PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jvirolPermissionsJournals.ASM.orgJournalJV ArticleJournal InfoAuthorsReviewers
 
J Virol. 2012 September; 86(18): 10015–10027.
PMCID: PMC3446614

Kinetics of Immune Responses in Deer Mice Experimentally Infected with Sin Nombre Virus

Abstract

Deer mice are the principal reservoir hosts of Sin Nombre virus, the etiologic agent of most hantavirus cardiopulmonary syndrome cases in North America. Infection of deer mice results in persistence without conspicuous pathology, and most, if not all, infected mice remain infected for life, with periods of viral shedding. The kinetics of viral load, histopathology, virus distribution, and immune gene expression in deer mice were examined. Viral antigen was detected as early as 5 days postinfection and peaked on day 15 in the lungs, hearts, kidneys, and livers. Viral RNA levels varied substantially but peaked on day 15 in the lungs and heart, and antinucleocapsid IgG antibodies appeared in some animals on day 10, but a strong neutralizing antibody response failed to develop during the 20-day experiment. No clinical signs of disease were observed in any of the infected deer mice. Most genes were repressed on day 2, suggesting a typical early downregulation of gene expression often observed in viral infections. Several chemokine and cytokine genes were elevated, and markers of a T cell response occurred but then declined days later. Splenic transforming growth factor beta (TGF-β) expression was elevated early in infection, declined, and then was elevated again late in infection. Together, these data suggest that a subtle immune response that fails to clear the virus occurs in deer mice.

INTRODUCTION

Hantavirus cardiopulmonary syndrome (HCPS) is characterized by vascular leakage that leads to cardiogenic shock (38) and a fatality rate of 35%. Most cases of HCPS are caused by Sin Nombre virus (SNV) in North America and Andes virus (ANDV) in South America (32). Capillary endothelial cells, without conspicuous cytopathic effect, are the principal target of infection (38). Several proinflammatory cytokines have been detected in autopsy and serum samples of patients with HCPS, including tumor necrosis factor (TNF), lymphotoxin (LT), gamma interferon (IFN-γ), interleukin-2 (IL-2), IL-4, IL-1α, and IL-6, suggesting the disease may be the result of inflammatory immunopathology (2, 30).

Throughout the Americas, HCPS has caused several thousands of deaths, and globally hantaviruses kill tens of thousands of people each year. Pathogenic hantaviruses are hosted by a variety of rodents, usually with a single species reservoir for a particular virus species (6, 26). Hantaviruses that cause HCPS have a conserved immunoreceptor tyrosine activation motif (ITAM) in the GN (or G1) glycoprotein (14), and while this ITAM has biological activity in vitro, it has not been demonstrated to affect immune responses in vivo.

In contrast, rodent reservoirs of hantaviruses do not appear to exhibit conspicuous pathology or pulmonary inflammation and remain persistently infected, perhaps for life, and shed virus in their excrement (3, 24). In rats (Rattus norvegicus) infected with Seoul virus (SEOV), CD4+CD25+FoxP3+ regulatory T (Treg) cells that secrete transforming growth factor beta (TGF-β) are elevated in the lungs, and depletion of CD25+ cells results in inflammation (10). In addition, some chemokines are elevated in the spleens of infected rats, including Ccl2 and Ccl5, but less so in the lungs (9).

CD4+ T cell lines generated from outbred deer mice experimentally infected with SNV express Th1, Th2, and Treg cell cytokines 10 days postinfection (dpi), but T cells from persistently infected deer mice (45 dpi) predominantly express the Treg cytokine TGF-β, suggesting a transition to a Treg-like immune response that limits inflammatory pathology but allows virus persistence (34). Deer mouse T cells do not appear to be susceptible to infection with SNV, as viral RNA cannot be detected from these lines after 25 days in culture.

Considering the substantial immune activity in reservoir deer mice and rats infected with their hantaviruses, it is evident that active immune responses that prevent inflammatory disease yet fail to clear virus occur in these species. We have thus examined the kinetics of viral infection, histology, antibody responses, and cytokine gene expression in deer mice during early infection to examine the reservoir host-virus interaction. No pathology was apparent in any of the deer mice, despite the presence of virus in various organs. In spleens, inflammatory chemokine, cytokine, and transcription factor genes were elevated midway in the infection, but late in infection many of these genes were downregulated in favor of expression of TGF-β. However, the expression of these genes was substantially less in the lungs, suggesting that the lungs have less immunological activity during infection. By 20 dpi, TGF-β expression levels were elevated in both tissues of most animals. Collectively, these data suggest that active immune responses that differ in the lungs and spleens but fail to clear the virus occur in deer mice.

MATERIALS AND METHODS

Deer mouse infections.

