Differential lymphocyte and antibody responses in deer mice infected with Sin Nombre hantavirus or Andes hantavirus.

UNLABELLED: Hantavirus cardiopulmonary syndrome (HCPS) is a rodent-borne disease with a high case-fatality rate that is caused by several New World hantaviruses. Each pathogenic hantavirus is naturally hosted by a principal rodent species without conspicuous disease and infection is persistent, perhaps for life. Deer mice (Peromyscus maniculatus) are the natural reservoirs of Sin Nombre virus (SNV), the etiologic agent of most HCPS cases in North America. Deer mice remain infected despite a helper T cell response that leads to high-titer neutralizing antibodies. Deer mice are also susceptible to Andes hantavirus (ANDV), which causes most HCPS cases in South America; however, deer mice clear ANDV. We infected deer mice with SNV or ANDV to identify differences in host responses that might account for this differential outcome. SNV RNA levels were higher in the lungs but not different in the heart, spleen, or kidneys. Most ANDV-infected deer mice had seroconverted 14 days after inoculation, but none of the SNV-infected deer mice had. Examination of lymph node cell antigen recall responses identified elevated immune gene expression in deer mice infected with ANDV and suggested maturation toward a Th2 or T follicular helper phenotype in some ANDV-infected deer mice, including activation of the interleukin 4 (IL-4) pathway in T cells and B cells. These data suggest that the rate of maturation of the immune response is substantially higher and of greater magnitude during ANDV infection, and these differences may account for clearance of ANDV and persistence of SNV. IMPORTANCE: Hantaviruses persistently infect their reservoir rodent hosts without pathology. It is unknown how these viruses evade sterilizing immune responses in the reservoirs. We have determined that infection of the deer mouse with its homologous hantavirus, Sin Nombre virus, results in low levels of immune gene expression in antigen-stimulated lymph node cells and a poor antibody response. However, infection of deer mice with a heterologous hantavirus, Andes virus, results in a robust lymph node cell response, signatures of T and B cell maturation, and production of antibodies. These findings suggest that an early and aggressive immune response to hantaviruses may lead to clearance in a reservoir host and suggest that a modest immune response may be a component of hantavirus ecology.

Assessing prevention measures and Sin Nombre hantavirus seroprevalence among workers at Yosemite National Park.

BACKGROUND: During 2012, a total of 10 overnight visitors to Yosemite National Park (Yosemite) became infected with a hantavirus (Sin Nombre virus [SNV]); three died. SNV infections have been identified among persons with occupational exposure to deer mice (Peromyscus maniculatus). METHODS: We assessed SNV infection prevalence, work and living environments, mice exposures, and SNV prevention training, knowledge, and practices among workers of two major employers at Yosemite during September-October, 2012 by voluntary blood testing and a questionnaire. RESULTS: One of 526 participants had evidence of previous SNV infection. Participants reported frequently observing rodent infestations at work and home and not always following prescribed safety practices for tasks, including infestation cleanup. CONCLUSION: Although participants had multiple exposures to deer mice, we did not find evidence of widespread SNV infections. Nevertheless, employees working around deer mice should receive appropriate training and consistently follow prevention policies for high-risk activities.

The adaptive immune response does not influence hantavirus disease or persistence in the Syrian hamster.

Pathogenic New World hantaviruses cause severe disease in humans characterized by a vascular leak syndrome, leading to pulmonary oedema and respiratory distress with case fatality rates approaching 40%. Hantaviruses infect microvascular endothelial cells without conspicuous cytopathic effects, indicating that destruction of the endothelium is not a mechanism of disease. In humans, high levels of inflammatory cytokines are present in the lungs of patients that succumb to infection. This, along with other observations, suggests that disease has an immunopathogenic component. Currently the only animal model available to study hantavirus disease is the Syrian hamster, where infection with Andes virus (ANDV), the primary agent of disease in South America, results in disease that closely mimics that seen in humans. Conversely, inoculation of hamsters with a passaged Sin Nombre virus (SNV), the virus responsible for most cases of disease in North America, results in persistent infection with high levels of viral replication. We found that ANDV elicited a stronger innate immune response, whereas SNV elicited a more robust adaptive response in the lung. Additionally, ANDV infection resulted in significant changes in the blood lymphocyte populations. To determine whether the adaptive immune response influences infection outcome, we depleted hamsters of CD4(+) and CD8(+) T cells before infection with hantaviruses. Depletion resulted in inhibition of virus-specific antibody responses, although the pathogenesis and replication of these viruses were unaltered. These data show that neither hantavirus replication, nor pathogenesis caused by these viruses, is influenced by the adaptive immune response in the Syrian hamster.

