Sin Nombre Research Articles

Long‐Term Patterns of Immune Investment by Wild Deer Mice Infected with Sin Nombre Virus

Immunocompetence of animals fluctuates seasonally, However, there is little consensus on the cause of these fluctuations. Some studies have suggested that these patterns are influenced by changes in reproductive condition, whereas others have suggested that differences result from seasonal variations in energy expenditures. The objective of our study was to examine these contrasting views of immunity by evaluating seasonal patterns of immune response and reproduction in wild populations of deer mice Peromyscus maniculatus exposed to Sin Nombre virus (SNV). Over three consecutive fall (September, October, November) and three consecutive spring (March, April, May) sampling periods, we used titration enzyme-linked immunosorbent assay (ELISA) to quantify virus-specific antibody production in 48 deer mice infected with SNV. Levels of reproductive hormones were quantified using ELISA. SNV antibody titers reached their lowest level during November (geometric mean titer [GMT] = 420) and their highest levels during September (GMT = 5,545) and May (GMT = 3,582), suggesting that the immune response of deer mice to SNV has seasonal patterns. The seeming decrease in antibody titer over winter coupled with the consistency in body masses suggests that during winter, immunocompetence may be compromised to offset the energetic costs of maintenance functions, including those associated with maintaining body mass. Deer mice showed distinct sex-based differences in SNV antibody production, with males producing higher antibody titers (GMT = 3,333) than females (GMT = 1,477). Levels of reproductive hormones do not appear to influence antibody production in either males or females, as there was no correlation between estradiol concentrations and SNV antibody titer in female deer mice (r² = 0.26), nor was there a significant relationship between levels of testosterone and SNV antibody titers in males (r² = 0.28). Collectively, this study demonstrates that immunocompetence of wild deer mice is seasonally variable; however, reproduction is not the primary stressor responsible for this variation. Rather, the data suggest that deer mice may compromise immunocompetence during winter to offset other maintenance costs during this period.

Ecology of Hantaviruses and Their Hosts in North America

Since the 1993 discovery of a highly pathogenic hantavirus associated with the North American deer mouse (Peromyscus maniculatus), intensive ecological studies have led to many advances in our understanding of the natural history of New World hantaviruses as it relates to human disease. Seventeen named hantaviruses have been identified in North America. Field and laboratory studies of Sin Nombre and other hantaviruses have delineated host associations, geographical distributions, mechanisms of transmission, temporal infection dynamics of these viruses in host populations, and environmental factors that influence these dynamics. Using data from these studies, preliminary predictive models of the risk of hantavirus infection to humans have been developed. Improved models using satellite-derived data are under development. Multidisciplinary collaboration, integration of field and laboratory studies, and establishment and maintenance of long-term monitoring studies will be critical to continued advancement in the understanding of hantavirus-host ecology and disease prevention in humans.

Sampling Frequency Differentially Influences Interpretation of Zoonotic Pathogen and Host Dynamics: Sin Nombre Virus and Deer Mice

Reports of novel emerging and resurging wildlife and zoonotic diseases have increased. Consequently, integration of pathogen sampling into wildlife monitoring programs has grown. Sampling frequency influences interpretations of coupled host-pathogen dynamics, with direct implication to human exposure risk, but has received little empirical attention. To address this, a 15-year study, based on monthly sampling, of deer mouse (Peromyscus maniculatus) populations and Sin Nombre virus (SNV; a virulent disease in humans) dynamics was evaluated. Estimates of deer mouse abundance, number infected with SNV, and SNV prevalence from sampling less frequently than each month (achieved by deletion of months and recalculation of these parameters) were compared to monthly sampling frequencies. Deer mouse abundance was underestimated (10%-20%), SNV prevalence was overestimated when prevalence was high (>15%), and fewer annual extremes of abundance and infection were detected when sampling frequency was less than monthly. Effort necessary to detect temporal dynamics of SNV differed from effort to detect demographic patterns in deer mouse abundance. Findings here are applicable to sampling strategies for other host-pathogen dynamics and have direct implications for allocation of public health resources and intervention programs.