All methods were approved by the University of Northern Colorado or University of New Mexico Institutional Animal Care and Use Committees and were conducted in accordance with the Animal Welfare Act according to a set of strict, written standard operating procedures (5). Outbred deer mice, of both sexes and of 6 to 10 weeks of age, were reared at the UNM Animal Care Facility. Animals for these experiments were transferred and housed in sheltered outdoor nest boxes at the Sevilleta National Wildlife Refuge and allowed to acclimated as previously described (3). They were placed into 6 groups of 10; in each group, 5 were inoculated intramuscularly with 20 animal 50% infective doses (ID50) of SNV 77734 and 5 were sham inoculated. The groups of deer mice were euthanized by cervical dislocation at the refuge site on days 2, 5, 7, 10, 15, and 20, and tissue was quick-frozen on dry ice for transport to the University of New Mexico Department of Pathology for tissue processing in biosafety cabinets.

Serology.

Blood was collected from each deer mouse at the time of euthanasia and tested for IgG antibodies (33). Plates were coated overnight with a truncated nucleocapsid antigen containing a dominant B cell epitope diluted in phosphate-buffered saline (PBS) (pH 7.2) onto Falcon PVC 96-well enzyme immunoassay (EIA) plates. The plates were washed with PBS-Tween 20 and blocked in PBS containing 0.25% gelatin (Sigma) for 1 h. Samples were diluted 1:100 in PBS and incubated for 1 h at room temperature. IgG was detected with a protein A/G-horseradish peroxidase (HRP) conjugate (Pierce) diluted 1:5,000 in PBS and incubated for 45 min at room temperature. Activated ABTS (KPL) was added for 15 min, and optical density at 405 nm (OD405) was recorded. Samples were considered positive if the OD was greater than 0.200 above the mean from a negative-control deer mouse sample. For titrations, log2 dilutions were made from 1:200 to 1:12,800; endpoint titers were determined from the reciprocal of the greatest dilution with an OD of 0.100 above that of the negative control.

Plaque reduction neutralization test.

Neutralizing antibody titers were performed as previously described (3). Serum samples were heat inactivated at 56°C for 30 min, serially diluted starting at 1:10, and incubated with 50 PFU of SNV strain 77734 for 1 h at 37°C. The samples were then adsorbed on Vero E6 cells for 4 h at 37°C, and medium in 1.2% methylcellulose was overlaid for 7 days. Cultures were fixed with methanol with 0.5% H2O2, followed by detection of nucleocapsid with rabbit antiserum (1:5,000), peroxidase-conjugated goat anti-rabbit IgG, and diaminobenzoin metal substrate (Pierce). Neutralization endpoint titers were determined from wells that had 80% reduction in PFU.

RNA preparation and virus quantitation.

RNA was extracted from lungs and spleens using an RNeasy minikit (Qiagen, Valencia, CA) and a BeadBeater (BioSpec Products, Inc., Bartlesville, OK), and the presence of the viral S segment was verified by reverse transcription (RT)-PCR. The TaqMan assay (Applied Biosystems, Foster City, CA) has been previously described (3, 4). Briefly, cDNA was made from the S segment, followed by real-time PCR amplification of samples and plasmid standards of 106, 104, and 102 copies per reaction (see Table 2). Viral RNA copy number was determined by regression analysis of the standard.

Table 2
Primers used in this work (5′ to 3′)

Real-time PCR detection of immune gene expression.

Real-time PCR (primers listed in Table 2) was performed in triplicate in 25-μl volumes by using the iQ SYBR green kit (Bio-Rad) for 40 cycles with an MyiQ real-time thermal cycler (Bio-Rad) as previously described (31). Expression levels for each gene from each sample were normalized against GAPDH. The means for each gene from the infection groups (n = 5) were divided by the means of the same gene from the uninfected groups (n = 5) to determine fold increase (above 1) or decrease (below 1) of expression (ΔΔCT). Means and 95% confidence intervals were calculated and plotted to compare expression levels between groups of infected and uninfected deer mice on each day and to compare means among days in infected mice. Fold changes were compared between infected versus sham-infected deer mice on each day, and significant differences were manifested as nonoverlapping 95% confidence intervals. Infected mice whose fold change values had overlapping 95% confidence intervals among days were placed into groups designated with a letter. Groups with different letter designations had significantly (P < 0.05) different fold change values.

Cluster analysis of gene expression.