Kinetics of immune responses in deer mice experimentally infected with Sin Nombre virus.

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-ß) 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.

Characterization of monoclonal antibodies against hantavirus nucleocapsid protein and their use for immunohistochemistry on rodent and human samples.

Monoclonal antibodies are important tools for various applications in hantavirus diagnostics. Recently, we generated Puumala virus (PUUV)-reactive monoclonal antibodies (mAbs) by immunisation of mice with chimeric polyomavirus-derived virus-like particles (VLPs) harbouring the 120-amino-acid-long amino-terminal region of the PUUV nucleocapsid (N) protein. Here, we describe the generation of two mAbs by co-immunisation of mice with hexahistidine-tagged full-length N proteins of Sin Nombre virus (SNV) and Andes virus (ANDV), their characterization by different immunoassays and comparison with the previously generated mAbs raised against a segment of PUUV N protein inserted into VLPs. All of the mAbs reacted strongly in ELISA and western blot tests with the antigens used for immunization and cross-reacted to varying extents with N proteins of other hantaviruses. All mAbs raised against a segment of the PUUV N protein presented on chimeric VLPs and both mAbs raised against the full-length AND/SNV N protein reacted with Vero cells infected with different hantaviruses. The reactivity of mAbs with native viral nucleocapsids was also confirmed by their reactivity in immunohistochemistry assays with kidney tissue specimens from experimentally SNV-infected rodents and human heart tissue specimens from hantavirus cardiopulmonary syndrome patients. Therefore, the described mAbs represent useful tools for the immunodetection of hantavirus infection.

A temporal dilution effect: hantavirus infection in deer mice and the intermittent presence of voles in Montana.

The effect of intermittently occurring, non-reservoir host species on pathogen transmission and prevalence in a reservoir population is poorly understood. We investigated whether voles, Microtus spp., which occur intermittently, influenced estimated standing antibody prevalence (ESAP) to Sin Nombre hantavirus (SNV, Bunyaviridae: Hantavirus) among deer mice, Peromyscus maniculatus, whose populations are persistent. We used 14 years of data from central Montana to investigate whether ESAP among deer mice was related to vole presence or abundance while controlling for the relationship between deer mouse abundance and ESAP. We found a reduction in deer mouse ESAP associated with the presence of voles, independent of vole abundance. A number of studies have documented that geographic locations which support a higher host diversity can be associated with reductions in pathogen prevalence by a hypothesized dilution effect. We suggest a dilution effect may also occur in a temporal dimension at sites where host richness fluctuates. Preservation of host diversity and optimization of environmental conditions which promote occurrence of ephemeral species, such as voles, may result in a decreased ESAP to hantaviruses among reservoir hosts. Our results may extend to other zoonotic infectious diseases.

Sin Nombre virus-specific immunoglobulin M and G kinetics in hantavirus pulmonary syndrome and the role played by serologic responses in predicting disease outcome.

BACKGROUND: Sin Nombre virus (SNV) is the primary cause of hantavirus pulmonary syndrome (HPS) in the United States. Although other studies have demonstrated a possible association between neutralizing antibody titers and the severity of HPS, the exact nature of serologic responses and their association with outcomes have not been fully characterized. METHODS: We examined immunoglobulin M (IgM) and immunoglobulin G (IgG) serologic responses in 94 clinical samples from 81 patients with confirmed HPS. We further compared a subset of 31 patients with fatal HPS and 20 surviving patients for whom samples were available within a week after the onset of HPS. RESULTS: SNV-specific IgM antibodies displayed a trend suggesting an early peak, whereas IgG antibody values peaked later. Among individuals with samples from the first week after the onset of HPS, all surviving patients had SNV-specific IgG responses, compared with <50% of patients with fatal HPS, and the distribution of IgG responses was significantly higher in surviving patients. CONCLUSIONS: Production of SNV-specific IgM antibodies occurs early during the clinical course of HPS, whereas production of IgG antibodies may be more protracted. The presence and overall distribution of higher IgG antibody titers in surviving patients with HPS suggests that production of SNV-specific IgG may be a strong predictor of favorable outcomes.

