STUDIES ON CORONAVIRUS

Federally Funded Research on Coronavirus

The U.S. Government supports a variety of research studies relating to coronavirus. These

studies are tracked by the Office of Extramural Research at the National Institutes of

Health.2 CRISP (Computerized Retrieval of Information on Scientific Projects) is a searchable

database of federally funded biomedical research projects conducted at universities,

hospitals, and other institutions.

Search the CRISP Web site at http://crisp.cit.nih.gov/crisp/crisp_query.generate_screen.

You will have the option to perform targeted searches by various criteria, including

geography, date, and topics related to coronavirus.

For most of the studies, the agencies reporting into CRISP provide summaries or abstracts.

As opposed to clinical trial research using patients, many federally funded studies use

animals or simulated models to explore coronavirus. The following is typical of the type of

information found when searching the CRISP database for coronavirus:

Project Title: 2-5A ANTISENSE FOR DEGRADATION OF SARS CORONAVIRUS

RNA

Principal Investigator & Institution: Cramer, Hagen; Ridgeway Biosystems, Inc. 9500

Euclid Ave, Nd-50 Cleveland, Oh 44195

Timing: Fiscal Year 2004; Project Start 01-APR-2004; Project End 31-OCT-2004 Coronavirus

Summary: (provided by applicant): Severe acute respiratory syndrome (SARS) is a

newly recognized illness that has rapidly spread throughout Asia, North America and

Europe. SARS has been found to be a highly infectious disease with high mortality,

considerable occupational hazard, and no specific therapy. On July 5, the World Health

Organization (WHO) finally declared that SARS has been contained around the world.

However, it seems likely that SARS will return. Therefore, the development of effective

drugs to treat SARS infection is imperative. Ridgeway Biosystems, Inc. is developing

novel drugs for targeting cancer and infectious diseases using its proprietary 2-5A

antisense technology. 2-5A antisense exploits a ubiquitous cellular ribonuclease L

(RNase L), which is activated by binding to 2-5A. By linking 2-5A to antisense, RNase L

is directed to a specific RNA molecule, in this case to SARS RNA. The efficiency of 2-5A

antisense is many times superior to corresponding antisense lacking 2-5A and it avoids

adverse non-specific effects associated with RNAi. We propose to develop an effective

and marketable 2-5A antisense drug for the treatment of SARS infection.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

Project Title: A SARS-COV SPIKE PROTEIN VACCINE

Principal Investigator & Institution: Sim, B Kim Lee.; Director; Protein Potential Llc 308

Argosy Dr Gaithersburg, Md 20878

Timing: Fiscal Year 2004; Project Start 01-APR-2004; Project End 31-MAR-2006

Summary: (provided by applicant): Severe acute respiratory syndrome (SARS) has been

reported in Asia, North America, and Europe. In March 2003, a novel coronavirus

(SARS-CoV) was discovered in association with cases of SARS. SARSCoV is a nonsegmented, single-stranded, (+) sense RNA, which was 29.74 kb in one of the first

sequenced isolates. There are no drugs or vaccine for treating or preventing SARS. We

aim to develop a vaccine to control infection with SARS-CoV by targeting a surface

accessible protein. The correlate of protective immunity for virtually all effective

vaccines against viruses is the titer of antibodies that neutralizes virus invasion into cells

and/or development of the virus within the cells. For antibodies to neutralize virus they

in general are directed against proteins or glycoproteins that are accessible on the

surface of viruses when they are extracellular. The "spike" (also called S or E2)

glycoprotein of SARS-CoV is such a target. It is also attractive as a target, because it is

thought to mediate invasion of the virus into host cells. The spike protein of one isolate

is 1256 amino acids encoded by 3768 base pairs. Based on our experience with

identifying biologically active regions of proteins that mediate invasion into cells, and

are good targets for antibody mediated protective immunity, we selected a region of the

SARS-CoV spike protein which we have designated Region I. It is represented by 2421

base pairs and codes for a protein of 807 amino acids. Our data indicate that Ngycosylation of such proteins reduces expression and immunogenicity of the proteins.

Region I was selected in part because of paucity of N-glycosylation sites. However, there

are 9 such sites in the native sequence. We therefore altered the the known sequence of

Region 1 to eliminate these 9 potential N-glycosylation sites, and produced a synthetic

gene based on this altered sequence. Our experience with expression of proteins in

Pichia pastoris indicates that altering codon usage based on our proprietary information

leads to enhanced expression of recombinant proteins and in vivo expression of proteins

by DNA vaccines. We therefore will use the Region I Spike Protein SARS-CoV native no

glycosylation (RI-native-NG) sequence as the basis for creating a second synthetic gene

in which we will optimize the gene sequence to alter codon usage. This sequence is

designated Region I Spike Protein SARS-CoV codon optimized no glycosylation (RIoptimized-NG). These synthetic DNA sequences, RI-native NG and RI-optimized-NG, Studies 5

will be used to construct DNA plasmids and to produce recombinant proteins in P.

Pastoris that will be optimized for production and purification and used as

immunogens. Mice and rhesus monkeys will be immunized with 3 doses of

recombinant protein in adjuvant or a sequential (prime-boost regimen) of a DNA

plasmid expressing the SARS-CoV spike protein followed by recombinant protein in

adjuvant. Anti-sera will be assessed for antibodies to spike protein by ELISA and

capacity to neutralize virus invasion of Vero cells in vitro. Successful completion of

Phase I will be achieved when we have generated a robust method for producing and

purifying high quality at high yield Region I rec. protein, and have demonstrated that

anti-sera elicited by immunization neutralizes viral activity in vitro. Phase II will include

a monkey immunization and challenge study and scale up and process development for

cGMP production and clinical trials. Our long term goal of commercializing a SARS

vaccine is enhanced by this collaboration among Protein Potential (recombinant protein

and DNA plasmid development expertise), CDC (viral neutralization assays and

knowledge of the disease), and NMRC (immunization of mice and monkeys, and

clinical trials).

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

Project Title: ANALYSIS OF THE SARS VIRUS S GLYCOPROTEINS

Principal Investigator & Institution: Bates, Paul F.; Associate Professor; Microbiology;

University of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104

Timing: Fiscal Year 2004; Project Start 01-APR-2004; Project End 31-MAR-2006

Summary: (provided by applicant): Severe Acute Respiratory Syndrome (SARS) is a

rapidly emerging infectious disease. First observed in the Guangdong Province in China

in late 2002, SARS has spread rapidly around the world with predominant SARS cases

documented in Hong Kong, Beijing, Guangdong, and Toronto. At present well over

3000 cases are reported with ongoing epidemics in Taiwan and Toronto. It appears that

the mortality rate for SARS is between 5-10% for the entire population with

progressively higher rates in older age groups reaching nearly 80% in patients over 65

years. A novel coronavirus has been identified and shown to be responsible for SARS.

This virus called SARS-associated coronavirus (SCV) is the object of this study.

Coronaviruses are enveloped RNA viruses that depend upon glycoproteins on the

virion surface to enter host cells. The Spike or S glycoprotein of coronaviruses is

responsible for both recognition of host receptors and also for membrane fusion to

deliver the virus inside the host cell. Viral glycoproteins are also the targets for humoral

immune responses and antibodies to S glycoproteins effectively neutralize other

coronaviruses. The goal of this application is to develop a system for comprehensive

analysis of the SARS coronavirus S glycoproteins. Toward that end, we propose in

Specific Aim 1 to develop retroviral pseudotypes that carry the SCV spike and to utilize

these pseudotypes to begin a preliminary analysis of the S glycoprotein. Specific Aim 2

will identify the cellular receptor(s) for SCV S using a variety of independent genetic

and biochemical strategies. Once identified the receptor(s) and its interaction with S

glycoproteins will be studied. Achieving these Specific Aims will not only significantly

enhance our understanding of the SARS virus, but will likely aid in the development of

vaccines for SARS and in design of therapeutics that block SARS S function or interfere

with S glycoprotein-receptor interactions.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

Project Title: CELL CULTURE ANIMAL MODELS FOR SARS-COV

Principal Investigator & Institution: Pekosz, Andrew S.; Assistant Professor; Molecular