Cluster analysis is a multivariate statistical technique that we used to identify genes with similar expression profiles over the course of the experiment. Levels of gene expression in 5 infected mice were measured at six time points (2, 5, 7, 10, 15, and 20 days postinfection). These were standardized with respect to expression levels in 5 uninfected mice on the same days. The average level of standardized expression was calculated for each day. Means were entered into a 6 (days)-by-31 (genes) matrix. The Pearson's correlation coefficient (ρ) among means was calculated between each pair of genes to generate a symmetrical 31-by-31 correlation matrix. For cluster analysis, a second matrix of 1 − ρ values was calculated to generate a distance matrix. If mean expression levels were perfectly correlated, then ρ = 1 and 1 − ρ = distance = 0, but if means were uncorrelated, then ρ = 0 and 1 − ρ = distance = 1. Conversely, if genes had opposite expression patterns, then ρ = −1, 1 − ρ = distance = 2. The 31-by-31 distance matrix was subjected to an unweighted pair group method with arithmetic mean (UPGMA) (35) cluster analysis using NEIGHBOR in PHYLIP3.6.7 (13) to generate a dendrogram.

Histopathology and immunohistochemistry.

Tissue preparation was performed as previously described (3). Tissues from freshly euthanized deer mice were collected into buffered formalin, embedded in paraffin, sectioned to 5 μm, and coated onto slides. For histopathology, slides were stained with hematoxylin and eosin. For IHC, slides were stained with rabbit anti-SNV nucleocapsid after antigen retrieval, followed by biotinylated goat anti-rabbit IgG, horseradish peroxidase-avidin conjugate, and amino-ethyl carbozole chromogen. Hematoxylin was used for counterstaining. A scoring regimen of 0 (no reactivity) to 4 (substantial reactivity) was used for evaluation of pathology and antigenic load.

RESULTS

Kinetics of viral RNA load in lungs and hearts.

Examination of viral RNA levels in lungs and heart by TaqMan real-time PCR (3) revealed substantial diversity in viral loads between animals in the infected groups (Table 1, Fig. 1), while none of the sham-inoculated deer mice had detectable viral RNA (data not shown). This diversity has been observed in our previous studies in which infection occurs in either a restricted or disseminated pattern (4). One deer mouse (number 7086) had a very high copy number in the lungs when euthanized on day 10 postinfection (>31,000 copies/mg) despite having seroconverted to nucleocapsid. Discounting this animal, mean viral RNA levels for each tissue gradually increased and peaked on day 15 and subsided on day 20. Viral RNA was detected in the lungs of two of five deer mice in the 2-dpi group and in all deer mice in subsequent lung groups. None of the 2-dpi deer mouse hearts had detectable viral RNA, and only one of the five in the 5-dpi group had detectable viral RNA. Only by day 15 did most deer mice in a group have detectable viral RNA in the hearts (four of five deer mice), and on day 20 all five deer mice had detectable viral RNA in the hearts.

Table 1
Viral RNA load and antibody responses in deer mice infected with SNV
Fig 1
Kinetics of viral RNA load in lungs and hearts of infected deer mice. Total RNA was extracted from deer mouse hearts and lungs on the indicated days and quantified by TaqMan real-time PCR. Virus copy number was determined from a standard curve, and the ...

Antibody responses.

We examined sera from each deer mouse for antibodies using enzyme-linked immunosorbent assay (ELISA) (nucleocapsid) and neutralization (PRNT). None of the sham-inoculated deer mice seroconverted (data not shown). On day 10, two infected deer mice had seroconverted with low titers (200 and 800) by ELISA (28), all five day-15 deer mice had seroconverted with low to high titers (400 to 6,400), and three of the day-20 deer mice had seroconverted with high titers (6,400) (Table 1). However, none of the deer mice had neutralizing antibody titers of greater than 10, the minimum dilution of the PRNT assay, and all of those were in the first 7 days of the experiment.

Histopathology and immunohistochemistry.

Previous work (3) examined tissues from deer mice at 21 dpi and no pathology was found. We intensified scrutiny by examining tissues from deer mice at 2, 5, 7, 10, 15, and 20 dpi. None of the infected deer mice exhibited signs of tissue pathology (Fig. 2). Immunohistochemistry revealed virus in tissues as early as day 5 dpi (Fig. 3). Four of five deer mice in the day-7 group, three of five in the day-10 group, five of five of the day-15 group, and two of five in the day-20 group had detectable virus. The lungs and livers were most frequently positive for viral antigen (11 of 30 animals each) with fewer animals positive in the kidneys (7) and hearts (4). Virus was found in the endothelia of lungs, alveolar walls, hepatic sinusoids, myocardial interstitium, and glomeruli.

Fig 2
Tissue samples from uninfected or infected deer mice. Sections from uninfected (A to D) or infected (E to H) deer mice collected 10 days postinoculation were stained with hematoxylin-eosin (200× magnification). No differences were found in lung ...
Fig 3
Immunohistochemical detection of SNV antigen. Sections from uninfected mice (A to D) or 10 days postinfection (E to H) were stained with rabbit anti-SNV IgG (400× magnification). Lung (A, E), kidney (B, F), heart (C, G), or liver (D, H).

Modulation of immune gene expression.