Sin Nombre Virus–Specific Immunoglobulin M and G Kinetics in Hantavirus Pulmonary Syndrome and the Role Played by Serologic Responses in Predicting Disease Outcome

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. 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. 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. 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.

New World Hantaviruses Activate IFNλ Production in Type I IFN-Deficient Vero E6 Cells

BackgroundHantaviruses indigenous to the New World are the etiologic agents of hantavirus cardiopulmonary syndrome (HCPS). These viruses induce a strong interferon-stimulated gene (ISG) response in human endothelial cells. African green monkey-derived Vero E6 cells are used to propagate hantaviruses as well as many other viruses. The utility of the Vero E6 cell line for virus production is thought to owe to their lack of genes encoding type I interferons (IFN), rendering them unable to mount an efficient innate immune response to virus infection. Interferon λ, a more recently characterized type III IFN, is transcriptionally controlled much like the type I IFNs, and activates the innate immune system in a similar manner.Methodology/Principal FindingsWe show that Vero E6 cells respond to hantavirus infection by secreting abundant IFNλ. Three New World hantaviruses were similarly able to induce IFNλ expression in this cell line. The IFNλ contained within virus preparations generated with Vero E6 cells independently activates ISGs when used to infect several non-endothelial cell lines, whereas innate immune responses by endothelial cells are specifically due to viral infection. We show further that Sin Nombre virus replicates to high titer in human hepatoma cells (Huh7) without inducing ISGs.Conclusions/SignificanceHerein we report that Vero E6 cells respond to viral infection with a highly active antiviral response, including secretion of abundant IFNλ. This cytokine is biologically active, and when contained within viral preparations and presented to human epithelioid cell lines, results in the robust activation of innate immune responses. We also show that both Huh7 and A549 cell lines do not respond to hantavirus infection, confirming that the cytoplasmic RNA helicase pathways possessed by these cells are not involved in hantavirus recognition. We demonstrate that Vero E6 actively respond to virus infection and inhibiting IFNλ production in these cells might increase their utility for virus propagation.

Convalescent Pulmonary Dysfunction Following Hantavirus Pulmonary Syndrome in Panama and the United States

The objective of this study was to document persistent pulmonary symptoms and pulmonary function abnormalities in adults surviving hantavirus pulmonary syndrome (HPS). Acute infection by most hantaviruses result in mortality rates of 25–35%, while in Panama the mortality rate of 10% is contrasted by an unusually high incidence. In all types of HPS, the viral prodrome, cardiopulmonary phase due to massive pulmonary capillary leak syndrome, and spontaneous diuresis are followed by a convalescent phase with exertional dyspnea for 3–4 weeks, but the frequency of persistent symptoms is not known. In this observational study of a convenience sample, 14 survivors of HPS caused by Choclo virus infection in Panama and 9 survivors of HPS caused by Sin Nombre virus infection in New Mexico completed a questionnaire and pulmonary function tests up to 8 years after infection. In both groups, exertional dyspnea persisted for 1–2 years after acute infection in 43% (Panama) and 77% (New Mexico) of survivors surveyed. Reduction in midexpiratory flows (FEF25–75%), increased residual volume (RV), and reduced diffusion capacity (DLCO/VA) also were common in both populations; but the severity of reduced expiratory flow did not correlate with exertional dyspnea. Symptoms referable to previous hantavirus infection had resolved within 3 years of acute infection in most but not all patients in the Panama group. Temporary exertional dyspnea and reduced expiratory flow are common in early convalescence after HPS but resolves in almost all patients.