Longitudinal Assessment of Cytokine Expression and Plasminogen Activation in Hantavirus Cardiopulmonary Syndrome Reveals Immune Regulatory Dysfunction in End-Stage Disease.

Pathogenic New World orthohantaviruses cause hantavirus cardiopulmonary syndrome (HCPS), a severe immunopathogenic disease in humans manifested by pulmonary edema and respiratory distress, with case fatality rates approaching 40%. High levels of inflammatory mediators are present in the lungs and systemic circulation of HCPS patients. Previous studies have provided insights into the pathophysiology of HCPS. However, the longitudinal correlations of innate and adaptive immune responses and disease outcomes remain unresolved. This study analyzed serial immune responses in 13 HCPS cases due to Sin Nombre orthohantavirus (SNV), with 11 severe cases requiring extracorporeal membrane oxygenation (ECMO) treatment and two mild cases. We measured viral load, levels of various cytokines, urokinase plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1). We found significantly elevated levels of proinflammatory cytokines and PAI-1 in five end-stage cases. There was no difference between the expression of active uPA in survivors' and decedents' cases. However, total uPA in decedents' cases was significantly higher compared to survivors'. In some end-stage cases, uPA was refractory to PAI-1 inhibition as measured by zymography, where uPA and PAI-1 were strongly correlated to lymphocyte counts and IFN-γ. We also found bacterial co-infection influencing the etiology and outcome of immune response in two cases. Unsupervised Principal Component Analysis and hierarchical cluster analyses resolved separate waves of correlated immune mediators expressed in one case patient due to a sequential co-infection of bacteria and SNV. Overall, a robust proinflammatory immune response, characterized by an imbalance in T helper 17 (Th17) and regulatory T-cells (Treg) subsets, was correlated with dysregulated inflammation and mortality. Our sample size is small; however, the core differences correlated to survivors and end-stage HCPS are instructive.

Seasonal variation in Sin Nombre virus infections in deer mice: preliminary results.

The proportion of deer mice (Peromyscus maniculatus) with recently acquired Sin Nombre virus (SNV) infections is an indicator of epizootic intensity and may be key in predicting outbreaks of hantavirus cardio-pulmonary syndrome in humans. We investigated whether incidence of recent infections was related to season, sex, reproductive status, or habitat disturbance. In May and September, 2006, we sampled 912 deer mice at six sites in Utah. We determined SNV antibody prevalence and estimated the number of recent infections with an avidity enzyme-linked immunosorbent assay. Antibody prevalence in adults (n = 735) was 22%, and putative maternal antibody prevalence in juveniles (n = 177) was 7%. Sampling period explained a significant amount of the variance in the probability of recent infections, which were two times more common in May versus September. Additionally, prevalence of high-avidity maternal antibodies (i.e., from dams with older infections) in juveniles did not correspond to the antibody avidity patterns in adult females. In May, no juveniles had high-avidity antibodies compared to adult females (49%); in September, avidity could not be measured in juveniles because none were seropositive, despite large sample sizes (n = 84) and an 11% seroprevalence in adult females. Based on the results, coupled with those from the literature, we speculate that the majority of new infections may occur predominantly in the spring and that SNV may impair reproductive output of females.

A Tale of Two Valleys: Disparity in Sin Nombre Virus Antibody Reactivity Between Neighboring Mojave Desert Communities.

INTRODUCTION: Hantaviruses are a group of globally distributed rodent-associated viruses, some of which are responsible for human morbidity and mortality. Sin Nombre orthohantavirus, a particularly virulent species of hantavirus associated with Peromyscus spp. mice, is actively monitored by the Department of Public Health in California (CDPH). Recently, CDPH documented high (40%) seroprevalence in a potentially novel reservoir species, the cactus mouse (Peromyscus eremicus) in Death Valley National Park. METHODS: This study was performed in the extremely isolated Mojave Desert Amargosa River valley region of southeastern Inyo County, California, 105 km from Death Valley, approximately over the same time interval as the CDPH work in Death Valley (between 2011 and 2016). Similar rodent species were captured as in Death Valley and were tested for select hantaviruses using serology and RT-PCR to assess risk to human health and the conservation of the endemic endangered Amargosa vole. RESULTS: Among 192 rodents tested, including 56 Peromyscus spp., only one seropositive harvest mouse (Reithrodontomys megalotis) was detected. DISCUSSION: These data highlight the heterogeneity in the prevalence of hantavirus infection even among nearby desert communities and suggest that further studies of hantavirus persistence in desert environments are needed to more accurately inform the risks to public health and wildlife conservation.