We examined 31 cytokine and other immune-related genes (Table 2) by real-time PCR (31, 34) for differences in expression levels in infected versus uninfected groups of deer mice. Most genes from groups of infected deer mice exhibited substantial variation; however, genes from uninfected groups had very little variation, suggesting the genes are under tight control until infection.

Many groups had expression levels more than 4-fold higher than those of uninfected control groups. Most genes were repressed on day 2, suggesting an antiviral state had been initiated by the cells in the lungs and spleens.

Splenic Ccl3 (MIP-1) was significantly elevated on days 2, 5, and 15 but near background on days 7, 10, and 15 (Fig. 4). Ccl5 (RANTES) was also biphasic, elevated 2-fold on day 2 but declined until day 15, where it was elevated 4-fold and then near background on day 20. Ccl2 was not elevated until day 7 but remained high until day 20. Ccl4 (MIP-1β) and Cxcl2 (MIP-2α) expressions were not elevated. In the lungs, the Cxcl2 level was elevated on day 15 and Ccl3 and Ccl5 levels were elevated on day 20.

Fig 4
Chemokine gene expression levels in deer mice infected with SNV. Real-time PCR was used to assess gene expression levels in spleens and lungs in groups of deer mice infected with SNV. For a given gene, the mean from each group (n = 5) was normalized against ...

Expression of several inflammatory cytokines occurred in the spleens by day 7, including IL-12p35, IL-21, and IL-23, all of which had subsided by day 15 (Fig. 5A). TGF-β expression was biphasic, first appearing on day 5 and then again on days 15 and 20, while granulocyte-macrophage colony-stimulating factor (GM-CSF) expression was elevated only on day 10. In the lungs, none of the genes were significantly elevated, although most were repressed on day 2 (Fig. 5B).

Fig 5Fig 5
Cytokine gene expression. Most cytokines were repressed in spleens and lungs on day 2, and none were at elevated levels. On day 5, only splenic TGF-β expression was elevated but declined until days 15 and 20. IL-12p35 expression, a surrogate for ...

In spleens, CD4, CD8α, and T cell receptor beta (TCRβ) expressions were significantly elevated by day 5; however, by day 10, CD4 expression had declined to baseline, while CD8α and TCRβ expressions remained elevated (Fig. 6). None of the other cell surface markers (lymphotoxin beta [LTB], IgM, TNF receptor-associated factor 2 [TRAF2]) were significantly elevated. Most of these markers were repressed in the lungs, and none were significantly elevated.

Fig 6
Cellular marker gene expression. Splenic T cell markers were initially elevated on day 5 but CD4 expression declined by day 10, while CD8α and TCRβ expressions declined by day 15. IgM expression was repressed in the spleen most days but ...

As with other gene products, transcription factors were generally repressed 2 dpi in spleens and lungs (Fig. 7). Only T-bet, a Th1 transcription factor, was significantly elevated and only on day 15 in both the spleens and lungs.

Fig 7
Transcription factor gene product expression. Transcription factors were repressed on day 2 postinfection. However, on day 5, splenic T-bet and STAT4, both of which are essential for Th1 differentiation, expressions were slightly elevated, followed by ...

The cluster analysis provided expression groupings for many genes in the spleen (Fig. 8). Ccl2, IL-12 p35 and IL-23 p21 (both of which form heterodimers with the IL-12p40 subunit), IL-21, TCRβ, CD8α, and CD4 genes clustered together. Ccl3 and Ccl5 gene expression was also coordinated. T-bet, TGF-β, and GM-CSF genes were upregulated but did not cluster with other expressed genes. Only the Ccl5 gene was upregulated in the lungs.

Fig 8
Cluster analysis of gene product expression. UPGMA dendrograms arising from cluster analysis of the distance matrix among the 31 gene products. Gene products that cluster together exhibit similar expression patterns, suggesting coordinated expression. ...

DISCUSSION

Hantaviruses appear to infect their rodent reservoirs with little or no discernible pathology. For SNV and deer mice, and SEOV and rats, the immune responses at persistence strongly resemble that of a regulatory T cell response with prominent expression of TGF-β and lack of inflammatory signatures (9, 10, 34).

The work here provides the most detailed examination of the kinetics of infection and host response in deer mice infected with SNV. By day 2 postinfection, low levels of viral RNA were detected in the lungs of some animals. At day 5 and beyond, viral RNA was detected in lungs of all animals, with some animals exhibiting high viral loads (>1,000 copies/mg) while others had low viral loads (<100 copies/mg). The high degree of variation occurred both between groups and within groups. This difference is striking and suggests that polymorphisms within the deer mice may contribute to control of viral gene expression and replication levels. Additionally, some deer mice may be “super spreaders,” animals that, for unknown reasons, produce substantially more virus than other members of the species (36). Deer mouse 7086 may represent one such animal, although no studies have examined naturally infected deer mice for this characteristic. Excluding deer mouse 7086, viral RNA levels peaked on day 15 postinfection, compared to levels in rats infected with SEOV, which peaked on day 30 (9). Another limitation of the present work is the small sample size, with 5 deer mice per group. Larger groups may have reduced the variation observed in the present work.