Relationship of Human Behavior within Outbuildings to Potential Exposure to Sin Nombre Virus in Western Montana

Sin Nombre virus (SNV) causes hantavirus cardiopulmonary syndrome (HCPS) in humans. Transmission of SNV among the deer mouse (Peromyscus maniculatus) host predominates during spring and summer, and is greater in peridomestic than sylvan settings where, protected from UV light, SNV may survive longer. Incidence of HCPS reflects these times and settings and is associated with inhalation of mouse excreta. Little is known, however, about how human use of outbuildings contributes to potential exposure to SNV. Here, the frequency and seasonality of outbuilding use by humans was evaluated, via a survey of rural residents in western Montana, to quantify human behaviors and potential risk of exposure to SNV. Retrieving or return of tools and scooping feed/grain were the most frequently undertaken activities. Seasonal activities coinciding with seasons of highest HCPS incidence and times of potentially high viral shedding by deer mice, included retrieving or returning tools, calving or lambing, and, to a lesser extent, feeding livestock and sweeping or cleaning. Human behavior is a component of SNV transmission risk and this preliminary study provides a basis from which to further evaluate this route of exposure.

Serologic Survey of Hantavirus Infection, Brazilian Amazon

To the Editor: Since the etiology of hantavirus cardiopulmonary syndrome (HCPS) was recognized in 1993 in the United States (1), various hantaviruses have been associated with the syndrome in South America (2,3). Depending on the viral genotype involved, hantavirus infection can take a wide variety of forms, from asymptomatic or oligosymptomatic to the classic clinical form (4,5). The first cases of HCPS in Brazil were reported in the state of Sao Paulo in 1993 (6,7). In 2000, an outbreak of HCPS was reported in the municipality of Anajatuba in the state of Maranhao in the Maranhao western lowlands, a microregion in the Brazilian Amazon (8,9). An ecologic study in this region identified antibodies against hantaviruses in wild rodents (Oligoryzomys fornesi and Holochilus sciureus). Analysis of RNA from the viruses isolated from these 2 rodent species showed 2 new hantaviruses, which were named Anajatuba and Rio Mearim, respectively (10). The Maranhao western lowlands is a swampy region that consists of 21 municipalities and ≈400,000 inhabitants. A cross-sectional study was conducted from August 2004 through September 2006 to identify exposures and activities associated with hantavirus infection. A convenience sample comprising 6 of the 21 municipalities in the region was selected: Pinheiro in the north, Vitoria do Mearim in the south, Sao Bento and Sao Joao Batista in the east, and Penalva and Viana in the west. In each municipality, a village in the countryside was randomly chosen. All members of those communities were invited to participate. Persons who agreed to take part in the study were interviewed, and a standardized questionnaire was used to collect demographic information. Participants provided blood samples after giving written informed consent. ELISA to detect immunoglobulin (Ig) G antibodies against hantaviruses was performed in the Arbovirology and Hemorrhagic Fevers Department at the Evandro Chagas Institute (Belem, PA, Brazil) by using antigen from the Sin Nombre virus supplied by the Centers for Disease Control and Prevention (Atlanta, GA, USA). Samples were initially screened at 1:100, and all positive results were confirmed in a serial dilution (starting at 1:400) test. Positive samples were those with titers >400 (10). Odds ratios (ORs) were estimated by logistic regression. Variables with p<0.20 in the univariate analysis were subjected to multivariate analysis. Our study comprised 1,386 persons. The seroprevalence of antihantavirus antibodies in the 6 municipalities was 4.7% (65/1,389), distributed as follows: Sao Joao Batista, 2.6% (8/307); Sao Bento, 3.1% (6/195); Penalva, 3.5% (5/145); Pinheiro, 4.8% (13/273); Viana, 5.6% (9/160); and Vitoria do Mearim, 7.8% (24/309). Most persons interviewed were farmers and illiterate and lived in rammed-earth huts. Killing rats in the fields was reported by 24.3% (337/1,389). According to univariate analysis, seeing rats in the dwelling or in the field, having killed rats in the dwelling or in the fields, being a farmer, being male, and being >20 years of age were associated with IgG against hantaviruses. No association was found between storage of grain inside a dwelling or presence of natural predators of rodents, e.g., domestic cats and snakes such as the red-tailed boa, and antibodies against hantaviruses (Table). Table Univariate analysis of risk factors associated with hantavirus infection in humans, Maranhao western lowlands, Brazil, 2006* Because of colinearity between rat exposure variables, only “having killed rats in the field” was submitted to adjusted analysis. In the multivariate analysis, being >20 years of age and having killed rats in the field remained associated with hantavirus infection. In the model that did not consider sex and age, being a farmer (OR 2.63, 95% confidence interval [CI] 1.56%–4.41%) and having killed rats in the field (OR 2.30, 95% CI 1.38%–3.84%) were independently associated with hantavirus infection. Hantavirus infection is endemic to the Maranhao western lowlands. It has characteristics of an occupational infection associated with agricultural work, but age mostly explained the effect of being a farmer (OR for being a farmer 1.29, 95% CI 0.74%–2.24%, adjusted for sex and age). Exposure to rodents appeared to occur both in fields and dwellings. Although serologic evidence indicated that hantaviruses were circulating in the 6 municipalities studied, no cases of HCPS were reported in any of them. This finding suggests that either only mild or asymptomatic cases were occurring and that these were not recognized as HCPS or that cases of hantavirus were not being detected and reported. However, in a study in 2000 in Anajatuba in a cohort of 234 persons who did not initially have IgG against hantaviruses, 4 seroconversions to IgG against these viruses were detected; 2 of these infections were asymptomatic and 2 were self-limiting febrile illnesses that occurred after 24 months of follow-up (9). The present study reinforces the suspicion that mild or atypical cases are occurring in this region and may be the main reason classical hantavirus infection has not been identified.