Vesicular Stomatitis Virus-Based Vaccines Provide Cross-Protection against Andes and Sin Nombre Viruses.

Andes virus (ANDV) and Sin Nombre virus (SNV) are the main causative agents responsible for hantavirus cardiopulmonary syndrome (HCPS) in the Americas. HCPS is a severe respiratory disease with a high fatality rate for which there are no approved therapeutics or vaccines available. Some vaccine approaches for HCPS have been tested in preclinical models, but none have been tested in infectious models in regard to their ability to protect against multiple species of HCPS-causing viruses. Here, we utilize recombinant vesicular stomatitis virus-based (VSV) vaccines for Andes virus (ANDV) and Sin Nombre virus (SNV) and assess their ability to provide cross-protection in infectious challenge models. We show that, while both rVSVΔG/ANDVGPC and rVSVΔG/SNVGPC display attenuated growth as compared to wild type VSV, each vaccine is able to induce a cross-reactive antibody response. Both vaccines protected against both homologous and heterologous challenge with ANDV and SNV and prevented HCPS in a lethal ANDV challenge model. This study provides evidence that the development of a single vaccine against HCPS-causing hantaviruses could provide protection against multiple agents.

Cross-reactive and serospecific epitopes of nucleocapsid proteins of three hantaviruses: prospects for new diagnostic tools.

The diagnosis of infectious diseases is sometimes difficult because of extensive immunological cross-reactivity between related viral antigens. On the path of constructing sero-specific antigens, we have identified residues involved in sero-specific and cross-reactive recognition of the nucleocapsid proteins (NPs) of Puumala virus (PUUV), Seoul virus (SEOV), and Sin Nombre virus (SNV) using serum samples from 17 Nephropathia epidemica patients. The mapping was performed by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis on a panel of N protein derivatives and alanine-substitution mutants in the three different hantavirus backgrounds. Four regions with different serological profiles were identified encompassing the amino acids (aa) 14-17, 22-24, 26, and 35-38. One of the regions showed strong cross-reactivity and was important for the recognition of SEOV and SNV antigens, but not the PUUV antigen (aa 35-38). Two regions displayed perceivable SEOV characteristics (aa 14-17 and aa 22-24 and 26) and the combined result of the alanine replacements resulted in a synergetic effect against the PUUV antigen (aa 14-17, 22-24, 26).

Development of an ELISA to detect Sin Nombre virus-specific IgM from deer mice (Peromyscus maniculatus).

Peromyscus maniculatus (deer mouse) is the primary reservoir for Sin Nombre virus (SNV). Although the presence of IgG antibodies is often used as a marker of infection, it provides little information on active infections in a population but usually is an indicator of past infections. The presence of IgM antibodies is a much better marker for determining whether active infections are present in a population. A mu-capture SNV-specific IgM enzyme linked immunosorbent assay (ELISA) was developed. From live-trap and release studies a total of 68 rodent sera were studied for the presence of Sin Nombre virus-specific IgG and IgM antibodies. In these studies, IgM responses were detected in a number of animals. In some cases early SNV infection was determined through the presence of anti-SNV IgM before IgG antibodies could be detected. From the set of animals analyzed, it was concluded that the IgM response against SNV can persist anywhere from 1 to up to over 2 months, with a median of less than 1 month. Most importantly, it was demonstrated that anti-Sin Nombre virus IgM is an important tool for detection of early infections in rodents and should be considered as a key diagnostic tool.

Rapid field immunoassay for detecting antibody to Sin Nombre virus in deer mice.

We developed a 1-hour field enzyme immunoassay (EIA) for detecting antibody to Sin Nombre virus in deer mice (Peromyscus maniculatus). The assay specificity and sensitivity were comparable to those of a standard EIA. This test will permit identification of rodents with antibody to this and perhaps other hantaviruses.

Selection and characterization of scFv antibodies against the Sin Nombre hantavirus nucleocapsid protein.