IgG specific for nucleocapsid was first detected on day 10 at low titers in two of the five deer mice, and all five mice from this group had detectable viral RNA in the lungs, including deer mouse 7086, which had the highest copy number of viral RNA in the experiment. The other seropositive deer mouse, number 7040, had 594 copies/mg, which was lower than that of the other deer mice in the group that were seronegative. We previously determined that some experimentally infected deer mice can remain seronegative to nucleocapsid for up to 45 days (29). Surprisingly, neutralizing antibodies were detected only in the first 7 days postinfection and likely represent low-titer IgM. Presumably, some of these IgM-secreting B cells should undergo class switching to IgG (and presumably other isotypes) and affinity maturation; however, the data presented suggest that this does not occur within 20 days of infection. Because high-titer IgG to the nucleocapsid was produced, class-switching and affinity maturation do not appear to be impaired. The dramatic difference in antibody responses is likely due to multiple factors, including a greater abundance of nucleocapsid antigen for the immune response and the increased sensitivity of ELISA compared to plaque reduction assays. The lack of neutralizing antibody and the presence of viremia suggest that deer mice may secrete infectious virus for several weeks after infection, which could improve the likelihood of transmission to other deer mice, an important component of virus ecology.

Antigen load in tissues appears to be low to moderate, as has been previously reported in deer mice (3). In contrast, levels of antigen appear to be higher in Syrian golden hamsters infected with the human-pathogenic Andes, Maporal (MAPV), or Choclo (CHOV) hantaviruses (11, 18, 25), although only ANDV and MAPV are pathogenic in hamsters. SNV causes an apathogenic infection in hamsters and elicits a strong neutralizing antibody response by 28 dpi (18); thus, it may be informative to examine the kinetics of neutralizing antibodies in this model. The low levels of antigen in deer mice likely reduce antigenic stimulus and could be partially responsible for the lack of neutralizing antibodies by day 20. Cultivation of SNV nucleocapsid-specific CD4+ T cells requires 5 μg/ml, a relatively high concentration; thus, low tissue levels of antigen may not be sufficient for eliciting an aggressive helper T cell response that would be required for affinity maturation and class switching in B cells.

In terms of host immune modulation, several genes were expressed at severalfold increases in the spleens of infected deer mice. As with viral RNA copy number, the increases within infected groups varied substantially, with some animals exhibiting more than 4-fold increases, while others in the same group had expression levels similar to those of the sham-inoculated control groups. In contrast, the 95% confidence intervals of the sham-inoculated groups were small for all time points and all genes, suggesting that the variation observed in infected groups reflects diverse response kinetics in the outbred deer mice. Because many immune genes are expressed only for hours, it may be that expression of these genes in some animals is missed by collecting the RNA, which requires euthanasia of the deer mice, before or after gene expression. This could account for the substantial variation observed since samples were collected at 2- to 5-day intervals.

Splenic expression levels of Ccl2, Ccl3, and Ccl5 were elevated at different points. Ccl3 and Ccl5 are thought to play roles in stimulating inflammatory responses by recruiting neutrophils and T cells to sites of infection (8, 15, 39). Ccl2 can direct the development of Th2 cells (7, 16), and IL-4, but not IL-5, expression was elevated in some deer mice, which also has been attributed to Ccl2 expression (20, 22). In Long Evans rats infected with SEOV, splenic expression of Ccl2 and Ccl5 also occurs (9).

The expressions of IL-12 p35 (surrogate for IL-12), IL-21, and IL-23 p19 (surrogate for IL-23) (1) inflammatory cytokine genes were elevated in spleens of some infected deer mice on days 7 and 10. IL-21 contributes to B cell germinal center formation (21, 40), and its peak expression correlated with the earliest appearance of IgG to nucleocapsid on day 10. TGF-β expression was elevated on day 5 but declined until day 15 and persisted at day 20. Its biphasic expression has been noted in other infections in which dendritic cells are thought to express it early, followed later by regulatory T cells (19, 37), a pattern that appears to occur in deer mice infected with SNV. In Long Evans rats infected with SEOV, TGF-β expression is detected on days 3 and 15 postinfection in the spleens and then subsides; however, it is unknown if its expression is reduced between days 3 and 15 (9). In contrast, in deer mice, pulmonary TGF-β is elevated only on day 20 in only some animals but is elevated in all rats on days 3, 15, and 30 (9). It is noteworthy that while Long Evans rats are outbred, their genetic diversity is limited compared to that of the deer mice used in these studies since the strain is derived from several Wistar Institute female rats and a single male wild rat. This may account for the more homogeneous response observed in these rats infected with SEOV.