Recognition of decay accelerating factor and αvβ3 by inactivated hantaviruses: Toward the development of high-throughput screening flow cytometry assays

Hantaviruses cause two severe diseases in humans: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). The lack of vaccines or specific drugs to prevent or treat HFRS and HCPS and the requirement for conducting experiments in a biosafety level 3 laboratory (BSL-3) limit the ability to probe the mechanism of infection and disease pathogenesis. In this study, we developed a generalizable spectroscopic assay to quantify saturable fluorophore sites solubilized in envelope membranes of Sin Nombre virus (SNV) particles. We then used flow cytometry and live cell confocal fluorescence microscopy imaging to show that ultraviolet (UV)-killed SNV particles bind to the cognate receptors of live virions, namely, decay accelerating factor (DAF/CD55) expressed on Tanoue B cells and αvβ3 integrins expressed on Vero E6 cells. SNV binding to DAF is multivalent and of high affinity (Kd∼26pM). Self-exchange competition binding assays between fluorescently labeled SNV and unlabeled SNV are used to evaluate an infectious unit-to-particle ratio of approximately 1:14,000. We configured the assay for measuring the binding of fluorescently labeled SNV to Tanoue B suspension cells using a high-throughput flow cytometer. In this way, we established a proof-of-principle high-throughput screening (HTS) assay for binding inhibition. This is a first step toward developing HTS format assays for small molecule inhibitors of viral–cell interactions as well as dissecting the mechanism of infection in a BSL-2 environment.

NATURAL HISTORY OF SIN NOMBRE VIRUS INFECTION IN DEER MICE IN URBAN PARKS IN OREGON

Sin Nombre virus (SNV), one of at least 45 hantaviruses described worldwide, is hosted by the deer mouse, Peromyscus maniculatus, a common species throughout most of North America. Herein, we describe general life-history characteristics of deer mice and the ways in which these factors relate to the incidence of SNV infections among populations of this host species in and around Portland, Oregon. In total, 3,175 deer mice were captured from October 2002 to September 2005. Transmission of SNV appears to be associated with male breeding behaviors, as more males and adults were infected than expected by capture rate; spring and summer had the highest infection prevalence, as well as scrotal male captures. Wounding rates between infected and uninfected deer mice were not different in any age or sex class. Capture rates were significantly and positively related to the interaction of temperature departure from normal, total precipitation, and number of clear days from two seasons previous (P=0.029), while infection prevalence was significantly and negatively related to the capture rate of juveniles from two seasons previous (P=0.029).