Microbiology; Washington University Lindell and Skinker Blvd St. Louis, Mo 63130

Timing: Fiscal Year 2004; Project Start 01-MAY-2004; Project End 30-APR-2006

Summary: (provided by applicant): In the spring of 2003, an outbreak of unknown

etiology began in South East Asia and quickly spread to several other countries. The

disease, termed severe acute respiratory distress syndrome or SARS, was identified and

contained by a massive public health effort orchestrated by the WHO and local

authorities. The etiological agent of this disease was identified as a new strain of

coronavirus, and given the name SARS-CoV. In order to gain additional knowledge as

to the replication, pathogenesis and immune responses induced by the virus, animal and

cell culture models are needed. The experiments described in this application are aimed

at establishing 1) in vitro tissue culture models for SARS-CoV infection using primary

respiratory epithelial cells 2) a mouse model for SARS-CoV infection using wild type as

well as immunodeficient mouse strains and 3) structure/function analysis systems for

SARS-CoV ORFs of unknown function. Successful completion of the aims outlined in

this application will establish systems for investigating the cell biology and pathogenesis

of SARS-CoV.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

Project Title: CONTROL OF MHV INFECTION IN THE CENTRAL NERVOUS

SYSTEM

Principal Investigator & Institution: Buchmeier, Michael J.; Professor; Scripps Research

Institute Tpc7 La Jolla, Ca 92037

Timing: Fiscal Year 2002; Project Start 01-FEB-1998; Project End 31-JAN-2004

Summary: (Adapted from Applicant's Summary): Multiple sclerosis (MS) is the most

common autoimmune neurodegenerative disease of adults in the United States,

affecting approximately 250,000 individuals. Available evidence suggests that MS

etiology and pathogenesis may involve two events, the first being exposure to an

environmental agent, likely a virus, early in life, followed by a second event in early

adulthood which triggers disease. Links with host genetics, particularly MHC-class II

antigens and gender have been shown, but a definitive pathogenetic sequence linking

the early and late events has not been shown, therefore animal model systems are of

value in providing insight into the pathogenesis of MS. This project seeks to understand

the mechanisms of CD4+ T cell mediated immune responses in the pathogenesis of CNS

demyelinating disease, the signals which trigger influx of inflammatory cells, and the

state and sites of virus persistence within the CNS. Intracerebral inoculation of C57B1/6

mice with the neurotropic murine coronavirus MHV-JHM and variants such as V5A13.1

derived from it results in a reproducible encephalomyelitis which usually resolves

within 7-14 days but is followed by acute or chronic episodes of demyelination.

Restriction of virus replication and spread within the brain is controlled by elements of

the T cell response, and is accompanied by induction of multiple cytokine and

chemokine mRNAs in the CNS compartment. Evidence suggests that CD4+ T cell

responses are central to both control of infection and demyelinating disease, hence Dr.

Buchmeier proposes three specific aims to elucidate details of this virus-host interaction.

These are: 1) to investigate in CD4 knockout mice the requirements for demyelination; 2)

to investigate in C57B1/6 and B6CD4 knockout mice the pathogenesis of virus-induced

acute and chronic demyelinating disease and to seek evidence of an antiself response

against components of the myelin sheath triggered by virus infection; and 3) to analyze