Splenic CD4, CD8α, and TCRβ expressions were elevated on day 5, but CD4 expression declined to background levels on day 10, suggesting that T cell responses were limited. The modulation of these levels could represent an increased expression within given cells, clonal expansion, or a combination of both, and we interpret this modulation as an indicator of T cell activation. Without CD4+ T cells, sustained CD8+ CTL response are impaired and result in persistent infection of laboratory mice (Mus musculus) with lymphocytic choriomeningitis virus (23). The lack of a strong helper T cell response in deer mice may contribute to persistence of SNV. IgM expression in the spleens was repressed on days 7 and 10 but elevated in some deer mice on day 15 before declining on day 20. Its detection represents expansion of B cells and/or increased synthesis of IgM heavy chain mRNA. Its decline on day 20 may signify class-switching events to other isotypes since on that day seropositive animals have high IgG titers to the nucleocapsid. TRAF2, a signal transduction protein involved in several pathways, including the NF-κB, MAPK, and caspase 8 pathways, was slightly elevated on day 5 in the lungs of some deer mice; however, we did not detect increased levels of TNF expression, so it is unclear what its elevation means in this context. TRAF2 is a frequent evasion target of viruses (17) and may be manipulated by SNV in deer mice.

Cluster analysis revealed that several genes involved in T cell mobilization were coordinately expressed (Fig. 8), including those for selection and activation of effector T cells (Ccl2, IL-12 p35, IL-21, IL-23, CD4, CD8, and TCRβ genes) and inflammation (Ccl3 and Ccl5 genes), but most subsided by day 20. These data suggest that in the spleen, a subtle to modest inflammatory T cell immune response is initiated but is not sustained. Correlating with this decline is the expression of the TGF-β gene, which can have anti-inflammatory activities and is prominently expressed by T cells from persistently infected deer mice and rats (10, 34). Despite the inflammatory gene expression signature, no histological evidence of inflammation was noted in any of the deer mouse tissues in this study. It may be that translational regulation prevents inflammation or that insufficient amounts of inflammatory proteins are made.

In the lungs, chemokine gene expression was not detected until 20 dpi, when Ccl2 and Ccl5 were elevated. Despite the expression of these genes, we did not have evidence that recruitment or activation of T cells occurred in the lungs. This is in agreement with previous work in which pulmonary tissue histology failed to show marked leukocytic infiltrates in the lungs of infected deer mice (3). At all other time points in the lungs, cytokine and chemokine gene expressions were at baseline, except for expressions of Ccl2, Ccl5, and TGF-β genes, which were elevated on day 20 in some animals. The expression of these three genes is seemingly paradoxical considering one is involved in Th2 responses (Ccl2 gene), one in inflammatory responses (Ccl5 gene), and one in anti-inflammatory responses (TGF-β gene). Evaluation of deer mice beyond 20 dpi may clarify the roles of these genes.

The expression of transcription factors was significantly repressed in both lungs and spleens on day 2 postinfection. While the increase of expression of most of these genes is low, transcription factors are enzyme-like in their abilities to initiate transcription of genes. The pattern of expression suggests differentiation of both Th1 and Th2 cells and correlates with initial expansion of T cells on day 5 postinfection. We did not observe elevation in Fox-p3 expression in either the lungs or spleens of infected deer mice. Our previous work demonstrated that SNV-specific CD4+ T cell lines established from most deer mice 42 days postinfection expressed Fox-p3. It is possible that the low sensitivity (i.e., signal-to-noise ratio) of using whole tissues, in which only a small percentage of the T cells are likely virus specific, versus homogeneous antigen-specific T cell lines, or that the fact that in the present work the T cells are from days 20 and before, may account for this difference.

In our previous work with SNV-specific CD4+ T cell lines (34), we detected severalfold increases of IFN-γ, IL-4, IL-5, and IL-10. Only increased expression of IL-4 was detected in spleens of some deer mice in the present study. Levels of TNF and LT, both of which are found in autopsy specimens of HCPS patients (30), generally were not elevated at any time point in the experiment, suggesting that the roles for these cytokines are minimal during infection. The low-level expression also suggests that in hantavirus reservoir models, immune responses are subdued relative to pathogenic models of viral infections (9, 12). With T cell lines, this problem is likely obviated since all the T cells in the culture are virus specific and are harvested after 2 days of cultivation with antigen. Unfortunately, a better comparison would be pathogenic hantavirus models in Syrian golden hamsters; however, immunologic reagents and molecular and cellular methodologies are not available for such an assessment in this species.