The effect of seasonality, density and climate on the population dynamics of Montana deer mice, important reservoir hosts for Sin Nombre hantavirus

1. Since Sin Nombre virus was discovered in the U.S. in 1993, longitudinal studies of the rodent reservoir host, the deer mouse (Peromyscus maniculatus) have demonstrated a qualitative correlation among mouse population dynamics and risk of hantavirus pulmonary syndrome (HPS) in humans, indicating the importance of understanding deer mouse population dynamics for evaluating risk of HPS. 2. Using capture-mark-recapture statistical methods on a 15-year data set from Montana, we estimated deer mouse survival, maturation and recruitment rates and tested the relative importance of seasonality, population density and local climate in explaining temporal variation in deer mouse demography. 3. From these estimates, we designed a population model to simulate deer mouse population dynamics given climatic variables and compared the model to observed patterns. 4. Month, precipitation 5 months previously, temperature 5 months previously and to a lesser extent precipitation and temperature in the current month, were important in determining deer mouse survival. Month, the sum of precipitation over the last 4 months, and the sum of the temperature over the last 4 months were important in determining recruitment rates. Survival was more important in determining the growth rate of the population than recruitment. 5. While climatic drivers appear to have a complex influence on dynamics, our forecasts were good. Our quantitative model may allow public health officials to better predict increased human risk from basic climatic data.

Sin Nombre Virus Infection in Field Workers, Colorado, USA

We report 2 cases of Sin Nombre virus (SNV) infection in field workers, possibly contracted through rodent bites. Screening for antibodies to SNV in rodents trapped in 2 seasons showed that 9.77% were seropositive. Quantitative real-time PCR showed that 2 of 79 deer mice had detectable titers of SNV RNA.

Roles of human disturbance, precipitation, and a pathogen on the survival and reproductive probabilities of deer mice

Climate change, human disturbance, and disease can have large impacts on the dynamics of a species by affecting the likelihood of survival and reproduction of individuals. We investigated the roles of precipitation, off-road vehicle (ORV) alteration of habitat, and infection with Sin Nombre virus on the survival and reproductive probabilities of deer mice (Peromyscus maniculatus). We used generalized linear mixed models to estimate the effects of these factors and their interactions by fitting capture-recapture data collected seasonally from 2002 to 2007 at 17 sites in the Great Basin Desert of central Utah, USA. During periods with high precipitation, we found no difference in survival and reproductive probabilities between seasons, but during drier periods, we found a reduction of overwinter survival and fall reproductive activity. Precipitation also interacted with disturbance to affect survival probabilities and female reproduction; in periods with low precipitation, deer mice on highly disturbed sites had extremely low survival probabilities and low reproductive probabilities of females compared to those of individuals from low-disturbance sites. However, high precipitation ameliorated the effect of disturbance on both parameters. Deer mice from sites with high impact of ORV disturbance also had low survival over summer. Additionally, male reproductive probabilities were diminished on highly disturbed sites in both seasons; in contrast, they were reduced only in the fall on low-disturbance sites. Density had an overall negative effect on survival and reproductive probabilities of deer mice. For females, the negative effect on reproductive activity was amplified in highly disturbed sites. We found no effect of hantavirus infection on survival probabilities of deer mice. Overall, this study revealed complexity in the determinants of deer mouse survival and reproduction given by the effects of a number of significant interactions among explanatory variables. Thus, factors that may not appear to have a strong effect when investigated alone can still be influential by modulating the effect of a different factor.

Environmental monitoring to enhance comprehension and control of infectious diseases

In a world of emerging and resurging infectious diseases, dominated by zoonoses, environmental monitoring plays a vital role in our understanding their dynamics and their spillover to humans. Here, we critically review the ecology, epidemiology and need for monitoring of a variety of directly transmitted (Sin Nombre virus, Avian Influenza) and vector-borne (Ross River virus, West Nile virus, Lyme disease, anaplasmosis and babesiosis) zoonoses. We focus on the valuable role that existing monitoring plays in the understanding of these zoonoses, the demands for new monitoring, and how improvements can be made to existing monitoring. We also identify the fruitful outcomes which would result from implementation of the monitoring demands we have highlighted. This review aims to promote improvements in our understanding of zoonoses, their management, and public health by encouraging discussion among researchers and public health officials.

