News and Publications

Emerging Virus Research Center

M.J. Buchmeier, K. Smith, F. Rodriguez , D.R. Burton, P.W.H.I. Parren, T. Maruyama, M. Kaneko, T.C. Bohlman,* S. St. Jeor,* K. McGwire,* A. Sette,** S. Southwood,** C.J. Peters,*** P.B. Jahrling****

* University of Nevada, Reno, NV
** Epimmune Corp., San Diego, CA
*** Centers for Disease Control and Prevention, Atlanta, GA
**** U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD

Established in 1996, the TSRI-UNR Emerging Virus Research Center is one of 3 centers sponsored by the National Institutes of Health to address fundamental problems related to the epidemiology of and immune responses to emerging viruses. In the past year, the center has supported work in 4 specific project areas related to the arenavirus, filovirus, and hantavirus groups.

Lassa fever virus is an important human pathogen that causes substantial morbidity and mortality among humans in West Africa and has been transmitted accidentally to persons in Western Europe and the United States. In a project directed by M. Buchmeier, TSRI, in collaboration with A. Sette and S. Southwood, Epimmune Corp, peptide epitopes for cytoxic T-lymphocyte (CTL) responses specific for Lassa fever virus in humans are being determined. The protocol includes a predictive alogrithm to detect potential MHC-binding motifs, competitive binding assays to determine molar binding affinities for purified MHC class I molecules, and CTL assays with lymphocytes obtained from patients seropositive for Lassa fever virus.

In the past year, we completed MHC-binding studies on the HLA A2, A3, and B7 families of alleles and detected 34 peptide epitopes that bind with high affinity to multiple MHC suballeles within a family. Identification of such superepitopes is a priority, because they represent a broader range of human CTL responses than those associated with other epitopes.

In addition to mapping the Lassa-specific CTL epitopes, we have collaborated with J. Whitton, Department of Neuropharmacology, to synthesize DNA vaccines for Lassa fever virus. Four DNA constructs have been made that express the gene for either the viral glycoprotein or the viral nucleocapsid protein with and without ubiquitin, which is incorporated to promote proteolysis and processing by the proteosome. In the coming year, we will begin animal studies to determine the efficiency and effectiveness of immunization with these constructs.

Recent outbreaks of infections caused by hantavirus and Ebola virus in Argentina and Central Africa, respectively, and heightened concern about the potential use of emerging viruses as bioterrorist weapons have increased public awareness of the threats posed by these agents. In a project led by D. Burton, Department of Immunology, specimens from humans who recovered from infections with hantavirus or Ebola virus were used to procduce libraries of recombinant human antibodies to these 2 viruses. Neutralizing monoclonal antibodies have been generated against Ebola virus, and screening of hantavirus libraries is under way.

The availability of neutralizing antibodies may provide specific antiviral countermeasures useful in the event of naturally occurring or deliberately produced outbreaks involving these pathogens. In the next year, the Burton laboratory and P. Jahrling, U.S. Army Medical Research Institute of Infectious Diseases, will test the efficacy of a neutralizing human monoclonal antibody against Ebola virus in protecting against infection in vivo in animal models of disease.

Two investigators at the University of Nevada, Reno, S. St. Jeor and K. McGwire, are collaborating in field studies designed to understand the ecology of hantaviruses in the natural host and the route of transmission between rodents. Remote sensing and geographic information system components have been developed to monitor fluxes in rodent populations and viral presence in the Walker River Basin of California and Nevada. In 1997, more than 100 sampling sessions were done of new field sites and of several sites that were sampled repeatedly. On nearly every site in the study area, the density of rodent hosts was less than 1995--1996 levels (85% lower, on average); the cause of this decrease is unknown.

Although we previously found no linear site-by-site relationship between host density and prevalence of antibody to hantavirus, this large-scale reduction in density enabled us to assess the effects of synchronized, temporal fluctuations in population density. We found that mean antibody prevalence in deer mice (Peromyscus maniculatus) decreased from 18% in 1995--1996 to 13% in 1997. Additionally, in 1997, a greater proportion of sites showed no evidence of active or recent hantavirus infections. Although we have no data on the effects of large-scale temporal increases in host density, our results suggest that such large-scale fluctuations may play an important role in determining the likelihood of outbreaks in humans. Work over the next year will reveal the effects of the 1998 El Niño on the density of the rodent poplulation and on infection rates.

A second goal of this work is to understand the biological characteristics of genetic variants of hantaviruses. A number of genotypes of hantaviruses have been detected and are being sequenced. A comparison of isolates from different geographic areas showed that Sin Nombre virus is genetically diverse. Deer mice in a natural setting were captured and blood samples obtained multiple times during a period of months. Blood from these mice was processed, and viral RNA was isolated. After amplication and purification, sequences were analyzed for nucleotide and amino acid changes to determine the extent of genomic diversity, which reflects viral quasi species in individual animals.

Initial results indicated limited genetic diversity within individual deer mice. Nonsynonymous mutations accounted for more than 50% of the changes detected, and 92% of the changes were transitions. Ongoing catch-and-release studies of deer mice will provide more samples to determine if the level of quasi species changes with time.

Mechanisms of Virus-Induced Demyelination

T.E. Lane, A. Paoletti, V. Asencio, M.J. Buchmeier

Multiple sclerosis is the most common neurodegenerative disease in the United States; it affects 200,000--250,000 persons. This chronic disease of the CNS is characterized by multifocal regions of inflammation and myelin destruction. Typically, multiple sclerosis has a protracted clinical course of several decades, and episodes of exacerbation are often followed by variable periods of remission. The disease is particularly devastating because it strikes young adults (20--30 years old) and has calamitous effects on the quality of life. The etiology of multiple sclerosis most likely is multifactorial, and epidemiologic evidence supports the idea that an infectious agent, such as a virus, acquired early in life contributes to the development of the disease in genetically susceptible persons. We use virus-induced demyelination in mice as a model for multiple sclerosis in an attempt to understand the complex pathologic mechanisms that contribute to demyelination.