The dramatic repression of gene expression 2 dpi suggests antiviral effector mechanisms are active in deer mice infected with SNV. Considering that many of these proteins are highly conserved, it should be possible to use antibodies specific to these proteins in other species to dissect the events that occur during cellular infection since SNV may have transcriptional activity (2729). Propagation of deer mouse cells susceptible to infection should be a high priority so that further scrutiny of the early events of infection can be performed. Additional effort should also focus on developing antibodies to detect the cytokines that are expressed now that evidence exists that they are involved in the host response to SNV.

ACKNOWLEDGMENTS

We thank Sabra Klein for helpful discussions regarding data analysis and Aaron Philips for assistance with statistical analysis.

Funding was provided by National Institutes of Health (NIH) contract AI25489 and NIH grant AI054461 (to T.S.); NIH grants 2 U19 AI45452, U01 AI 56618, and AI45452 (to B.H.); the βββ Biological Honor Society (S.F., S.P.); Fogarty International Center Research grant D43 TW007131 (M.A.-R., F.T.-P.), and the University of Northern Colorado (T.S.).

Footnotes

Published ahead of print 11 July 2012

REFERENCES

1. Agnello D, et al. 2003. Cytokines and transcription factors that regulate T helper cell differentiation: new players and new insights. J. Clin. Immunol. 23:147–161. [PubMed]
2. Borges AA, et al. 2008. Role of mixed Th1 and Th2 serum cytokines on pathogenesis and prognosis of hantavirus pulmonary syndrome. Microbes Infect. 10:1150–1157. [PubMed]
3. Botten J, et al. 2000. Experimental infection model for Sin Nombre hantavirus in the deer mouse (Peromyscus maniculatus). Proc. Natl. Acad. Sci. U. S. A. 97:10578–10583. [PubMed]
4. Botten J, et al. 2003. Persistent Sin Nombre virus infection in the deer mouse (Peromyscus maniculatus) model: sites of replication and strand-specific expression. J. Virol. 77:1540–1550. [PMC free article] [PubMed]
5. Botten J, Ricci R, Hjelle B. 2001. Establishment of a deer mouse (Peromyscus maniculatus rufinus) breeding colony from wild-caught founders: comparison of reproductive performance of wild-caught and laboratory-reared pairs. Comp. Med. 51:314–318. [PubMed]
6. Calisher CH, Mills JN, Root JJ, Beaty BJ. 2003. Hantaviruses: etiologic agents of rare, but potentially life-threatening zoonotic diseases. J. Am. Vet. Med. Assoc. 222:163–166. [PubMed]
7. Chensue SW, et al. 1996. Role of monocyte chemoattractant protein-1 (MCP-1) in Th1 (mycobacterial) and Th2 (schistosomal) antigen-induced granuloma formation: relationship to local inflammation, Th cell expression, and IL-12 production. J. Immunol. 157:4602–4608. [PubMed]
8. Cocchi F, et al. 1995. Identification of RANTES, MIP-1 alpha, and MIP-1 beta as the major HIV-suppressive factors produced by CD8+ T cells. Science 270:1811–1815. [PubMed]
9. Easterbrook JD, Klein SL. 2008. Seoul virus enhances regulatory and reduces proinflammatory responses in male Norway rats. J. Med. Virol. 80:1308–1318. [PubMed]
10. Easterbrook JD, Zink MC, Klein SL. 2007. Regulatory T cells enhance persistence of the zoonotic pathogen Seoul virus in its reservoir host. Proc. Natl. Acad. Sci. U. S. A. 104:15502–15507. [PubMed]
11. Eyzaguirre EJ, Milazzo ML, Koster FT, Fulhorst CF. 2008. Choclo virus infection in the Syrian golden hamster. Am. J. Trop. Med. Hyg. 78:669–674. [PMC free article] [PubMed]
12. Favre D, et al. 2009. Critical loss of the balance between Th17 and T regulatory cell populations in pathogenic SIV infection. PLoS Pathog. 5:e1000295 doi:10.1371/journal.ppat.1000295. [PMC free article] [PubMed]
13. Felsenstein J. 2005. PHYLIP (Phylogeny Inference Package), 3.6 ed Department of Genome Sciences, University of Washington, Seattle, WA.
14. Geimonen E, et al. 2003. Hantavirus pulmonary syndrome-associated hantaviruses contain conserved and functional ITAM signaling elements. J. Virol. 77:1638–1643. [PMC free article] [PubMed]
15. Glass WG, Rosenberg HF, Murphy PM. 2003. Chemokine regulation of inflammation during acute viral infection. Curr. Opin. Allergy Clin. Immunol. 3:467–473. [PubMed]