by in situ hybridization, immunohistochemistry and PCR the state and cellular sites of viral persistence within the CNS following infection. Coronaviruses are widespread upper respiratory and enteric pathogens in man and animals, and coronavirus RNA has recently been described in the brains of human multiple sclerosis patients. The studies proposed will reveal basic information in interpreting the host-virus relationship in coronavirus infections, how they cause persistent infections, and the mechanisms of pathogenesis of demyelinating disease. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: CORE--NEUROPATHOLOGY Principal Investigator & Institution: Lavi, Ehud; University of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104 Timing: Fiscal Year 2002 Summary: The core facility will consolidate all the morphological services for the use of the various projects. These services include histological preparations such as tissue processing, embedding sectioning and staining. Following histological processing the core will provide evaluation and interpretation of histopathological changes in tissue sections. These include evaluation and quantification of demyelination during coronavirus infection in mice, evaluation of virus-induced cerebrovascular disease in murine leukemia virus infection in mice, and analysis of HSV receptor expression in human brain. Computerized morphometric analysis will be offered as necessary. Additional morphological services or assistance will be provided for studies which require immunohistochemistry, immunofluorescence, in situ hybridization, in situ-PCR, and electron microscopy. Thus the core facility services are essential to the success of the fundamental goal of the project. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: CORE--NEUROPATHOLOGY FACILITY Principal Investigator & Institution: Weiner, Leslie P.; University of Southern California 2250 Alcazar Street, Csc-219 Los Angeles, Ca 90033 Timing: Fiscal Year 2002 Summary: A DI vector core is a new addition to this program. It will support the work described in the individual sections and will be headed by Dr. Lai. DI vectors are proposed to explore the issues of altered pathogenesis via expression of viral structural proteins, cytokines and inhibitors of the immune response. The lack of an infectious cDNA clone for any coronavirus, which would allow stable expression of these factors, provides the basic rationale for DI expression. We demonstrated the high rate of coronavirus recombination; however, the use of recombinant or variant viruses with limited numbers of mutations are significantly hampered by the requirement to completely sequence the 31 kb genome to demonstrate that any alterations in pathogenesis are indeed due to the mutation under study. A centralized core to produce the DI vector pools will provide both uniformity and quality control. Experiments examining the pathogenesis of MHV-A59 using the DI vector to express the JHMV HE protein were carried out over a three year period and are a summary of over 10 separate experiments. We noted no variation in any of the four pools prepared by Dr. Zhang; however, consistency in preparation, construction of the vector RNA, monitoring the DI gene expression as well as routine titer determinations will prevent any loss of time and effort within the individual projects. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen 8 Coronavirus • Project Title: CORONAVIRUS-RECEPTOR INTERACTIONS Principal Investigator & Institution: Holmes, Kathryn V.; Professor; Microbiology; University of Colorado Hlth Sciences Ctr P.O. Box 6508, Grants and Contracts Aurora, Co 800450508 Timing: Fiscal Year 2002; Project Start 01-APR-1988; Project End 30-JUN-2004 Summary: Human coronaviruses cause 15 to 30 percent of common colds, yet there has been little investigation of these human pathogens. Our long term goal is to develop anti-viral drugs to prevent and/or treat human coronavirus infections of the upper respiratory tract. We anticipate that these drugs would be used in combination with drugs to prevent or treat colds due to rhinoviruses which are now being developed in other laboratories. By covering the viruses that cause most common colds, such combination drugs would make it unnecessary to identify the type of common cold virus causing early symptoms before initiating therapy. Our strategy for developing anti-coronavirus drugs is to use information about viral spike protein and receptor structure and function to identify small molecules that may block interactions of the viral spike glycoprotein with its cellular receptor glycoprotein. Our lab has identified receptors for human, murine and feline coronaviruses. We have expressed and purified soluble recombinant receptor glycoproteins, and are testing them for receptor activities. We will use mutagenesis to identify the amino acids and domains of the viral spike proteins and receptor glycoproteins that interact, and test the mutant proteins for binding activity and the ability to induce or undergo conformational changes that lead to membrane fusion and virus entry. We will analyze the structures of the spike proteins and receptor glycoproteins using cryo-electron microscopy and X-ray crystallography. The resulting structural models will be used to develop small molecules that block virus binding and/or receptor induced conformational changes in the spike protein. These molecules will be tested in cell culture model systems for toxicity and for the ability to block virus infection. Non-toxic drugs with anti-viral activity in cultures will be tested in animal model systems for the ability to block coronavirus infections of the respiratory tract. Our experimental models are the interactions of two coronaviruses, mouse hepatitis virus (MHV) and human coronavirus 229E (HCoV-229E), with their receptors, MHVR and hAPN, respectively. The murine model is more advanced than the human model, and will provide information and strategies that will facilitate our studies on human coronavirus-receptor interactions. The MHV model will also provide fundamental information on how an immunoglobulin-related receptor initiates infection with an enveloped virus. The 229E model will elucidate how the enzyme APN acts as a virus receptor. Inhibition of HCoV-229E respiratory infection by candidate drugs will be studied in transgenic mice that express the HCoV-229E receptor, hAPN, in the respiratory epithelium. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: DECIPHERING -1 FRAMESHIFT CIS ACTING DOMAINS IN BYDV Principal Investigator & Institution: Staplin, William R.; Plant Pathology; Iowa State University Ames, Ia 500112207 Timing: Fiscal Year 2003; Project Start 23-JUN-2003; Project End 22-JUN-2006 Summary: (provided by applicant): Frameshift is relevant to both plant and mammalian viruses, including Human Immunodeficiency Virus Type 1 (HIV-1) and Human Coronavirus (HCV). This frameshift allows for the translation of the RNA dependent RNA polymerase (Rd-RP). BYDV is a well-characterized plant virus with several unique translational processes, including a long distance base pairing which allows -1 Studies 9 frameshift. Nucleotides on the bulge of the adjacent downstream stem loop (ADSL) base pair to the long distance frameshift element (LDFE), four kilobases downstream. This novel mechanism requires further characterization since other cis acting components both distal and proximal to the frameshift site were found to influence frameshift. These regions will be assessed through structural analysis, site directed mutagenesis and in vitro and in vivo frameshift assays within wheat and oat protoplast transfection systems to quantitatively measure frameshift efficiencies. These assays will delineate the responsible RNA domains, necessary for frameshift. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: DEVELOPING A RECOMBINANAT VACCINE FOR SARS Principal Investigator & Institution: Zhang, Xuming; Associate Professor; Microbiology and Immunology; University of Arkansas Med Scis Ltl Rock Little Rock, Ar 72205 Timing: Fiscal Year 2004; Project Start 01-APR-2004; Project End 31-MAR-2006 Summary: (provided by applicant): The current epidemic of the severe acute respiratory syndrome (SARS) is caused by a novel coronavirus, SARS-CoV. To date, the global confirmed SARS cases have reached >8,000 with L800 deaths in 30 countries. With an ease of transmission and severity of the disease, SARS poses a great threat to public health and causes significant economic loss. The long-term goal of this proposed research is to develop an efficacious vaccine for preventing future SARS epidemic. While various types of vaccines have been developed for different illnesses, current information obtained from studies on animal coronaviruses and their respective hosts suggests that the most promising vaccine for SARS would be a coronavirus-based live vaccine. However, the development of an attenuated, live SARS-CoV vaccine is a longterm approach with an unpredictable outcome. Previously, the P.I. has isolated a human enteric coronavirus (HECoV) associated with acute, mild diarrhea but with no other severe clinical symptoms. Based on the tenet of a common mucosal immune system, antigenic stimulation by oral immunization is usually very effective in inducing immunity to respiratory pathogens. Therefore, the P.I. proposes (i) to develop a recombinant human enteric coronavirus expressing the spike protein of the SARS-CoV as a vaccine for SARS, and (ii) to characterize the biological properties of the recombinant coronavirus. These studies can be accomplished within the proposed, short period of time and will provide crucial information about the utility of the recombinant coronavirus as an expression vector and as a candidate live vaccine for SARS. They will facilitate future clinical trials in animals and humans. Such a recombinant system will not only be important for the development of SARS vaccine but may provide potential avenues for the development of vaccines for other severe infectious diseases such as AIDS. It also provides a powerful molecular tool for studying pathogenesis and immunity of SARS-CoV. The practical impact of this research will be undoubtedly enormous. This research thus represents the exploratory nature of the R21 mechanism. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: DEVELOPMENT OF VLP VACCINES FOR SARS PREVENTION Principal Investigator & Institution: Compans, Richard W.; Professor and Chair; Microbiology and Immunology; Emory University 1784 North Decatur Road Atlanta, Ga 30322 Timing: Fiscal Year 2004; Project Start 01-JUL-2004; Project End 30-JUN-2006 Summary: (provided by applicant): The focus of this project will be to develop novel virus-like particle (VLP)-based immunogens for eliciting strong neutralizing antibody 10 Coronavirus responses against severe acute respiratory syndrome (SARS) coronavirus. Previous studies with coronaviruses have shown that co-expression of the M and E glycoproteins can lead to the formation of virus-like particles and their release from the cell. Furthermore, when the spike (S) protein is expressed along with the M and E proteins, it can be incorporated into the coronavirus VLPs. We plan to achieve our goals in three phases. First, based on the results obtained in our preliminary studies and our experience in VLP design and production, we will develop "first generation" SARS CoV VLPs consisting of its M, E and S proteins or "first generation" chimeric VLPs formed by SIV Gag proteins that incorporate high levels of SARS CoV S proteins. Second, we will investigate whether VLPs formed by other viral core proteins will provide a better framework for the development of VLP-based vaccines against SARS. Third, we will explore alternative approaches to develop "second generation" phenotypically mixed VLPs to enhance their immunogenicity. Specifically, we will focus on two promising strategies: 1) To design phenotypically mixed VLPs for targeting to antigen-presenting cells to augment immune responses (ligand for fit3, mannose receptor); and 2) To design phenotypically mixed VLPs for targeting to mucosal surfaces to enhance mucosal immune responses (inclusion of HA, HN). Through this systematic approach, we will explore different strategies to design and produce novel SARS VLPs, compare their efficacies in inducing immune responses in small animal models and determine optimal immunization routes (mucosal immunization by intranasal route vs. systemic immunization via intramuscular injection), with the aim to develop candidate vaccines for large-scale production. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: ENTRY OF CORONAVIRUSES INTO HOST CELLS Principal Investigator & Institution: Whittaker, Gary R.; Assistant Professor; Microbiology and Immunology; Cornell University Ithaca Office of Sponsored Programs Ithaca, Ny 14853 Timing: Fiscal Year 2004; Project Start 01-JUN-2004; Project End 31-MAY-2006 Summary: (provided by applicant): Entry of coronaviruses into host cells is an underexplored area of virology research. In this application, we propose to use infectious bronchitis virus (IBV) of chickens as a model that would be directly applicable to highly pathogenic human coronaviruses, such as SARS-CoV. The major goal of this application is to define basic information regarding the fusion mechanism and route of internalization of IBV. Certain important gaps remain in our understanding of coronavirus entry: specifically 1) the fact that, to date, no virus--cell fusion assays have been employed to study coronavirus entry, and 2) although coronaviruses are generally considered to undergo cell--cell fusion at neutral pH, previous data also suggest a pHdependent route of entry through endosomes. To address these issues, we have two specific aims: 1 - To develop a virus-cell fusion assay for coronaviruses. Using protocols in our laboratory to study influenza virus--cell fusion, a principal goal is to develop a fluorescence-based virus-cell fusion assay for the coronavirus IBV. 2 - To determine the internalization route of IBV into host cells. We have previously characterized the entry of other viruses using a combination of molecular, pharmacological and morphological approaches. Using similar techniques, a second principal goal is to define the role of endocytosis during IBV entry. Our work is designed not only to elucidate the basic entry mechanism of IBV, but also to serve as a model for coronaviruses in general--especially highly pathogenic human coronaviruses, such as SARS-CoV. IBV would serve as an excellent model system to understand coronavirus entry and fusion, and facilitate the development of future anti-viral drugs. Studies 11 Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: IMMUNITY AND VIRUS DISEASE Principal Investigator & Institution: Welsh, Raymond M.; Professor; Pathology; Univ of Massachusetts Med Sch Worcester Office of Research Funding Worcester, Ma 01655 Timing: Fiscal Year 2004; Project Start 01-JUL-1980; Project End 31-MAR-2009 Summary: (provided by applicant): Despite many advances in biomedical research, viral infections remain a major threat to human health. In the past 20 years we have witnessed either the emergence or the definition of acute and persistent viral infections caused by agents such as HIV, HTLV, hepatitis viruses, ebola virus, hantavirus, papillomavirus, herpesviruses 6-8, the SARS coronavirus, and monkeypox, and we continue to live with the specter of the re-emergence of a 1918-like strain of influenza virus or the release of variola (small pox) virus by bioterrorism. There are few effective antiviral drugs to combat these viruses, and protection from these agents mainly rests on public health containment measures and on the use and development of vaccines. It is now thought that attenuated viruses which induce CD8 T cell responses, as well as antibody, make better, longer lasting vaccines. Thus, it is important to understand factors contributing to the maintenance or loss of CD8 memory in an environment in which a host is continually challenged by pathogens that may sometimes become persistent. The CD8 T cell response to viral infections is highly dynamic, beginning with a cytokine driven apoptotic loss (lymphopenia) in memory CD8 T cell number in the early stages of infection, followed by a dramatic expansion in number of virus-specific T cells and then by a second decline or silencing phase, associated with T cell apoptosis and dissemination into peripheral tissues. The activated CD8 T cells during the acute response also become sensitized to undergo Fas/FasL-mediated activation-induced cell death (AICD) on strong signaling through their TCR, and this may contribute to the transient immune deficiencies seen during viral infections and to the clonal exhaustion of T cells under conditions of antigen excess. My lab has made the unique observations that (1) there is a cytokine-driven apoptosis early in infection, that (2) apoptosis of CD8 T cells is differentially regulated, dependent on the tissue site, and that (3) there is substantial attrition of memory CD8 T cells occurring as a consequence of acute or persistent infections with unrelated viruses. Here we propose to continue our studies on CD8 T cell apoptosis in mice infected with LCMV, Pichinde, and vaccinia viruses, and determine (1) the mechanism and significance of the early cytokine-induced lymphopenia and apoptosis of memory CD8 T cells during viral infections, (2) the mechanisms regulating the tissue-dependent differences in apoptosis of virus-specific CD8 T cells, and (3) the mechanism and significance of memory T cell attrition following acute and persistent viral infections. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: IMMUNOPATHOGENESIS OF VIRUS-INDUCED DEMYELINATION Principal Investigator & Institution: Perlman, Stanley; Professor; Pediatrics; University of Iowa Iowa City, Ia 52242 Timing: Fiscal Year 2002; Project Start 01-SEP-2000; Project End 31-AUG-2004 Summary: C57B1/6 (B6) mice infected with the neurotropic coronavirus, mouse hepatitis virus, strain JHM, develop a demyelinating encephalomyelitis with clinical signs of hindlimb paralysis. The clinical and pathological features of this disease have many similarities to the human disease, multiple sclerosis. One important similarity between MS and the MHV-infected mouse is that the host immune response is critical 12 Coronavirus for the development of demyelination. The applicant has recently shown that MHV infection of mice that lack mature T and B cells due to a deficiency in recombinaseactivating gene activity (Rag1 -/- mice) do not develop demyelination unless splenocytes from immunocompetent B6 are adoptively transferred. Demyelination then occurs reproducibly in 6-7 days. In addition, he has adapted methods for identifying antigen-specific CD4 and CD8 T cells (soluble MHC/peptide tetramer assays and assays for measuring intracellular interferon-gamma production) to the direct ex vivo analysis of lymphocyte harvested from mice with MHV-induced neurological disease. The central hypothesis of this proposal is that adoptively transferred CD4 or CD8 T cells secrete a factor or perform a function (or both) that is critical for the induction and/or propagation of the demyelinating process in Rag -/- mice. This hypothesis will be approached in the following three specific aims. 1. To determine if antigen-specific CD4 or CD8 T cells in the absence of the other subset are sufficient to induce and propagate the demyelinating process. The contribution of Fas/FasL interactions and of specific chemokines and cytokines will also be determined. 2. To investigate the role of nitric oxide and axonal degeneration, two potentially key components of the pathogenic process, in the adoptive transfer model of demyelination. 3. To determine whether MHV antigen-nonspecific CD4 T cells become activated and contribute to demyelination in virus-infected mice. These experiments will take advantage of recent advances in our ability to detect antigen-specific CD4 T cells in the MHV-infected CNS. Demyelination is rapid and reproducible after adoptive transfer into the MHV-infected Rag1 -/- mice, making this a unique system for investigating the pathogenesis of virus-induced demyelination and answering questions about virus-induced demyelination. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: INTRACELLULAR ASSEMBLY OF THE CORONAVIRUS, IBV Principal Investigator & Institution: Machamer, Carolyn E.; Associate Professor; Cell Biology; Johns Hopkins University 3400 N Charles St Baltimore, Md 21218 Timing: Fiscal Year 2002; Project Start 01-APR-2002; Project End 31-MAR-2006 Summary: (provided by applicant): All enveloped viruses exploit the cellular secretory pathway for biosynthesis of their membrane proteins. The best studied enveloped viruses assemble by budding from the plasma membrane, where their membrane proteins accumulate. Assembly of enveloped viruses at intracellular membranes is less well understood, although these viruses must also accumulate their membrane proteins at the budding site. Intracellular assembly of the avian coronavirus infectious bronchitis virus (IBV) in the cis Golgi network will be studied. Coronaviruses are ubiquitous in vertebrates, and in humans cause mild respiratory disease (responsible for about 20 percent of common colds). Coronaviruses are conveniently studied in cell culture systems, and are thus an ideal model for intracellular virus assembly. Understanding the intracellular assembly of enveloped viruses is important because several virus families that cause significant human disease assembly at intracellular membranes. These include Bun yaviridae and Flaviviridae. The long term goals of the proposed experiments are to elucidate the mechanism and advantages of intracellular assembly of enveloped viruses, and to identify unique strategies to interfere with assembly and infection by this subset of viruses. Specifically, the experiments are designed to test the following hypotheses: (1) the IBV RNA 3 proteins (3a, 3b, and E) play important roles in virus assembly and infection; (2) the IBV E protein has an additional function in infected cells to slow membrane traffic, allowing S to accumulate at the budding site and possibly preventing virus antigen presentation to the immune system; and (3) distinct Studies 13 envelope lipids (derived from the cis Golgi network) provide an advantage for the virus during subsequent rounds of infection by promoting fusion with susceptible cells. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: MECHANISMS OF CORONAVIRUS RNA AMPLIFICATION Principal Investigator & Institution: Brian, David A.; Professor; Pathobiology; University of Tennessee Knoxville Knoxville, Tn 37996 Timing: Fiscal Year 2002; Project Start 01-JUL-1977; Project End 31-MAY-2007 Summary: (provided by applicant): Coronavirus-induced diseases in humans and animals are widespread and include acute respiratory disease, gastroenteritis, hepatitis, nephritis, and acute and chronic encephalitis. This proposal seeks to characterize five cis-acting higher-order RNA elements and one cis-acting process (translation) required for the replication of a coronavirus helper-dependent minireplicon (Dl RNA). These features are presumed requirements for the viral genome as well and are postulated to function in the formation of the RdRp replication complex. They also specifically address the enigma of how it is that coronavirus subgenomic mRNAs, synthesized on mRNA-length double-stranded intermediates and possessing termini identical to those on the genome (minimally 65 5' and 1633 3' nts), fail to replicate following transfection into helper virus-infected cells. The Dl RNA replicon is a 2.2 kb fusion product of the virus genomic termini and differs from mRNA 7 by only 421 nts of additional contiguous 5'-proximal sequence. It is postulated that sgmRNAs lack 3 of the necessary 5'-proximal signals for replication. The study has three specific aims: (1) To characterize the role of the phylogenetically-conserved 5'-proximal stem-loops Ill, IV, and V, including identification of the viral and cellular protein(s) that bind stem-loop III and the cellular protein(s) that binds stem-loop IV. (2) To characterize the cis-acting translation requirements for the (fused) partial la and entire N ORFs. (3) To characterize the 3' UTR cis-acting features of the phylogenetically conserved pseudoknot and its associated upstream bulged stem-loop, and the phylogenetically conserved octamer and its associated heptameric helix. Analyses will employ RNA structure probing, sitedirected mutagenesis, mass-spectrometric identification of binding proteins, and assays for DI RNA minus-strand synthesis, replication and packaging. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: MICROMECHANICAL SENSORS FOR VIRUS DETECTION Principal Investigator & Institution: Bashir, Rashid; Associate Professor; Electrical Engineering; Purdue University West Lafayette West Lafayette, in 479072040 Timing: Fiscal Year 2002; Project Start 30-SEP-2002; Project End 31-AUG-2004 Summary: (provided by applicant): The recent technological advances in nanotechnology and micromachining of semi-conductor materials present themselves with new opportunities for cheap, small, and sensitive diagnostic devices capable of rapid and highly accurate detection of infectious agents. Surface derivitized cantilever structures have successfully been applied to the detection of DNA, proteins and cells, yet still fully realized at the levels of sensitivity required for practical applications. In addition, detection of viruses has not been fully explored with these technologies. The application of this technique in to detection of aerosolized virus particles is the main goal of the current proposal. This proposal brings together a group of truly interdisciplinary researchers from the fields of micro/nano-systems technology, molecular biology and virology, and bio-separations engineering to develop microcantilever-based virus detection techniques and systems which promises performance 14 Coronavirus characteristics exceeding the sensitivity and specificity of PCR amplification assays and ELISAs. Calculated limits of detection of our approach are 10-17 to 10 -18 gm of mass change on the cantilever surface. This translates to the mass of single virus particles. When this method is coupled to currently available monoclonal antibodies against viruses, its specificity could surpass ELISAs since our technique doesn't rely on enzymatic reaction kinetics as does the former. The ability to detect and monitor-in realtime and continual basis- of viruses and their subtypes, particularly the most contagious viruses and bioterrorism agents, can have drastic implications in the confinement and management of the viral epidemics. The long-term objective of this application is to develop a micro-scale, robust, real-time monitoring device, based on micro-machined ultrathin cantilever arrays for the rapid and sensitive detection of infectious agents, particularly bioterrorism agents in field setting and in primary-patient care facilities. The array will be specific for specific pathogens and will have the sensitivity to detect a single virus or toxin molecule. During Phase I, the proposed effort aims to develop dielectrophoresis-based infectious agent trapping, separation and concentration device and a proof-of-principle demonstration for the detection of an air-borne virus on functionalized micro-scale cantilever. The performance value of the devices for trapping, separation, concentration and detection of aerosolized coronavirus particles will be assessed. During Phase II, this sensor design and manufacturing capabilities will be extended and scaled-up to other infectious agents in the form of integrated sensor arrays with capability for on-board signal processing. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: MOLECULAR ANALYSIS OF CORONAVIRUS ASSEMBLY Principal Investigator & Institution: Hogue, Brenda G.; Associate Professor; Microbiology; Arizona State University P.O. Box 873503 Tempe, Az 852873503 Timing: Fiscal Year 2003; Project Start 15-SEP-2003; Project End 29-FEB-2008 Summary: (provided by applicant): Coronaviruses (CVs) are widespread, medically important respiratory and enteric pathogens of humans and many domestic animals, causing a significant portion of human upper respiratory infections. These enveloped viruses contain a positive(+)-sense, single-stranded RNA genome that is the largest (approximately 30 kb) of all the RNA viruses. Many questions remain to be answered about the molecular details of assembly of enveloped RNA viruses. The studies proposed herein focus on the mechanism of assembly of mouse hepatitis virus (MHV) that acquire their envelopes by a nucleocapsid independent manner at membranes of the endoplasmic reticulum Golgi intermediate compartment (ERGIC). Virus-likeparticles (VLPs) assemble when only the small envelope (E) and membrane (M) proteins are coexpressed. We hypothesize that E performs its role through its interplay with M and the ERGIC lipid membranes and possibly host proteins. Previous studies addressing the role of E and M in virus assembly relied on virus-infected cells, VLPs, and targeted RNA recombination. With the availability of a new MHV infectious clone we are uniquely positioned to directly manipulate the virus genome to study the mechanism by which E and M function in virion assembly. In Aim 1 we will focus on understanding the role of the E and M proteins in targeting assembly to intracellular membranes. We will precisely identify the site of intracellular localization for E. The retention signal in E and domains involved in potential co-localization with M will be identified. The role of E, M and host proteins will be studied to determine their roles in assembly/budding at intracellular membranes. Aims 2 and 3 are to understand the roles of the E and M proteins in virion formation using VLPs, the MHV infectious clone and replicons. We will use biochemical and microscopic analyses of chimerics and site- Studies 15 specific mutants of E and M expressed from various vectors and the MHV infectious clone to study the proteins, subcellular fractions and assembly of wild-type and altered VLPs and viruses. Information gained from this study can be used to (i) increase our understanding of fundamental mechanisms by which viruses of medical importance acquire their envelopes at intracellular membranes, (ii) identify major targets for antiviral drug development, (iii) assist in development of CV heterologous gene expression vectors for vaccine use, and (iv) contribute to understanding of proteinprotein and protein-membrane interactions and transport. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: MOLECULAR DISSECTION OF THE CORONAVIRUS SPIKE Principal Investigator & Institution: Gallagher, Thomas M.; Associate Professor; Microbiology and Immunology; Loyola University Chicago Lewis Towers, 13Th Fl Chicago, Il 60611 Timing: Fiscal Year 2003; Project Start 15-DEC-1993; Project End 30-JUN-2008 Summary: (provided by applicant): The enveloped, RNA-containing murine coronaviruses include a large collection of strains. Each strain infects different tissues and thus causes a distinct disease such as hepatitis, gastroenteritis, or chronic encephalomyelitis; the latter serves as a model for human neurodegenerative diseases. This proposal will elucidate coronavirus entry mechanisms, and in doing so will explain how very similar strains cause such dramatically different diseases. A key determinant of coronavirus tropism is the spike (S), a protein that carries out essential virus entry functions. S proteins bind to carcinoembryonic antigen-related cell adhesion molecule (CEACAM) receptors, which cause conformational changes culminating in virus-cell membrane fusion. The first aim will characterize the structural changes following interactions between S and CEACAM. We will specifically focus on novel biochemical features of the S proteins, such as their thiol-disulfide reactivities, as they relate to structural changes required for virus entry. We expect to provide new insights into protein-mediated membrane fusion reactions. The second aim will determine how cholesterol operates as a cofactor to support coronavirus entry. We will develop in vitro assays for virus fusion and use them to identify cholesterol-dependent stages of the Smediated fusion process. The third aim will connect our biochemical studies of coronavirus entry with in vivo investigations of disease. This will be accomplished by comparing viruses of variable neurovirulence in controlled in vitro measurements of SCEACAM binding and S-induced membrane fusion. Our results will relate the biochemical properties of the coronaviruses with their pathogenesis. Our collective findings will expand current knowledge of virus entry, and help set the stage for future antiviral drug developments. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: MOLECULAR STUDIES OF CORONAVIRUS REPLICATION Principal Investigator & Institution: Makino, Shinji; Professor; Microbiology and Immunology; University of Texas Medical Br Galveston 301 University Blvd Galveston, Tx 77555 Timing: Fiscal Year 2002; Project Start 01-JUL-1990; Project End 30-NOV-2006 Summary: (provided by applicant): Coronaviruses cause disease in man and animals. This group of single-stranded, positive-sense, enveloped RNA viruses causes a range of serious gastrointestinal and upper respiratory tract infections. Our overall objective is to learn how the parts of this viral pathogen assemble, using mouse hepatitis virus (MHV) 16 Coronavirus as a coronavirus model system. Some experiments will ask whether a virus protein if not involved in assembly exerts an effect on infectivity. The packaging signal (PS) of MHV is a short RNA sequence that is necessary and sufficient for MHV to package RNA into virion. We observed a specific interaction between MHV envelope protein M and the MHV PS, which occurred in the absence of N protein. This observation suggested a previously unforeseen possibility that specific binding of the PS to the M membrane glycoprotein is the initiator of MHV's precise RNA packaging. We propose to describe the MHV viral RNA packaging initiation mechanism, and will start by determining whether or not the PS directly binds M. Assuming M directly binds the PS, then we will identify exactly which M region binds to the PS. We will investigate the possibilities that RNA with the PS drives N protein-M protein interaction, and that binding of M to multiple regions outside of the PS on the genome-length MHV mRNA occurs in infected cells. Coexpression of E and M proteins results in the production of virus-like particles (VLP), and this was shown to be temperature sensitive (ts). This ts phenotype of VLP production will be used to prove that M interacts with E protein and to determine whether or not M oligomerizes at MHV budding sites during VLP production; the biological importance of these putative processes will be investigated. We will also study whether N is needed for RNA packaging, because we speculate that it is dispensable for packaging and central to infectivity. If our speculation is correct, then whether or not VLPs lacking the N are infectious will be determined. The case that VLPs lacking the N are not infectious would mean that an N domain(s) is important for viral infectivity, and these will be identified. How packaged RNA and M interact in the presence of N will be studied. If, however, the case turns out to be that N is necessary for RNA packaging, then we will look at the importance of N protein-M protein interaction in RNA packaging. Any N domain(s) affecting viral RNA packaging will be investigated. Finally, we will study whether homotypic interaction of N exists in the viral nucleocapsid, and if it does, we will look at whether the PS or M protein triggers this. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: MURINE CORONAVIRUS RNA SYNTHESIS Principal Investigator & Institution: Lai, Michael Mc.; Distinguished Professor; Molecular Microbiol and Immun; University of Southern California 2250 Alcazar Street, Csc-219 Los Angeles, Ca 90033 Timing: Fiscal Year 2002; Project Start 01-AUG-1982; Project End 30-MAY-2005 Summary: (Adapted from the Investigator's abstract): The overall objective of this grant application is to understand the mechanism of regulation of the murine coronavirus mouse hepatitis virus (MHV). This virus has the largest RNA genome (31 kb) and a complex mechanism of RNA transcription; thus, it potentially serves as a model for RNA viruses in general. Dr. Lai has previously identified two cellular proteins, heterogeneous nuclear ribonuclear protein (hnRNP A1) and polypyrimidine tractbinding protein (PTB), which bind to the cis-acting regulatory sequences of MHV RNA. In this project, the roles of these cellular proteins in MHV RNA transcription and the mechanism of formation of the transcription complex will be studied. The specific aims are: 1.Determination of the functional roles of PTB and hnRNP A1 in MHV RNA synthesis: Using permanent cell lines expressing wild-type and dominant negative mutants of PTB and hnRNP A1, the composition of RNP complex involving cellular and viral proteins will be characterized. 2.Characterization of additional cellular factors involved in MHV RNA synthesis: Other cellular proteins interacting with components of viral transcription complex will be examined by yeast two hybrid screening. Studies 17 3.Characterization of proteins involved in MHV RNA synthesis: The role of viral polymerases will be determined by expressing dominant negative mutants of various gene 1 protein products and by performing protein depletion studies. To complement these studies, targeted recombination within gene 1 will be performed. 4.Study of the effects of hnRNP A1 and PTB on viral translation and cellular RNA splicing: The potential effects of these cellular proteins on regulation of viral translation will be studied. Finally, the potential effects of viral RNA synthesis on the cellular RNA processing, with particular emphasis on the possible alterations of alternative RNA splicing will be examined. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: PATHOGNESIS OF MOUSE HEPATITIS CORONAVIRUS Principal Investigator & Institution: Weiss, Susan R.; Professor; University of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104 Timing: Fiscal Year 2002 Summary: Indirect evidence implicates a virus in the etiology of Multiple Sclerosis (MS) and more direct evidence shows that viruses can cause other human demyelinating diseases. Coronavirus, mouse hepatitis virus (MHV), strain A59 infection of mice is a good animal model system for the study of virus induced demyelination. After the initial acute encephalitis, virus becomes impossible to detect during the chronic stages of disease while viral nucleic acids may be detected in white matter. Furthermore, MHVA59 causes a persistent, productive, but non-lytic infection in primary glial cells in culture. The long term goal of this project is to determine the molecular basis for MHVA59 persistence in the mouse central nervous system (CNS) and to determine the relationship between viral persistence and chronic demyelination. In this proposal we plan to continue to analyze a weakly demyelinating mutant of MHV-A59 (C12), that we have isolated from persistently infected glial cell cultures. We will compare the pathogenesis of wild type MHV-A59 with C12, to understand the differences between these viruses that result in the weakly demyelinating phenotype of the mutant. More specifically we will compare these viruses as far as persistence in the CNS and the ability to induce demyelination and determine whether persistence is necessary for demyelination. The C12 mutant has five amino acids substitutions in the entire genome compared to wild type virus, tow in the spike protein and three in the replicase. We have demonstrated that the weakly demyelinating phenotype correlates with the presence of Q159L in a receptor binding of the spike protein. We will use in vitro assays to ask whether this mutation alters the interaction if virus with several forms of the MHV receptor or with several CNS cell types. We have recently used targeted recombination to introduce each of the two spike mutations, Q159L and H716D, into recombinant viruses. These will be used to determine whether Q159L is sufficient to result in the weakly demyelinating phenotype. More generally, the recombination technology represent an important step forward in the analysis of the pathogenesis of MHV-A59 in allowing us for the first time to introduce specific mutations into the spike protein and determine the relationship between sequence and pathogenesis. In the final portion of this proposal we will use this approach to introduce the spike gene of the more neurovirulent MHV-4 (JHM) into the A59 genome. This will allow us to determine whether the properties of increase virulence and panencephalitis of MHV-4 are due entirely to the spike protein and if so where they map within the protein. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen 18 Coronavirus • Project Title: PATHOLOGICAL MECHANISMS OF DEMYELINATION Principal Investigator & Institution: Lane, Thomas E.; Assistant Professor; Molecular Biology and Biochem; University of California Irvine Irvine, Ca 926977600 Timing: Fiscal Year 2002; Project Start 01-FEB-1998; Project End 31-JAN-2003 Summary: (Applicant's Abstract): The underlying mechanisms involved in the pathogenesis of the human demyelinating disease multiple sclerosis (MS) are not well understood. Available evidence indicates that the cause of MS is multifactorial and includes both the genetic background of the host as well as environmental influences. Infection of susceptible strains of mice with the coronavirus mouse hepatitis virus (MHV), a positive-stranded RNA virus, leads to a chronic, progressive, demyelinating disease with many clinical and histologic similarities with MS. Animals often develop partial to complete hind-limb paralysis. Although viral RNA persists in the CNS, the host immune response is thought to be the key factor in contributing to demyelination. T-lymphocytes and macrophages are associated with demyelinating plaque lesions in both MS patients as well as MHV-infected mice. The long range goal of this research proposal is to define the cellular mechanisms mediating demyelination in MHV-infected mice. First, emphasis will be placed on the role of the CD4+ T lymphocytes. These cells appear to be crucial in participating in the demyelinating process most likely by release of soluble factors which activate both inflammatory macrophages and resident glia to release cytotoxic factors. A second goal of this project is to study the signals i.e. chemokines which function in attracting CD4+ T cells as well as macrophages to the CNS to sites of viral persistence. Finally, this proposal will analyze the contributions of cytotoxic factors tumor necrosis factor alpha (TNF-a) and nitric oxide (NO) in MHVinduced demyelination. Together, these studies will provide insight into the complex immunological mechanisms involved in CNS demyelinating disease and allow a better understanding of the pathological mechanisms which exist in human demyelinating disease. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: POLYMERASE PROTEINS IN CORONAVIRUS REPLICATION Principal Investigator & Institution: Denison, Mark R.; Associate Professor; Pediatrics; Vanderbilt University 3319 West End Ave. Nashville, Tn 372036917 Timing: Fiscal Year 2002; Project Start 30-SEP-1991; Project End 29-FEB-2004 Summary: The goal of our research is to define the functions of the coronavirus gene 1 proteins in viral replication. Mouse hepatitis virus (MHV) contains a 32 kb singlestranded, positive-sense RNA genome and synthesizes its polymerase gene products by translation and processing of an 803 kDa polyprotein from gene 1 of the input genome RNA. Many of the proteins processed from the gene 1 polyprotein have been identified, and the importance of the gene 1-encoded 3C-like proteinase in polyprotein processing has been established. However, many of the identified and predicted polyprotein cleavage products, including the putative RNA-dependent RNA-polymerase and helicase have not been experimentally confirmed. Furthermore, the importance of gene 1 proteins in formation and function of the MHV replication complex is not understood. This proposal describes a research program that will define the translation, processing, and localization of gene 1 proteins in virus infected cells. In addition, the gene 1 proteins in the viral replication complex will be identified. In Aim 1, the complete pattern of expression and processing of the gene 1 polyprotein will be determined in virus-infected cells, and the precise termini of the processed proteins will be identified. In Aim 2 immunofluorescence and laser confocal microscopy will be used to define the location of Studies 19 gene 1 proteins and their association with sites of MHV RNA synthesis and MHV structural proteins during MHV infection. In Aim 3 the viral protein, RNA, and membrane components of MHV replication complexes will be isolated from infected cells and the interactions of gene 1 proteins in the complexes will be defined. These experiments will precisely identify mechanisms by which coronaviruses regulate the availability and interactions of proteins essential for RNA transcription and replication and will determine how MHV proteins target intracellular sites of viral replication. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: PORCINE RESPIRATORY CORONAVIRUS AS A SARS MODEL Principal Investigator & Institution: Saif, Linda J.; Professor; Food Animal Hlth Research Prog; Ohio State University 1960 Kenny Road Columbus, Oh 43210 Timing: Fiscal Year 2004; Project Start 01-JUL-2004; Project End 30-JUN-2008 Summary: (provided by applicant): Severe acute respiratory syndrome (SARS) is a newly emerging global disease of humans with a major economic impact and significant bioterrorism potential caused by a new strain of coronavirus (CoV). The lung is the target organ related to the disease manifestations, although diarrhea occurs in some patients. Unresolved questions related to SARS pathogenesis include the mechanisms for "superspreaders" and the atypical pneumonia and variable diarrhea induced and the role of polymicrobial infections in the variable severity of SARS. Host immune factors, especially proinflammatory cytokines may play a role in the severe pulmonary damage, as observed in our studies of respiratory disease in pigs. The widespread use of steroids and IFNs for treatment of SARS patients without a clear understanding of their impact on respiratory disease, necessitates studies of their impact in an animal model susceptible to respiratory CoV infection. Although primates are susceptible to SARS CoV, their limited availability and expense hampers comprehensive studies of SARS pathogenesis. In mouse models, the clinicopathological manifestations of CoV or influenza viral infections differ from in humans whereas in pigs they mimic the human disease. The anatomy, physiology and immune system of the pig respiratory tract closely resembles that of man, providing a unique animal model for the study of viral respiratory disease of humans. The porcine respiratory CoV (PRCV), a spike deletion mutant of the enteric CoV transmissible gastroenteritis virus (TGEV), shows striking pathogenetic similarities to SARS CoV in its primary replication in lung. Of interest, PRCV invariably induces similar lung lesions with atypical pneumonia, even in asymptomatic pigs. Our studies suggest that polymicrobial co-infections influence the severity of PRCV infection, lesions and disease via multiple mechanisms. These include the repertoire of proinflammatory cytokines or the cell infiltrates induced in lung, and the multiple cell types infected. Therefore our aim is to determine the influence of steroids and coinfections with respiratory viruses or bacterial derived components (and the cytokines induced) on the severity of a SARS-like respiratory coronavirus (PRCV) infection of swine. Our Specific Aims are: 1) To assess if corticosteroid treatment of PRCV-infected pigs has an impact on cytokines induced by PRCV or acquired immunity to PRCV and the subsequent course of PRCV infection and disease (mimic impact of steroids on SARS patients); 2) To investigate the impact of prior infection with a distantly related (Nidovirales) low pathogenic respiratory viral pathogen (arterivirus, PRRSV) on subsequent PRCV infection and disease (mimic dual SARS CoV and distinct respiratory CoV infections); 3) To explore the impact of initial infection with PRCV followed by subsequent infection with the respiratory viral pathogen swine influenza virus on PRCV infection and disease (mimic dual infections with SARS CoV and influenza); 4) To determine the impact of concurrent infection of pigs with two 20 Coronavirus antigenically related coronaviruses with distinct tissue tropisms (PRCV, respiratory and TGEV, enteric) on generation of PRCV/TGEV recombinants and coronavirus infection and disease (mimic SARS superspeaders with diarrhea); 5) To examine the impact of sequential inoculation of pigs with PRCV followed by bacterial cell wall components on cytokine production and disease (mimic impact of bacterial coinfections on bacterial coinfections on SARS). Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: RAPID NAT SYSTEM FOR POINT-OF-CARE SARS DIAGNOSIS Principal Investigator & Institution: Wang, Zihua; Iquum, Inc. 214 Lincoln St, Ste 300 Allston, Ma 02134 Timing: Fiscal Year 2004; Project Start 01-APR-2004; Project End 31-MAR-2005 Summary: (provided by applicant): The goal of this research application is to develop a rapid, easy to use, integrated nucleic acid test for severe acute respiratory syndrome (SARS) based on our proprietary lab-in-a-tube (Liat TM) platform. The Liat system enables the integration of sample preparation, multiplex nucleic acid amplification and real-time quantification in a single closed tube system. The recent SARS epidemic showed that more sensitive, rapid, and automated nucleic acid testing capability is needed at the local level to better diagnose patients and improve quarantine effectiveness. When completed, the proposed system will be capable of taking raw plasma or sputum samples and performing fully automated sample-to-answer SARS associated coronavirus (SCV) nucleic acid testing at the point-of-care in less than 1 hour. In meeting these requirements, the system will have broad applications in bio-defense and civilian healthcare. To enable the Phase I goals several SCV-specific minor groove binding (MGB)-TaqMan probes will be designed and validated. Viral RNA isolation using silica magneti6 beads or nucleic acid hybridization methodologies will be integrated into the lab-in-a-tube testing platform. Following probe design and RNA enrichment, the system will be validated for SCV detection sensitivity and SNP analysis in less than 1 hour from sample-to-answer using reverse transcription-polymerase chain reaction in our closed system Liar Molecular Analyzer prototype. We believe this proposed research will contribute significantly to two of NIAID's top priorities: 1) improving our nation's bio-defense capability, and 2) enhancing the diagnosis and treatment of infectious diseases. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: RECOMBINANT & LIVE ORAL SALMONELLA TYPHI HYBRID VACCINES Principal Investigator & Institution: Levine, Myron Max.; Professor; Medicine; University of Maryland Balt Prof School Baltimore, Md 21201 Timing: Fiscal Year 2004; Project Start 01-APR-1990; Project End 28-FEB-2009 Summary: (provided by applicant): In November 2002 in China, an outbreak of atypical pneumonia occurred in which a proportion of cases were very severe or fatal, and a high lethality was seen among elderly patients. The clinical syndrome began with fever, dry cough, myalgia and sore throat and progressed to atypical pneumonia. Outbreaks followed thereafter in 2003 in Vietnam, Hong Kong, Singapore, Canada, and Taiwan. Extraordinary characteristics of this global epidemic of "Severe Acute Respiratory Syndrome" (SARS) include the rapid isolation of the etiologic agent (a novel coronavirus; SARS-CoV), elucidation of the complete sequence of the viral genome, accelerated development of diagnostic tests, and rapid global exchange of clinical, Studies 21 epidemiologic and microbiologic information via the Internet by scientists and health officials in many countries. Investigators in the USA and Hong Kong were first to isolate from patients the novel coronavirus that is distinct from previously recognized groups of coronavirus. The underlying hypothesis of this research plan is that by appropriate manipulation of attenuated Salmonella enterica serovar Typhi (S. Typhi) and Shigella live vectors it will be possible to develop a mucosally-administered "prime-boost" vaccination strategy to prevent SARS. We will utilize attenuated S. Typhi or Shigella flexneri 2a live vector vaccine strains to deliver (via mucosal immunization) a Sindbis eukaryotic DNA replicon encoding the S (spike) and M (membrane) glycoproteins and the N nucleocapsid protein of the Urbani strain of the SARS-CoV to prime the immune system to recognize these coronavirus antigens. We will then boost the immune response by mucosally administering proteosomes (meningococcal outer membrane protein vesicles) to which the same SARS proteins are adsorbed (along with a lipopolysaccharide adjuvant). Virus-like Particles and attenuated S. Typhi expressing SARS peptide epitopes will serve as back-up boosting strategies. We will study whether these constructs can elicit the relevant immune responses, first in mice, then in cynomolgus monkeys, and finally in clinical trials in humans (the latter under separate funding). The induction of B and T cell memory pools will also be examined in monkeys. This approach aims to mimic the strong and broad immunity elicited by live virus vaccines with the inherent safety factor of not having to use putative attenuated live SARS virus derivatives. If the proposed vaccination strategy can indeed elicit broad, balanced and long-lasting immune responses in cynomolgus monkeys, these studies can be followed by a challenge (under respiratory pathogen biosafety level 3 containment) to assess the efficacy of the vaccine against wild type SARS-CoV. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: REVERSE GENETICS WITH A CORONAVIRUS INFECTIOUS CONSTRUCT Principal Investigator & Institution: Baric, Ralph S.; Associate Professor; Epidemiology; University of North Carolina Chapel Hill Aob 104 Airport Drive Cb#1350 Chapel Hill, Nc 27599 Timing: Fiscal Year 2002; Project Start 01-MAY-2001; Project End 30-APR-2005 Summary: (Provided by the applicant): Coronaviruses contain a 30Kb single-stranded, positive polarity RNA genome. Using a novel strategy and six adjoining cDNA subclones, we have developed an approach to systematically assemble a full-length infectious construct of the coronavirus, transmissible gastroenteritis virus (TGE), an economically important pathogen in swine. T7 transcripts derived from the full-length TGE construct were infectious and progeny virions were serially passage in permissive host cells. The availability of this cDNA construct will allow us to address fundamental questions in the biology of coronaviruses, which were previously untenable. In this application, we will use reverse genetics and the full length TGE construct to systematically address the role of gene order, cis and trans acting regulatory sequences, and N gene function in coronavirus transcription and replication. Aim 1. We hypothesize that the highly ordered coronavirus genome structure is selectively maintained to safeguard coordinated levels of virus gene expression. We will study the phenotypic consequences of gene deletion, duplication and rearrangement on TGE transcription and replication in vitro and determine the minimal genome requirements for coronavirus transcription. These studies will also determine if RNA recombination functions to maintain the precise gene order of Nidoviruses. Aim2. We will use site specific mutagenesis to distinguish between the hypotheses that discontinuous 22 Coronavirus transcription is guided by base pairing during negative or positive strand RNA synthesis. These studies will also define the minimal promoter elements in the TGE leader RNA and intergenic sequence required for expression of heterologous genes like green fluorescent protein and the Norwalk capsid protein in vitro. Aim3. We will directly test the hypothesis that the N protein functions in virus transcription. It is not clear whether N is necessary for transcription or has some other ancillary role in mRNA, genome or negative strand synthesis. We will assemble TGE replicons either deficient in or encoding the TGE N genes and measure the effects on the replication and transcription of TGE replicon RNAs encoding green fluorescent protein (GFP) in vitro.The assembly of a full-length TGE construct is an important breakthrough for coronavirus research and will benefit all aspects of coronavirus pathogenesis, molecular biology, epidemiology and biochemistry. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: SARS CORONAVIRUS REVERSE GENETICS AND PATHOGENESIS Principal Investigator & Institution: Masters, Paul S.; Research Scientist; Wadsworth Center Empire State Plaza Albany, Ny 12237 Timing: Fiscal Year 2004; Project Start 01-JUL-2004; Project End 30-JUN-2006 Summary: (provided by applicant): Coronaviruses are a family of enveloped, singlestranded, positive-sense RNA viruses. The genomes of coronaviruses are the largest among all the RNA viruses, which has made their genetic manipulation a formidable problem. Our laboratory developed the first reverse genetic system for coronaviruses, called targeted RNA recombination. This system has been used to answer fundamental questions about viral protein structure and function, host species specificity, virion assembly, and the unusually complex mechanism of coronavirus genome RNA synthesis. Targeted RNA recombination is a powerful and versatile method that, in principle, should be applicable to all species of coronaviruses. The major object of this application is to develop and apply this system to the coronavirus that is the causative agent for Severe Acute Respiratory Syndrome (SARS-CoV). To accomplish this, we will construct an interspecies chimeric coronavirus that will have gained the ability to infect mouse cells. This virus, designated mSARS-CoV, will serve as the cornerstone for a hostrange-based selection system for SARS-CoV reverse genetics. The tissue culture properties of mSARS-CoV and mutants of mSARS-CoV will be explored to gain insights into the potential roles of the eight unique genes in SARS-CoV that do not appear in other coronaviruses. The disease caused by these constructed viruses in the mouse host will be characterized and compared to that caused by the well-studied mouse coronavirus (MHV). Additionally, this genetic system will be used to answer basic questions about the components of SARS-CoV virion assembly and RNA synthesis and to create a mouse virus surrogate of SARS-CoV whose virion proteins are completely derived from MHV. The proposed work is expected to produce significant new information upon which more detailed future studies will be based. It will also generate valuable tools to test antiviral drugs, reveal targets for such drugs, and provide a means to manipulate SARS-CoV for vaccine design. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: STRUCTURE AND FUNCTION OF THE CORONAVIRUS REPLICASE Principal Investigator & Institution: Baker, Susan C.; Associate Professor; Microbiology and Immunology; Loyola University Chicago Lewis Towers, 13Th Fl Chicago, Il 60611 Timing: Fiscal Year 2002; Project Start 01-JAN-2001; Project End 31-DEC-2005 Studies 23 Summary: Coronaviruses are a family of RNA viruses that cause respiratory, gastrointestinal and neurological diseases in a variety of animals including humans. Coronavirus RNA synthesis is unusual because viral mRNAs are generated via a discontinuous mechanism during which a leader RNA and mRNA body sequence become contiguous. Furthermore, replicating viral RNAs undergo high frequency RNA recombination. To understand these unique mechanisms of viral RNA synthesis, we are characterizing the coronavirus replicase, the enzyme responsible for RNA synthesis and recombination. The corona virus replicase is synthesized as an 800 kDa polyprotein that is processed by viral proteinases. In this proposal, we will investigate how the replicase polyprotein is processed, assembled on intracellular membranes, and functions in viral RNA synthesis. We hypothesize that certain replicase products act as the scaffold for assembly of the replicase complex onto membranes, and that this assembly is essential for the generation of the functional replicase. Using a series of polyclonal antisera generated to individual replicase products, we will determine the subcellular localization of replicase products by confocal and immuno-electron microscopy. Biochemical methods and a system for the cytoplasmic expression of replicase products will be used to determine if individual replicase products act as integral membrane proteins or if their localization is dependent of other replication products. We will also use the cytoplasmic expression system to express replicase products in trans and determine if they can complement temperature sensitive mutants defective in viral RNA synthesis. These studies will provide new information of a viral replicase that mediates discontinuous mRNA synthesis and RNA recombination, events that contribute to the ability of viruses to rapidly evolve, evade the immune system and hamper vaccine development. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: STUDIES OF A SARS-CORONAVIRUS RECEPTOR Principal Investigator & Institution: Farzan, Michael R.; Instructor; Brigham and Women's Hospital 75 Francis Street Boston, Ma 02115 Timing: Fiscal Year 2004; Project Start 01-JUN-2004; Project End 31-MAY-2009 Summary: (provided by applicant): Spike (S) proteins of coronaviruses, including that which causes Severe Acute Respiratory Syndrome (SARS-CoV), associate with cellular receptors to mediate infection of their target cells. In Preliminary Data, we identify a metallopeptidase, angiotensin-converting enzyme 2 (ACE2), isolated from SARS-CoVpermissive Vero E6 cells, that efficiently binds the S1 domain of the SARS-CoV S protein. A soluble form of ACE2, but not that of a related enzyme, ACE1, blocked association of the S1 domain with Vero E6 cells. 293T cells transfected with ACE2, but not those transfected with HIV-1 receptors, formed syncytia with S-protein-expressing cells. Finally, SARS-CoV replicated efficiently on ACE2- but not mock-transfected 293T cells, and SARS-CoV replication could be blocked by anti-ACE2 but not anti-ACE1 antibodies. ACE2 is therefore a functional receptor for SARS-CoV. We propose to extend this work by (1) describing the role of ACE2 enzymatic activity in S-protein-mediated fusion; (2) assessing the ability of the S protein to modulate ACE2 activity; (3) describing the ability of various proteins, peptides, and small molecules that bind ACE2 to block S1 association, syncytia formation, and viral replication; (4) cloning ACE2 genes of a variety of animals, and describing their ability to associate with the S1 domain of the S protein and to support infection; (5) using chimeras of human and animal ACE2, and other ACE2 variants, to identify the S-protein-binding domain of ACE2; (6) identifying the minimum ACE2-binding domain of the S1 domain of the SARS-CoV S protein, and identifying residues within that domain essential for ACE2 association; and (7) 24 Coronavirus optimizing an infection system using SIV pseudotyped with the S protein. These studies will provide a comprehensive description of the S-protein/ACE2 association and of the role of ACE2 in S-protein-mediated fusion. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen • Project Title: THE ROLE OF CD4 CELLS IN THE TRAFFICKING OF CTLS IN CNS TISSUE Principal Investigator & Institution: Hinton, David R.; Professor; University of Southern California 2250 Alcazar Street, Csc-219 Los Angeles, Ca 90033 Timing: Fiscal Year 2002; Project Start 01-APR-2002; Project End 31-MAR-2003 Summary: Infection of the central nervous system (CNS) with the neurotropic murine coronavirus JHMV results in acute encephalomyelitis, primary demyelination , and persistent infection in susceptible strains. Complete elimination of virus during the acute infection by multiple effectors including CD8+ cytotoxic T lymphocytes (CTL), is critical in preventing viral persistence and chronic demyelination. We have recently shown that the absence of CD4+ T cells in JHMV infection results in decreased numbers of activated CTL in the brain and this is associated with a marked increase in the number of cells undergoing programmed cell death or apoptosis. As a direct extension of this work, we propose the general HYPOTHESIS that the infiltration and survival of CTL in CNS during JHMV infection is dependent, at least in part, upon CD4+ T cells. The first aim of the project will be to determine the mechanism by which CTL infiltrate into CNS tissue from the perivascular space and how this is modified by CD4+ cells. We suggest that CTL infiltration is increased by secretion of metalloproteinases (MMP-7, MMP-9) and in response to chemokine gradients (MIP-1alpha, RANTES) and that these processes are stimulated by the secreted products of CD4+ T cells. The second aim will be to determine the mechanism by which CTL undergo apoptosis in the CNS of JHMVinfected mice and how this is modulated by CD4+ T cells. We suggest apoptosis of CTL occurs secondary to deletion of the growth factor interleukin-2 (IL-2) OR by Fasmediated killing, processes associated with decreased levels of bcl-2 and that CD4+ T cell depletion further accentuates IL-2 depletion. Both brains and isolated CTL from normal mice and CD4-deficient mice will be studied as well as specific mutant and transgenic strains. Pathogenesis will be altered by over expression of specific MMP, chemokines of apoptosis- related molecules using a Defective Interfering vector system. The results of these experiments will provide a better understanding of the mechanisms involved in CTL trafficking within the specialized microenvironment of the brain. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen