Shedding and intracage transmission of Sin Nombre hantavirus in the deer mouse (Peromyscus maniculatus) model

The mechanism(s) by which Sin Nombre (SN) hantavirus is maintained in deer mouse populations is unclear. Field studies indicate that transmission occurs primarily if not exclusively via a horizontal mechanism. Using an experimental deer mouse infection model in an outdoor laboratory, we tested whether infected rodents shed SN virus in urine, feces, and saliva, whether infected mice transmit infection to na?ve cage mates, and whether infected dams are able to vertically transmit virus or antibody to offspring. Using pooled samples of urine, feces, and saliva collected from mice infected 8 to 120 days postinoculation (p.i.), we found that a subset of saliva samples, collected between 15 and 90 days p.i., contained viral RNA. Parallel studies conducted on wild-caught, naturally infected deer mice showed a similar pattern of intermittent positivity, also only in saliva samples. Attempts to isolate virus through inoculation of cells or na?ve deer mice with the secreta or excreta of infected mice were uniformly negative. Of 54 attempts to transmit infection by cohousing infected deer mice with seronegative cage mates, we observed only a single case of transmission, which occurred between 29 and 42 days p.i. Dams passively transferred antibodies to neonatal pups via milk, and those antibodies persisted for at least 2 months after weaning, but none transmitted infection to their pups. Compared to other hantavirus models, SN virus is shed less efficiently and transmits inefficiently among cage mates. Transmission of SN virus among reservoir rodents may require factors that are not required for other hantaviruses.     

Delayed density-dependent prevalence of Sin Nombre virus infection in deer mice (Peromyscus maniculatus) in central and western Montana

Understanding how transmission of zoonoses takes place within reservoir populations, such as Sin Nombre virus (SNV) among deer mice (Peromyscus maniculatus), is important in determining the risk of exposure to other hosts, including humans. In this study, we examined the relationship between deer mouse populations and the prevalence of antibodies to SNV, a system where the effect of host population abundance on transmission is debated. We examined the relationship between abundance of deer mice in late summer-early autumn and SNV antibody prevalence the following spring-early summer (termed delayed density-dependent [DDD] prevalence of infection) at both regional and local scales, using 12 live-trapping grids for 11-14 yr, across central and western Montana. When all trapping grids were combined (regional scale), there was a significant DDD relationship for individual months and when months within seasons were averaged. However, within individual grids (local scale), evidence of DDD prevalence of infection was observed consistently at only one location. These findings suggest that, although there is evidence of DDD prevalence of infection at regional scales, it is not always apparent at local scales, possibly because the regional pattern of DDD infection prevalence is driven by differences in abundance and prevalence among sites, rather than in autumn-spring delays. Transmission of SNV may be more complex than the original hypothesis of autumn-spring delayed density dependence suggests. This complexity is also supported by recent modeling studies. Empirical investigations are needed to determine the duration and determinants of time-lagged abundance and antibody prevalence. Our study suggests predicting local, human exposure risk to SNV in spring, based on deer mouse abundance in autumn, is unlikely to be a reliable public health tool, particularly at local scales.     

Population density and seasonality effects on Sin Nombre virus transmission in North American deermice (Peromyscus maniculatus) in outdoor enclosures

Surveys of wildlife host-pathogen systems often document clear seasonal variation in transmission; conclusions concerning the relationship between host population density and transmission vary. In the field, effects of seasonality and population density on natural disease cycles are challenging to measure independently, but laboratory experiments may poorly reflect what happens in nature. Outdoor manipulative experiments are an alternative that controls for some variables in a relatively natural environment. Using outdoor enclosures, we tested effects of North American deermouse (Peromyscus maniculatus) population density and season on transmission dynamics of Sin Nombre hantavirus. In early summer, mid-summer, late summer, and fall 2007-2008, predetermined numbers of infected and uninfected adult wild deermice were released into enclosures and trapped weekly or bi-weekly. We documented 18 transmission events and observed significant seasonal effects on transmission, wounding frequency, and host breeding condition. Apparent differences in transmission incidence or wounding frequency between high- and low-density treatments were not statistically significant. However, high host density was associated with a lower proportion of males with scrotal testes. Seasonality may have a stronger influence on disease transmission dynamics than host population density, and density effects cannot be considered independent of seasonality.     

