immunophenotyped mononuclear cell infiltrates from HCoV ted antigen methods anti OC43 is N Butler et al Virology 347 2006 410 421 413 day 9 we could clearly identify Purkinje cells that harbored virus antigen Fig 3E When we evaluated whether astrocytes Fig 3 Neurons are the primary target of HCoV OC43 NV in vivo Mice were inocula harvested 7 or 9 days p i Tissue samples were prepared for in situ hybridization standard light fluorescence or confocal microscopy as described in Materials and cords Panel A depicts immunohistochemical detection of HCoV OC43 using the demarcate the white matter C E Morphology of HCoV OC43 NV infected cells brain stems from mice harvested 7 9 days p i Sections were stained with O 4 3 followed for HCoV OC43 nucleocapsid RNA Cy3 labeled antisense probe red and immunohistoc Panel H is a merged image of panels F and G Original images are 20C2 magnifica OC43 NV infected mice 7 days postinfection we found that a large fraction consisted of CD4 and CD8 Tcells Fig 4A One intranasally with 30 LD 50 of HCoV OC43 NV and brains and spinal cords were staining or Luxol Fast Blue LFB staining and slides were examined with A B HCoV OC43 antigen is localized in the gray matter of spinal S hybridoma O 4 3 Panel B depicts a serial section stained with LFB to consistent with that of neurons Images are representative coronal sections of by Cy3 labeled goat anti mouse F H Combination in situ hybridization hemistry for the astrocyte marker GFAP FITC labeled anti GFAP green tion for panels A and B or 40C2 magnification for panels C H N Butler et al Virology414 HCoV OC43 specific CD4 T cell epitope recognized in C57BL 6 mice is known spanning residues 133 147 of the transmembrane M protein epitope M 133 Identification of this epitope was based on sequence homology with MHV We found that 1 3 of the infiltrating CD4 T cells at day 9 recognized this epitope Fig 4B By contrast 20 25 of CD4 T cells in the MHV infected CNS responded to epitope M133 at 7 days p i Haring et al 2001 No HCoV OC43 CD8 Tcell epitopes have been identified yet so the magnitude of the virus specific CD8 T cell response could not be determined To probe the role of the host adaptive immune response in pathogenesis we infected immunodeficient mice lacking normal T and B cell responses mice with genetic disruption of the recombination activating gene 1 RAG1 C0 C0 and monitored these mice for weight loss and survival HCoV OC43 NV infected RAG1 C0 C0 mice developed signs of enceph Fig 4 Tcell infiltration into the HCoV OC43 NV infected CNS contributes to morbidity OC43 NV infected mice Mononuclear cells were harvested 7 days p i surface stained methods Data from one representative mouse are shown B Two color FACS analysis OC43 specific CD4 T cells Whole brains were isolated from HCoV OC43 NV infected intracellularly stained for IFN g ex vivo after no stimulation left panel or stimulatio shown C HCoV OC43 NV infected RAG1 C0 C0 C57BL 6 mice lose weight left panel infected wild type mice Indicates P 0 05 D Relative burden of HCoV OC43 type and RAG1 C0 C0 mice Data in panel C are expressed as mean T SEM for 4 6 347 2006 410 421 alitis lethargy hunching and weight loss similar to those observed in infected wild type mice but with delayed kinetics Fig 4C left panel Infected RAG1 C0 C0 mice also survived longer than did their B6 counterparts Fig 4C right panel At the time of death virus loads were 5 7 fold higher than detected in moribund B6 mice when measured by real time RT PCR Fig 4D left panel or infectious virus titers Fig 4D right panel At this time viral antigen or RNA detected by immunohistochemistry or in situ hybridization was detected primarily in the brainstem although more cells were infected than in B6 mice As in wild type C57BL 6 mice neurons were the primary target for infection suggesting that the antiviral immune response was not responsible for the lack of infection of glial cells data not shown To confirm the pathological role of T cells we adoptively transferred HCoV OC43 immune cells to RAG1 C0 C0 mice that and mortality A Both CD4 and CD8 Tcells infiltrate the brains of HCoV for CD4 and CD8 and subjected to FACS analysis as described in Materials and of in vitro peptide stimulated mononuclear cells demonstrating infiltration of mice 7 days p i Mononuclear cells were surface stained for CD4 and n with M 133 peptide right panel Data from one representative mouse are and succumb to infection right panel with delayed kinetics relative to RNA left panel and infectious virus right panel in brains of moribund wild mice per group Indicates P 10 in HRT 18 cells Neurovirulence Uniformly lethal to adult mice Lethal only to young 21 days mice c Avirulent in mice d a S protein residue numbering is based on HCoV OC43 NV strain with the corresponding residues for HCoV OC43 QUE and HCoV OC43 TC in parenthe ses S1 and S2 encompass amino acids 1 759 and 760 1353 to 1365 respectively b The sequence for HCoV OC43 QUE corresponds to GenPept accession number AAT84362 submitted by St Jean et al St Jean et al 2004 c Jacomy and Talbot 2003 d Fig 1 genes encoding non structural 12 9 kDa the small envelope E the nucleocapsid N and the transmembrane M proteins and the 3Vuntranslated region revealed no nucleotide differ ences between HCoV OC43 NV and HCoV OC43 QUE Broad species and cell type tropism of HCoV OC43 TC and HCoV OC43 NV As described above HCoV OC43 differs from most strains of coronaviruses in that it readily crossed species barriers to infect mice McIntosh et al 1967 This observation raised that possibility that HCoV OC43 unlike most other coronaviruses is able to infect a wide variety of species without substantial adaptation To gain insight into this possibility we infected a variety of tissue culture cells from a wide range of species hamster pig human mouse rat monkey and cat with HCoV OC43 NV or HCoV OC43 TC As shown in Fig 5 immunocy tochemical staining for viral antigen revealed that the tissue culture adapted virus infected hamster pig human mouse monkey and cat cells but not FRT rat epithelium cells The neurovirulent strain also exhibited a wide host range specific ity infecting hamster pig human monkey cat and mouse cells For some cells lines