16. Handel TM, Domaille PJ. 1996. Heteronuclear (1H, 13C, 15N) NMR assignments and monocyte chemoattractant protein-1 (MCP-1). Biochemistry 35:6569–6584. [PubMed]
17. Hewitt EW. 2003. The MHC class I antigen presentation pathway: strategies for viral immune evasion. Immunology 110:163–169. [PubMed]
18. Hooper JW, Larsen T, Custer DM, Schmaljohn CS. 2001. A lethal disease model for hantavirus pulmonary syndrome. Virology 289:6–14. [PubMed]
19. Izcue A, Coombes JL, Powrie F. 2006. Regulatory T cells suppress systemic and mucosal immune activation to control intestinal inflammation. Immunol. Rev. 212:256–271. [PubMed]
20. Karpus WJ, et al. 1997. Differential CC chemokine-induced enhancement of T helper cell cytokine production. J. Immunol. 158:4129–4136. [PubMed]
21. Linterman MA, et al. 2010. IL-21 acts directly on B cells to regulate Bcl-6 expression and germinal center responses. J. Exp. Med. 207:353–363. [PMC free article] [PubMed]
22. Lukacs NW, et al. 1997. C-C chemokines differentially alter interleukin-4 production from lymphocytes. Am. J. Pathol. 150:1861–1868. [PubMed]
23. Matloubian M, Concepcion RJ, Ahmed R. 1994. CD4+ T cells are required to sustain CD8+ cytotoxic T-cell responses during chronic viral infection. J. Virol. 68:8056–8063. [PMC free article] [PubMed]
24. Meyer BJ, Schmaljohn CS. 2000. Persistent hantavirus infections: characteristics and mechanisms. Trends Microbiol. 8:61–67. [PubMed]
25. Milazzo ML, Eyzaguirre EJ, Molina CP, Fulhorst CF. 2002. Maporal viral infection in the Syrian golden hamster: a model of hantavirus pulmonary syndrome. J. Infect. Dis. 186:1390–1395. [PubMed]
26. Mills JN, Childs JE. 1998. Ecologic studies of rodent reservoirs: their relevance for human health. Emerg. Infect. Dis. 4:529–537. [PMC free article] [PubMed]
27. Mir MA, Brown B, Hjelle B, Duran WA, Panganiban AT. 2006. Hantavirus N protein exhibits genus-specific recognition of the viral RNA panhandle. J. Virol. 80:11283–11292. [PMC free article] [PubMed]
28. Mir MA, Panganiban AT. 2005. The hantavirus nucleocapsid protein recognizes specific features of the viral RNA panhandle and is altered in conformation upon RNA binding. J. Virol. 79:1824–1835. [PMC free article] [PubMed]
29. Mir MA, Panganiban AT. 2004. Trimeric hantavirus nucleocapsid protein binds specifically to the viral RNA panhandle. J. Virol. 78:8281–8288. [PMC free article] [PubMed]
30. Mori M, et al. 1999. High levels of cytokine-producing cells in the lung tissues of patients with fatal hantavirus pulmonary syndrome. J. Infect. Dis. 179:295–302. [PubMed]
31. Oko L, et al. 2006. Profiling helper T cell subset gene expression in deer mice. BMC Immunol. 7:18. [PMC free article] [PubMed]
32. Schmaljohn C, Hjelle B. 1997. Hantaviruses: a global disease problem. Emerg. Infect. Dis. 3:95–104. [PMC free article] [PubMed]
33. Schountz T, et al. 2007. Rapid field immunoassay for detecting antibody to Sin Nombre virus in deer mice. Emerg. Infect. Dis. 13:1604–1607. [PMC free article] [PubMed]
34. Schountz T, et al. 2007. Regulatory T cell-like responses in deer mice persistently infected with Sin Nombre virus. Proc. Natl. Acad. Sci. U. S. A. 104:15496–15501. [PubMed]
35. Sneath PHE, Sokal RR. 1963. Principles of numerical taxonomy. Freeman & Co, San Francisco, CA.
36. Stein RA. 2011. Super-spreaders in infectious diseases. Int. J. Infect. Dis. 15:e510–e513 doi:10.1016/j.ijid.2010.06.020. [PubMed]
37. Wahl SM. 2007. Transforming growth factor-beta: innately bipolar. Curr. Opin. Immunol. 19:55–62. [PubMed]
38. Zaki SR, et al. 1995. Hantavirus pulmonary syndrome. Pathogenesis of an emerging infectious disease. Am. J. Pathol. 146:552–579. [PubMed]
39. Zhao W, Pahar B, Borda JT, Alvarez X, Sestak K. 2007. A decline in CCL3-5 chemokine gene expression during primary simian-human immunodeficiency virus infection. PLoS One 2:e726 doi:10.1371/journal.pone.0000726. [PMC free article] [PubMed]
40. Zotos D, et al. 2010. IL-21 regulates germinal center B cell differentiation and proliferation through a B cell-intrinsic mechanism. J. Exp. Med. 207:365–378. [PMC free article] [PubMed]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)