Infection of susceptible strains of mice with mouse hepatitis virus (MHV) reproducibly causes a chronic, progressive, demyelinating disease characterized clinically by hind limb paralysis and histologically by infiltrates of mononuclear cells and demyelination of the CNS. MHV-induced demyelinating disease is considered a relevant animal model of multiple sclerosis because the 2 diseases have similar histologic and clinical courses. Currently available evidence indicates that demyelination requires persistent MHV infection of the CNS and that cellular elements of the immune system, including T cells, contribute to the disease. We have focused our efforts on determining the various mechanisms by which demyelination occurs in MHV-infected animals.

Mice in which either the CD4⁺ or the CD8⁺ subset of T cells was genetically eliminated (knockout strains) were infected with MHV, and the severity of demyelination was evaluated. Both CD4 and CD8 knockout mice had higher viral titers in their brains and greater mortality than did control C57BL/6 mice. At 12 and 21 days after infection, surviving CD4 knockout mice had much higher levels of persistent virus in the brain and spinal cord and yet had markedly less severe demyelination than did C57BL/6 mice and CD8 knockout mice (Fig. 1). These data indicate that CD4⁺ T cells play an important role in amplifying demyelination in MHV-infected mice.

In collaboration with I. Campbell, Department of Neuorpharmacology, we determined that MHV infection causes an orchestrated expression of chemokines in the CNS. The chemokines are low molecular weight, proinflammatory factors that function as important chemoattractants for distinct populations of leuokocytes during inflammation. Chronic demyelination in MHV-infected mice requires maintenance of an inflammatory response. During chronic demyelination in infected animals, the predominant chemokines expressed in the CNS are CRG-2/IP-10 and RANTES.

To determine whether expression of either chemokines or cytokines was correlated with demyelination, we analyzed chemokine and cytokine mRNA transcripts in the brains of MHV-infected C57BL/6, CD4 knockout, and CD8 knockout mice 12 days after infection. We found no significant differences in cytokine expression between the 3 strains of mice. However, both C57BL/6 and CD8 knockout mice (which had marked demyelination) had significantly higher levels of transcripts for the chemokine RANTES than did CD4 knockout mice (which had only limited demyelination). These data indicate that CD4⁺ T cells are the major source of RANTES in the CNS after infection with MHV. Furthermore, we think that CD4⁺ T cells and factors such as RANTES released from these cells are important in initiating demyelination.

We recently evaluated the contribution of nitric oxide generated by nitric oxide synthase type 2 in tissue damage in MHV-induced demyelination. Infected animals were treated daily with either aminoguanidine, a selective inhibitor of the activity of nitric oxide synthase type 2, or with phosphate-buffered saline, and the severity of clinical and histologic disease was examined. We found a significant decrease in the severity of clinical disease and in demyelination in animals treated with aminoguanidine. This finding suggests that restricting the activity of nitric oxide in the affected tissue may be beneficial. We also analyzed the chemokine mRNA transcripts and found that levels of MCP-1, a chemokine important in attracting monocytes and macrophages to sites of inflammation, were significantly lower in mice treated with aminoguanidine than in control mice. Together, these data suggest that nitric oxide generated by nitric oxide synthase type 2 may have a role in virus-induced demyelination; it may control inflammation by modulating chemokine expression in the CNS.

PUBLICATIONS

Buchmeier, M.J. Lymphocytic choriomeningitis virus. In: Encyclopedia of the Life Sciences. Atlas, R. (Ed.). MacMillan, New York, in press.

Buchmeier, M.J., Zajac, A. Lymphocytic choriomeningitis virus. In: Persistent Viral Infections. Ahmed, R., Chen, I. (Eds.). Wiley, New York, in press.

Clegg, J.C.S., Bowen, M.D., Buchmeier, M.J., Gonzalez, J.-P., Lukashevich, I.S., Peters, C.J., Rico-Hesse, R., Romanowski, V. Arenaviridae. In: Virus Taxonomy: Classification and Nomenclature of Viruses. Seventh Report of the International Committee on Taxonomy of Viruses. Springer-Verlag, New York, in press.

Geiger, K.D., Nash, T.C., Sawyer, S., Krahl, T., Patstone, G., Reed, J.C., Krajewski, S., Dalton, D., Buchmeier, M.J., Sarvetnick, N. Interferon-gamma protects against herpes simplex virus type 1-mediated neuronal death. Virology 238:189, 1997.

Lane, T.E., Asensio, V., Paoletti, A., Yu, N., Campbell, I., Buchmeier, M.J. Dynamic regulation of and ß chemokine expression in the central nervious system during mouse hepatitis virus-induced demyelinating disease. J. Immunol. 160:970, 1997.

Lane, T.E., Fox, H.S., Buchmeier, M.J. Inhibition of nitric oxide synthase type 2 reduces the severity of mouse hepatitis virus-induced demyelination: Implication for NOS2/NO regulation of chemokine expression and inflammation. J. Neurovirol., in press.

Lane, T.E., Perlman, S., Buchmeier, M.J. Coronaviruses: Hepatitis and central nervous system disease. In: Effects of Microbes on the Immune System. Cunningham, M., Fujinami, R. (Eds.). Lippincott-Raven, Philadelphia, in press.