Rodent-borne hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) in the old world and hantavirus cardio-pulmonary syndrome (HCPS) in the new. Most cases of HCPS in North America are caused by Sin Nombre Virus (SNV). Current viral detection technologies depend upon the identification of anti-viral antibodies in patient serum. Detection of viral antigen may facilitate earlier detection of the pathogen. We describe here the characterization of two single-chain Fv antibodies (scFvs), selected from a large naïve phage antibody library, which are capable of identifying the Sin Nombre Virus nucleocapsid protein (SNV-N), with no cross reactivity with the nucleocapsid protein from other hantaviruses. The utility of such selected scFvs was increased by the creation of an scFv-alkaline phosphatase fusion protein which was able to directly detect virally produced material without the need for additional reagents.

Increased permeability of human endothelial cell line EA.hy926 induced by hantavirus-specific cytotoxic T lymphocytes.

Hantavirus infection causes two human diseases, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. The typical feature of these diseases is increased permeability in microvascular beds in the kidneys and the lungs, respectively. The mechanism of capillary leakage, however, is not understood. Some evidence suggests that hantavirus disease pathogenesis is immunologically mediated by cytotoxic T lymphocytes and other immune cells in target organs producing inflammatory cytokines. In this study we examined the roles of virus-specific cytotoxic T lymphocytes in increased permeability of human endothelial cells infected with hantavirus. We used a human CD8(+) hantavirus-specific cytotoxic T lymphocyte line, 1A-E2, specific for the HLA-A24-restricted epitope in Sin Nombre and Puumala virus G2 protein, and the human endothelial cell line, EA.hy926 that expresses HLA-A24 molecule. The cytotoxic T lymphocyte line recognized and lysed target cells infected with Sin Nombre virus, and in transwell permeability assays increased permeability of EA.hy926 cell monolayer infected with Sin Nombre virus or recombinant adenovirus expressing the Sin Nombre virus G2 protein. These results suggest that cytotoxic T lymphocyte activity contribute to capillary leakage observed in patients with hantavirus pulmonary syndrome or hemorrhagic fever with renal syndrome.

Delayed density-dependent prevalence of Sin Nombre virus antibody in Montana deer mice (Peromyscus maniculatus) and implications for human disease risk.

American hantaviruses cause a severe respiratory disease known as hantavirus pulmonary syndrome (HPS). In the United States, Sin Nombre virus (SNV), carried by the deer mouse ( Peromyscus maniculatus), is the etiologic agent in the majority of HPS cases. The relationship between deer mouse population density and SNV infection prevalence in deer mice is poorly understood. Our purpose was to clarify this relationship by demonstrating the existence of delayed-density-dependent prevalence of SNV infection in populations of wild deer mice. We also explored the relationship between SNV infection in deer mouse populations and the incidence of human HPS. The study population was 3,616 deer mice captured on 10 mark-recapture grids in Montana during May and September, 1994-2004. Using multivariate logistic regression analysis, we found a strong association between deer mouse population density in fall (September) and SNV antibody prevalence in deer mice the following spring (May). Other characteristics associated with SNV infection in deer mice in spring were: (1) presence of at least one infected deer mouse in the population the previous fall, (2) male gender, (3) adult age class, (4) presence of scars, (5) grassland and logged habitats, and (6) elevations below 1,300 m. There was a strong association between concurrently measured SNV antibody prevalence in deer mice and probable exposure of human HPS cases during the same time period. Human cases were more likely to occur during seasons when SNV antibody prevalence was at least 10% in deer mouse populations. These findings suggest that fall rodent population parameters could be used to help guide prevention efforts the following spring.

A longitudinal study of hantavirus infection in three sympatric reservoir species in agroecosystems on the Argentine Pampa.

Prevalence of antibody reactive with Sin Nombre hantavirus (SNV) was evaluated from rodents captured over 31 months (March 1988 to September 1990) from six mark-recapture grids on the central Argentine Pampa. The most frequently infected rodents were: Akodon azarae (31/459), Necromys benefactus (8/141), and Oligoryzomys flavescens (10/281), which are known hosts of Pergamino, Maciel, and Lechiguanas hantaviruses, respectively. Relative population density and antibody prevalence varied seasonally and from year to year, population densities were highest in fall and prevalences were highest in spring. A positive association between antibody prevalence and body weight corroborated findings from other studies suggesting that hantaviruses are maintained in reservoir populations by horizontal transmission. In two of three host species, transmission was more frequent among male than among female mice. We found no evidence for a detrimental effect of hantavirus infection on host body weight, growth, longevity, movement, or reproductive preparedness. This analysis, based on cryopreserved specimens, represents the earliest conducted longitudinal, mark-recapture study of the dynamics of infection of autochthonous American hantaviruses in their sigmodontine host populations.