Deer mice (Peromyscus maniculatus) are the principal natural host of Sin Nombre virus (SNV), the etiologic agent of most hantavirus pulmonary syndrome (HPS) cases in humans in North America 123. Deer mice are found throughout most of North America, except for the eastern seaboard and southeast United States 4.

SNV predominantly infects capillary endothelial cells without discernible pathology 5. A prominent feature of HPS is capillary leakage and subsequent hypotension that causes death by cardiac failure. In addition to resident alveolar macrophages, lymphocytes infiltrate the lungs of patients and contribute to the pathology by secreting proinflammatory cytokines, including interferon-γ (IFNγ), interleukin-2 (IL-2), tumor necrosis factor (TNF), and lymphotoxin (LT) 6. These characteristics suggest HPS may be a cytokine-mediated immunopathology.

As is usual with most long-term reservoir hosts, deer mice exhibit little pathology during acute infection with SNV 7. The virus infects capillary endothelial cells of deer mouse lungs, but lymphocyte infiltration or inflammation are not observed. After acute infection, the virus establishes persistence in many tissues 8 and most, if not all, deer mice remain infected for the remainders of their lives.

The role of the immune response in infected deer mice is unclear because few reagents of defined specificity have been developed to characterize their immune function. A strong IgG antibody response occurs 79, suggesting that B cells and helper T cells participate in containment of the virus, but the role of cytokines has not been characterized. As part of our continuing efforts to develop the deer mouse as a model for SNV research 1011; (Green et al., in press; Richens et al., submitted), we established a colony of deer mice captured in a region where SNV is prevalent 1213. Splenocytes obtained from deer mice from this colony were used to clone several cytokine and chemokine cDNA sequences. These sequences represent several components of the immune response, including the transition from innate to adaptive responses (IL-12a, IL-21, IL-23a), type I (IL-2) and type II responses (IL-6, IL-13), a colony stimulating factor (GM-CSF), and chemokines (CCL2, CCL3, CCL4, CXCL2). The availability of such sequences will permit experimental examination of the roles of these cytokines in deer mice infected with SNV.

Pathogenic hantaviruses induce acute inflammatory responses in humans that contribute to HPS disease progression 56. Much of the pathology occurs because of the production of several inflammatory cytokines in the lungs, including IL-2, IFNγ, tumor necrosis factor (TNF) and lymphotoxin (LT). In addition, pulmonary T cell infiltrates are observed in patients that die from HPS. It is evident that immunopathology contributes to HPS progression, given that SNV does not cause discernible direct cytopathology.

We targeted several deer mouse cytokines important in different aspects of immune responses. Our approach was to identify highly conserved regions of orthologous sequences from rodents and humans and to design primers from them. In many instances no consensus was found, so degenerate primer sets were used. In our analysis we determined the approximate relative sizes of the fragments based upon the known length of M. musculus orthologs; it is likely that some deer mouse genes will be longer or shorter when full-length sequences become available.

We also used cells from ostensibly SNV-susceptible deer mice. It is plausible that, in response to coadaptation with the virus, some selective pressure has occurred over time on deer mouse cytokine genes.

IL-2 is the principal T cell growth factor and is secreted by T helper 1 (Th1) cells during immune responses 3031. It is also produced by pulmonary infiltrates in HPS patients 6 and probably plays an indirect role in inflammation by augmenting TNF expression 32. IL-21 is an IL-2 family member that is also secreted by Th1 cells. It was initially described as an activator of NK cells and an augmenter of B cell and T cell proliferation 22. More recently, it has been shown to play a role in the transition from innate to adaptive responses by limiting NK cell responses and activating cytotoxic T lymphocyte responses 33. It also skews the immune response towards a type I response by augmenting expression of the T-bet transcription factor, IFNγ and receptors for IL-2, IL-12 and IL-18 34.