Simultaneous Detection of CDC Category "A" DNA and RNA Bioterrorism Agents by Use of Multiplex PCR & RT-PCR Enzyme Hybridization Assays.

Assays to simultaneously detect multiple potential agents of bioterrorism are limited. Two multiplex PCR and RT-PCR enzyme hybridization assays (mPCR-EHA, mRT-PCR-EHA) were developed to simultaneously detect many of the CDC category “A” bioterrorism agents. The “Bio T” DNA assay was developed to detect: Variola major (VM), Bacillus anthracis (BA), Yersinia pestis (YP), Francisella tularensis (FT) and Varicella zoster virus (VZV). The “Bio T” RNA assay (mRT-PCR-EHA) was developed to detect: Ebola virus (Ebola), Lassa fever virus (Lassa), Rift Valley fever (RVF), Hantavirus Sin Nombre species (HSN) and dengue virus (serotypes 1–4). Sensitivity and specificity of the 2 assays were tested by using genomic DNA, recombinant plasmid positive controls, RNA transcripts controls, surrogate (spiked) clinical samples and common respiratory pathogens. The analytical sensitivity (limit of detection (LOD)) of the DNA asssay for genomic DNA was 1×100∼1×102 copies/mL for BA, FT and YP. The LOD for VZV whole organism was 1×10−2 TCID50/mL. The LOD for recombinant controls ranged from 1×102∼1×103copies/mL for BA, FT, YP and VM. The RNA assay demonstrated LOD for RNA transcript controls of 1×104∼1×106 copies/mL without extraction and 1×105∼1×106 copies/mL with extraction for Ebola, RVF, Lassa and HSN. The LOD for dengue whole organisms was ∼1×10−4 dilution for dengue 1 and 2, 1×104 LD50/mL and 1×102 LD50/mL for dengue 3 and 4. The LOD without extraction for recombinant plasmid DNA controls was ∼1×103 copies/mL (1.5 input copies/reaction) for Ebola, RVF, Lassa and HSN. No cross-reactivity of primers and probes used in both assays was detected with common respiratory pathogens or between targeted analytes. Clinical sensitivity was estimated using 264 surrogate clinical samples tested with the BioT DNA assay and 549 samples tested with the BioT RNA assay. The clinical specificity is 99.6% and 99.8% for BioT DNA assay and BioT RNA assay, respectively. The surrogate sensitivities of these two assays were 100% (95%CI 83–100) for FT, BA (pX02), YP, VM, VZV, dengue 2,3,4 and 95% (95%CI 75–100) for BA (pX01) and dengue 1 using spiked clinical specimens. The specificity of both BioT multiplex assays on spiked specimens was 100% (95% CI 99–100). Compared to other available assays (culture, serology, PCR, etc.) both the BioT DNA mPCR-EHA and BioT RNA mRT-PCR-EHA are rapid, sensitive and specific assays for detecting many category “A” Bioterrorism agents using a standard thermocycler.

SEASONAL DISPERSAL PATTERNS OF SYLVAN DEER MICE (PEROMYSCUS MANICULATUS) WITHIN MONTANA RANGELANDS

We examined seasonal dispersal patterns and timing of new infections of Sin Nombre virus (SNV), as determined by recent acquisition of antibodies (seroconversion), in deer mice (Peromyscus maniculatus) at two Montana rangeland study sites over three years, 2004-2007. One study site was located in grassland habitat, and the other was located in shrub-steppe. In Montana, both of these habitats are commonly associated with peridomestic environments (in and around buildings). Peridomestic environments are where most reported human cases of hantavirus pulmonary syndrome (HPS) likely originate. Furthermore, deer mice dispersing from sylvan habitats colonize peridomestic environments. Thus, a thorough understanding of deer mouse dispersal is needed to help predict when humans are most at risk for exposure to SNV. We trapped mice at each study site twice a month, accumulating 85,200 trap nights of effort and capturing 6,185 individual deer mice a total of 22,654 times. We documented 980 dispersing individuals over 3 yr. We found positive correlations between the number of dispersing mice and number captured at each site, but there were no statistically significant seasonal differences in the number of dispersing mice. However, we did find a spring/summer bias in mice that seroconverted and dispersed, suggesting that recently infected deer mice are most likely to enter settings where humans may be exposed to SNV during spring and summer.