Persistent Sin Nombre virus infection in the deer mouse (Peromyscus maniculatus) model: sites of replication and strand-specific expression

To address Sin Nombre (SN) virus persistence in deer mice, we sacrificed experimentally infected deer mice at eight time points from day 21 to day 217 postinoculation (p.i.) and examined their tissues for viral nucleocapsid (N) antigen expression and both negative-strand (genomic) and positive-strand (replicative/mRNA) viral S segment RNA titers. All the animals that we inoculated developed persistent infections, and SN virus could be isolated from tissues throughout the course of infection. The transition from an acute to a persistent pattern of infection appeared to occur between days 60 and 90 p.i. Beginning on day 60 p.i., the heart, brown adipose tissue (BAT), and lung retained antigen expression and genomic viral RNA the most frequently. We found a statistically significant association among the presence of replicative RNA in the heart, lung, and BAT, widespread antigen expression (in > or =5 tissues), and RNA viremia. Of these three tissues, the heart retained negative-strand RNA and viral N antigen the most consistently (in 25 of 26 animals). During persistence, there were two distinct patterns of infection: restricted versus disseminated tissue involvement. Mice with the restricted pattern exhibited N antigen expression in < or =3 tissues, an absence of viral RNA in the blood, neutralizing antibody titers of < or =1:1,280 (P = 0.01), and no replicative RNA in the heart, lung, or BAT. Those with the "disseminated" pattern showed N antigen expression in > or =5 tissues, neutralizing antibody titers of 1:160 to 1:20,480, replicative RNA in the heart, lung, and BAT at a high frequency, and RNA viremia. Virus could be isolated consistently only from mice that demonstrated the disseminated pattern. The heart, lung, and BAT are important sites for the replication and maintenance of SN virus during persistent infection.     

Human Exposure to Particulate Matter Potentially Contaminated with Sin Nombre Virus

The most common mechanism for human exposure to hantaviruses throughout North America is inhalation of virally contaminated particulates. However, risk factors associated with exposure to particulates potentially contaminated with hantaviruses are generally not well understood. In North America, Sin Nombre virus (SNV) is the most common hantavirus that infects humans, causing hantavirus pulmonary syndrome, which has a significant mortality rate (approximately 35%). We investigated human exposure to particulate matter and evaluated the effects of season, location (sylvan and peridomestic environment), and activity (walking and sweeping) on generation of particulates at the breathing zone (1.5 m above the ground). We found greater volumes of small inhalable particulates during the spring and summer compared to the fall and winter seasons and greater volumes of small inhalable particulates produced in peridomestic, compared to sylvan, environments. Also, greater volumes of particulates were generated at the breathing zone while walking compared to sweeping. Results suggest that more aerosolized particles were generated during the spring and summer months. Our findings suggest that simply moving around in buildings is a significant source of human exposure to particulates, potentially contaminated with SNV, during spring and summer seasons. These findings could be advanced by investigation of what particle sizes SNV is most likely to attach to, and where in the respiratory tract humans become infected.     

Temporal and Spatial Analysis of Sin Nombre Virus Quasispecies in Naturally Infected Rodents

Sin Nombre virus (SNV) is thought to establish a persistent infection in its natural reservoir, the deer mouse (Peromyscus maniculatus), despite a strong host immune response. SNV-specific neutralizing antibodies were routinely detected in deer mice which maintained virus RNA in the blood and lungs. To determine whether viral diversity played a role in SNV persistence and immune escape in deer mice, we measured the prevalence of virus quasispecies in infected rodents over time in a natural setting. Mark-recapture studies provided serial blood samples from naturally infected deer mice, which were sequentially analyzed for SNV diversity. Viral RNA was detected over a period of months in these rodents in the presence of circulating antibodies specific for SNV. Nucleotide and amino acid substitutions were observed in viral clones from all time points analyzed, including changes in the immunodominant domain of glycoprotein 1 and the 3' small segment noncoding region of the genome. Viral RNA was also detected in seven different organs of sacrificed deer mice. Analysis of organ-specific viral clones revealed major disparities in the level of viral diversity between organs, specifically between the spleen (high diversity) and the lung and liver (low diversity). These results demonstrate the ability of SNV to mutate and generate quasispecies in vivo, which may have implications for viral persistence and possible escape from the host immune system.     

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

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Dispersal of deer mice,Peromyscus maniculatus

Summary Dispersal of deer mice, Peromyscus maniculatus, was measured as immigration to and emigration from two control areas, and as immigration to a removal area. The number of mice dispersing was linearly related to the densities on the control areas, while the proportion of the population dispersing (rate of dispersal) was correlated primarily with the rate of increase of control populations. High rates of dispersal were also associated with a breakdown of the established social structure in the spring and fall. Dispersing animals were compared to residents with respect to sex ratio, weight, age, and breeding condition. The types of animals dispersing varied seasonally: light-weight, non-breeding males dispersed in the spring and summer; juveniles and breeding males dispersed at the end of the breeding season; and light-weight mice of both sexes dispersed over the winter. It is proposed that the animals that dominated the dispersal samples each season were moving in response to social pressure from residents, or local limitations of some resource, and thus, that dispersal was adaptive for the individuals concerned. Some tests of the hypotheses concerning resource limitation are suggested.     

A Lethal Disease Model for Hantavirus Pulmonary Syndrome in Immunosuppressed Syrian Hamsters Infected with Sin Nombre Virus

Sin Nombre virus (SNV) is a rodent-borne hantavirus that causes hantavirus pulmonary syndrome (HPS) predominantly in North America. SNV infection of immunocompetent hamsters results in an asymptomatic infection; the only lethal disease model for a pathogenic hantavirus is Andes virus (ANDV) infection of Syrian hamsters. Efforts to create a lethal SNV disease model in hamsters by repeatedly passaging virus through the hamster have demonstrated increased dissemination of the virus but no signs of disease. In this study, we demonstrate that immunosuppression of hamsters through the administration of a combination of dexamethasone and cyclophosphamide, followed by infection with SNV, results in a vascular leak syndrome that accurately mimics both HPS disease in humans and ANDV infection of hamsters. Immunosuppressed hamsters infected with SNV have a mean number of days to death of 13 and display clinical signs associated with HPS, including pulmonary edema. Viral antigen was widely detectable throughout the pulmonary endothelium. Histologic analysis of lung sections showed marked inflammation and edema within the alveolar septa of SNV-infected hamsters, results which are similar to what is exhibited by hamsters infected with ANDV. Importantly, SNV-specific neutralizing polyclonal antibody administered 5 days after SNV infection conferred significant protection against disease. This experiment not only demonstrated that the disease was caused by SNV, it also demonstrated the utility of this animal model for testing candidate medical countermeasures. This is the first report of lethal disease caused by SNV in an adult small-animal model.     

Sin Nombre Virus and Rodent Species Diversity: A Test of the Dilution and Amplification Hypotheses

Background

Species 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 Findings

We 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/Significance

While 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.     

Hemoglobin polymorphism in the deer mouse,Peromyscus maniculatus

Horizontal starch gel electrophoresis reveals three hemoglobin phenotypes in natural populations of the deer mouse, Peromyscus maniculatus,from northern Arizona. Phenotypic variation is controlled by a single locus. A sample of deer mice from Michigan was monomorphic for a multiple-banded phenotype. Hemoglobin polymorphism was also observed in Arizona samples of the species Peromyscus boylii.This investigation was supported by Public Health Service Research Grant R01-GM12190.     

Sin Nombre hantavirus decreases survival of male deer mice

How pathogens affect their hosts is a key question in infectious disease ecology, and it can have important influences on the spread and persistence of the pathogen. Sin Nombre virus (SNV) is the etiological agent of hantavirus pulmonary syndrome (HPS) in humans. A better understanding of SNV in its reservoir host, the deer mouse, could lead to improved predictions of the circulation and persistence of the virus in the mouse reservoir, and could help identify the factors that lead to increased human risk of HPS. Using mark-recapture statistical modeling on longitudinal data collected over 15 years, we found a 13.4% decrease in the survival of male deer mice with antibodies to SNV compared to uninfected mice (both male and female). There was also an additive effect of breeding condition, with a 21.3% decrease in survival for infected mice in breeding condition compared to uninfected, non-breeding mice. The data identified that transmission was consistent with density-dependent transmission, implying that there may be a critical host density below which SNV cannot persist. The notion of a critical host density coupled with the previously overlooked disease-induced mortality reported here contribute to a better understanding of why SNV often goes extinct locally and only seems to persist at the metapopulation scale, and why human spillover is episodic and hard to predict.     

Sixty polymorphic microsatellite markers for the oldfield mouse developed in Peromyscus polionotus and Peromyscus maniculatus

We isolated and characterized 60 novel microsatellite markers from the closely related oldfield mouse (Peromyscus polionotus) and deer mouse (Peromyscus maniculatus) for studies of conservation, ecological, quantitative and population genetics. We assessed all 60 markers in a wild population of Peromyscus polionotus rhoadsi (N = 20) from central Florida and found an average of nine alleles per marker and an observed heterozygosity (H_O) of 0.66 (range = 0.00–1.00). These polymorphic markers contribute to the growing number of genomic resources for Peromyscus, an emerging model system for ecological and evolutionary research.     

How Host Population Dynamics Translate into Time-Lagged Prevalence: An Investigation of Sin Nombre Virus in Deer Mice

Human cases of hantavirus pulmonary syndrome caused by Sin Nombre virus are the endpoint of complex ecological cascade from weather conditions, population dynamics of deer mice, to prevalence of SNV in deer mice. Using population trajectories from the literature and mathematical modeling, we analyze the time lag between deer mouse population peaks and peaks in SNV antibody prevalence in deer mice. Because the virus is not transmitted vertically, rapid population growth can lead initially to reduced prevalence, but the resulting higher population size may later increase contact rates and generate increased prevalence. Incorporating these factors, the predicted time lag ranges from 0 to 18 months, and takes on larger values when host population size varies with a longer period or higher amplitude, when mean prevalence is low and when transmission is frequency-dependent. Population size variation due to variation in birth rates rather than death rates also increases the lag. Predicting future human outbreaks of hantavirus pulmonary syndrome may require taking these effects into account.