Although GM-CSF is secreted by Th1 cells 31, it is commonly used to generate bone marrow-derived dendritic cells (DC). The receptor-binding region of deer mouse GM-CSF is highly conserved with respect to house mouse and rat GM-CSF. This suggested to us that recombinant house mouse GM-CSF (rGM-CSF), which is commercially available, might bind to the deer mouse GM-CSF receptor, and we have obtained preliminary evidence suggesting that it does (data not shown). Human DC have recently been shown to support the replication of Hantaan virus 35, which is related to SNV. It should be possible to generate deer mouse DC with rGM-CSF to determine whether they can be infected by SNV and to determine whether there is a functional consequence of infection.

Both IL-12a and IL-23a polypeptides form heterodimers with the IL-12b (p40) subunit to form IL-12 and IL-23, respectively 2436. IL-12 facilitates the transition from the innate response to a type I adaptive immune response, while IL-23 appears to sustain proliferation of Th1 cells during the course of an immune response 2437. Both are produced by macrophages and dendritic cells and influence T cell maturation towards a Th1 phenotype.

In addition to Th2 cells, IL-6 is expressed by many other cell types and has broad biological activity, including a major role in augmenting antibody production in activated B cells 38. IL-13 is closely linked to the IL-4 gene cluster in humans and house mice and has a role in class switching and controlling inflammatory responses 3940.

Chemokine production in humans infected with SNV has not been examined in detail. TNF is produced in the lungs of patients that die from HPS 6 and mononuclear infiltrates are present in pulmonary tissues 5. Since chemokines are potent recruiters of blood leukocytes into infected tissues, there must be a prominent role for these proteins in HPS. Deer mice exhibit no conspicuous recruitment of leukocytes into the lungs 7, thus comparison of chemokine responses in humans and deer mice infected with SNV may provide clues as to how their respective immune systems respond to the virus and how the virus evades the immune response in deer mice to establish persistence.

We did not find any conspicuous differences in the amino acid sequences of deer mouse cytokines or chemokines compared to rat or house mouse orthologs that would suggest a functional difference in the mechanism by which these molecules exert their effects in vivo. More likely, immunological decisions are made during acute infection in deer mice that lead to a qualitative difference in cytokine profiles. These sequences and others that we previously reported 11 will be useful in characterizing cytokine and chemokine responses in deer mice. By comparing the profiles of these responses in deer mice and humans, it is possible that therapeutic targets can be identified in humans infected with hantaviruses. For HPS, treatment strategies must function quickly because of the rapidity of patient decline; death can occur within hours after medical treatment is sought 541. Although antiviral drugs may be developed against the virus, it is likely that an immunomodulatory approach that prevents or reverses pulmonary inflammation would be useful in treating patients.

We believe that the primer sets developed in this work can be used to amplify orthologous sequences from a variety of rodent species. Deer mice are New World rodents, while house mice and rats (Rattus sp.) are Old World rodents. Deer mice are about 50 million years divergent (myd) from house mice, while house mice and the laboratory rat (R. rattus) are only 15–25 myd 42. We attempted to perform phylogenetic analyses; however, the short fragments representing most of the genes possessed insufficient data for meaningful interpretation. We are currently pursuing additional sequence data to address this deficiency (Palmer et al., manuscript in preparation).

We have cloned a number of deer mouse cytokine and chemokine cDNA sequences. These represent several components of immune responses, including transition from innate to adaptive immunity, type I and type II responses, chemotaxis, and a cytokine useful for producing mature monocytic cells from bone marrow. The availability of these sequences will allow the characterization of a portion of the cytokine and chemokine responses in deer mice acutely or persistently infected with SNV.

IL, interleukin; CC, CC chemokine; CXC, CXC chemokine; GM-CSF, granulocyte macrophage-colony stimulating factor; SNV, Sin Nombre virus; TNF, tumor necrosis factor; IFNγ, interferon-γ ; LT, lymphotoxin

TS provided conceptual efforts on establishment of the colony and the overall cloning strategy and cloned the IL-13, IL-23a, CCL2 and CXCL2 genes. RG cloned and sequenced the IL-6 gene. BD cloned and sequenced the GM-CSF gene. AB cloned the IL-2 and IL-21 genes. TH cloned and sequenced the IL-12a, CCL3 and CCL4 genes. JJR supervised the trapping and bled the deer mice. FD cared for the deer mice during the quarantine period. CHC and BJB performed diagnostic testing of the deer mouse serum samples for SNV infection.