Evaluation of an Enzyme-Linked Immunosorbent Assay Based on Araraquara Virus Recombinant Nucleocapsid Protein

Laboratory diagnosis of hantavirus cardiopulmonary syndrome (HCPS) in Brazil has been performed mostly by detection of IgM antibodies to recombinant antigen purified from Sin Nombre virus and Andes virus (ANDV). Recently, a recombinant nucleocapsid (rN) protein of Araraquara virus (ARAV), a Brazilian hantavirus, was obtained in Escherichia coli. To evaluate ARAV rN as antigen for antibody detection, serum samples from 30 patients from Argentina seropositive for hantavirus were tested. All samples were positive for IgG and IgM by enzyme-linked immunosorbent assay (ELISA) using either ARAV rN or ANDV rN antigens. In Brazil, six of 60 serum samples from patients with suspected HCPS (10%) were positive for IgM by ELISA using ARAV rN antigen and 7 were positive using ANDV rN antigen. For results obtained with 90 serum samples analyzed by IgM ELISA with ANDV rN antigen, the sensitivity of the IgM ELISA using ARAV rN antigen was 97.2%, the specificity was 100%, the positive predictive value was 100%, and the negative predictive value was 98.1%. The results show that ARAV rN is a suitable antigen for diagnosis of hantavirus infection in Brazil and Argentina.

Sin nombre virus and rodent species diversity: a test of the dilution and amplification hypotheses.

BackgroundSpecies diversity is proposed to greatly impact the prevalence of pathogens. Two predominant hypotheses, the “Dilution Effect” and the “Amplification Effect”, predict divergent outcomes with respect to the impact of species diversity. The Dilution Effect predicts that pathogen prevalence will be negatively correlated with increased species diversity, while the Amplification Effect predicts that pathogen prevalence will be positively correlated with diversity. For many host-pathogen systems, the relationship between diversity and pathogen prevalence has not be empirically examined.Methodology/Principal FindingsWe tested the Dilution and Amplification Effect hypotheses by examining the prevalence of Sin Nombre virus (SNV) with respect to diversity of the nocturnal rodent community. SNV is directly transmitted primarily between deer mice (Peromyscus maniculatus). Using mark-recapture sampling in the Spring and Fall of 2003–2005, we measured SNV prevalence in deer mice at 16 landscape level sites (3.1 hectares each) that varied in rodent species diversity. We explored several mechanisms by which species diversity may affect SNV prevalence, including reduced host density, reduced host persistence, the presence of secondary reservoirs and community composition. We found a negative relationship between species diversity and SNV prevalence in deer mice, thereby supporting the Dilution Effect hypothesis. Deer mouse density and persistence were lower at sites with greater species diversity; however, only deer mouse persistence was positively correlated with SNV prevalence. Pinyon mice (P. truei) may serve as dilution agents, having a negative effect on prevalence, while kangaroo rats (Dipodomys ordii), may have a positive effect on the prevalence of SNV, perhaps through effects on deer mouse behavior.Conclusions/SignificanceWhile previous studies on host-pathogen systems have found patterns of diversity consistent with either the Dilution or Amplification Effects, the mechanisms by which species diversity influences prevalence have not been investigated. Our study indicates that changes in host persistence, coupled with interspecific interactions, are important mechanisms through which diversity may influence patterns of pathogens. Our results reveal the complexity of rodent community interactions with respect to SNV dynamics.

Temporal and geographic evidence for evolution of Sin Nombre virus using molecular analyses of viral RNA from Colorado, New Mexico and Montana

BackgroundAll viruses in the family Bunyaviridae possess a tripartite genome, consisting of a small, a medium, and a large RNA segment. Bunyaviruses therefore possess considerable evolutionary potential, attributable to both intramolecular changes and to genome segment reassortment. Hantaviruses (family Bunyaviridae, genus Hantavirus) are known to cause human hemorrhagic fever with renal syndrome or hantavirus pulmonary syndrome. The primary reservoir host of Sin Nombre virus is the deer mouse (Peromyscus maniculatus), which is widely distributed in North America. We investigated the prevalence of intramolecular changes and of genomic reassortment among Sin Nombre viruses detected in deer mice in three western states.MethodsPortions of the Sin Nombre virus small (S) and medium (M) RNA segments were amplified by RT-PCR from kidney, lung, liver and spleen of seropositive peromyscine rodents, principally deer mice, collected in Colorado, New Mexico and Montana from 1995 to 2007. Both a 142 nucleotide (nt) amplicon of the M segment, encoding a portion of the G2 transmembrane glycoprotein, and a 751 nt amplicon of the S segment, encoding part of the nucleocapsid protein, were cloned and sequenced from 19 deer mice and from one brush mouse (P. boylii), S RNA but not M RNA from one deer mouse, and M RNA but not S RNA from another deer mouse.ResultsTwo of 20 viruses were found to be reassortants. Within virus sequences from different rodents, the average rate of synonymous substitutions among all pair-wise comparisons (πs) was 0.378 in the M segment and 0.312 in the S segment sequences. The replacement substitution rate (πa) was 7.0 × 10-4 in the M segment and 17.3 × 10-4 in the S segment sequences. The low πa relative to πs suggests strong purifying selection and this was confirmed by a Fu and Li analysis. The absolute rate of molecular evolution of the M segment was 6.76 × 10-3 substitutions/site/year. The absolute age of the M segment tree was estimated to be 37 years. In the S segment the rate of molecular evolution was 1.93 × 10-3 substitutions/site/year and the absolute age of the tree was 106 years. Assuming that mice were infected with a single Sin Nombre virus genotype, phylogenetic analyses revealed that 10% (2/20) of viruses were reassortants, similar to the 14% (6/43) found in a previous report.ConclusionAge estimates from both segments suggest that Sin Nombre virus has evolved within the past 37–106 years. The rates of evolutionary changes reported here suggest that Sin Nombre virus M and S segment reassortment occurs frequently in nature.

Bayou virus detected in non-oryzomyine rodent hosts: an assessment of habitat composition, reservoir community structure, and marsh rice rat social dynamics

In the United States, Bayou virus (BAYV) ranks second only to Sin Nombre virus (SNV) in terms of hantavirus pulmonary syndrome (HPS) incidents, having been confirmed in cases from Texas and Louisiana since its discovery in 1994. This study on BAYV infection among sympatric, non-oryzomyine rodents ("spillover") in Freeport, TX, is the first to link patterns of hantavirus interspecific spillover with the spatiotemporal ecology of the primary host (marsh rice rat, Oryzomys palustris). Mark-recapture and/or harvest methods were employed from March 2002 through May 2004 in two macrohabitat types. Rodent blood samples were screened for the presence of IgG antibody to BAYV antigen by IFA after which Ab-positive blood, saliva, and urine were analyzed for the presence of viral RNA by nested RT-PCR. From 727 non-oryzomyine captures, five seropositive (but not viral RNA positive) individuals were detected: one each of Baiomys taylori, Peromyscus leucopus, and Reithrodontomys fulvescens; and two Sigmodon hispidus. Spillover hosts were not associated with macrohabitat where O. palustris abundance, density, or seroprevalence was highest. Rather, spillover occurred in the macrohabitat indicative of greater overall disturbance (as indicated by grazing and exotic plant diversity) and overall biodiversity. Spillover occurred during periods of high seroprevalence detected elsewhere within the study region. Spillover locations differed significantly from all other capture locations in terms of percent water, shrub, and grass cover. Although greater habitat and mammal diversity of old-fields may serve to reduce seroprevalence levels by tempering intraspecific contacts between rice rats, greater diversity also may create an ecologically opportunistic setting for BAYV spillover. Impacts of varying levels of disturbance and biodiversity on transmission dynamics represent a vastly uncharacterized component of the evolutionary ecology of hantaviruses.