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.     

Autophagic clearance of Sin Nombre hantavirus glycoprotein Gn promotes virus replication in cells

Hantavirus glycoprotein precursor (GPC) is posttranslationally cleaved into two glycoproteins, Gn and Gc. Cells transfected with plasmids expressing either GPC or both Gn and Gc revealed that Gn is posttranslationally degraded. Treatment of cells with the autophagy inhibitors 3-methyladenine, LY-294002, or Wortmanin rescued Gn degradation, suggesting that Gn is degraded by the host autophagy machinery. Confocal microscopic imaging showed that Gn is targeted to autophagosomes for degradation by an unknown mechanism. Examination of autophagy markers LC3-I and LC3-II demonstrated that both Gn expression and Sin Nombre hantavirus (SNV) infection induce autophagy in cells. To delineate whether induction of autophagy and clearance of Gn play a role in the virus replication cycle, we downregulated autophagy genes BCLN-1 and ATG7 using small interfering RNA (siRNA) and monitored virus replication over time. These studies revealed that inhibition of host autophagy machinery inhibits Sin Nombre virus replication in cells, suggesting that autophagic clearance of Gn is required for efficient virus replication. Our studies provide mechanistic insights into viral pathogenesis and reveal that SNV exploits the host autophagy machinery to decrease the intrinsic steady-state levels of an important viral component for efficient replication in host cells.     

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.     

Transmission study of Andes hantavirus infection in wild sigmodontine rodents

Our study was designed to contribute to an understanding of the timing and conditions under which transmission of Andes hantavirus in Oligoryzomys longicaudatus reservoir populations takes place. Mice were caged in test habitats consisting of steel drums containing holding cages, where seronegative rodents were exposed to wild seropositive individuals by freely sharing the same cage or being separated by a wire mesh. Tests were also performed for potential viral transmission to mice from excrement-tainted bedding in the cages. Andes virus transmitted efficiently; from 130 attempts with direct contact, 12.3% resulted in virus transmission. However, if we consider only those rodents that proved to be infectious, from 93 attempts we obtained 16 infected animals (17.2%). Twelve of them resulted from intraspecies O. longicaudatus encounters where male mice were differentially affected and 4 resulted from O. longicaudatus to Abrothrix olivaceus. Experiments using Abrothrix longipilis as receptors were not successful. Transmission was not observed between wire mesh-separated animals, and mice were not infected from excrement-tainted bedding. Bites seemed not to be a requisite for oral transmission. Genomic viral RNA was amplified in two out of three saliva samples from seropositive rodents, but it was not detected in urine samples obtained by vesicle puncture from two other infected rodents. Immunohistochemistry, using antibodies against Andes (AND) hantavirus proteins, revealed strong reactions in the lung and salivary glands, supporting the possibility of oral transmission. Our study suggests that AND hantavirus may be principally transmitted via saliva or saliva aerosols rather than via feces and urine.     

Study of hantavirus infection in captive breed colonies of wild rodents

Wild sigmondontine rodents are known to be the reservoir of several serotypes of New World hantaviruses. The mechanism of viral transmission is by aerosol inhalation of the excreta from infected rodents. Considering that the captive breed colonies of various wild mammals may present a potential risk for hantaviral transmission, we examined 85 specimens of Thrichomys spp. (Echimyidae) and 17 speciemens of Nectomys squamipes (Sigmodontinae) from our colony for the presence of hantavirus infections. Blood samples were assayed for the presence of antibodies to Andes nucleocapsid antigen using enzyme-linked immunosorbent assay (ELISA). Additionally, serum samples from workers previously exposed to wild rodents, in the laboratories where the study was conducted, were also tested by ELISA to investigate prevalence of anti-hantavirus IgG antibodies. All blood samples were negative for hantavirus antibodies. Although these results suggest that those rodent's colonies are hantavirus free, the work emphasizes the need for hantavirus serological monitoring in wild colonized rodents and secure handling potentially infected rodents as important biosafety measures.     

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.     

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

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Temporal analysis of Andes virus and Sin Nombre virus infections of Syrian hamsters

Andes virus (ANDV) and Sin Nombre virus (SNV) are rodent-borne hantaviruses that cause a highly lethal hemorrhagic fever in humans known as hantavirus pulmonary syndrome (HPS). There are no vaccines or specific drugs to prevent or treat HPS, and the pathogenesis is not understood. Syrian hamsters infected with ANDV, but not SNV, develop a highly lethal disease that closely resembles HPS in humans. Here, we performed a temporal pathogenesis study comparing ANDV and SNV infections in hamsters. SNV was nonpathogenic and viremia was not detected despite the fact that all animals were infected. ANDV was uniformly lethal with a mean time to death of 11 days. The first pathology detected was lymphocyte apoptosis starting on day 4. Animals were viremic and viral antigen was first observed in multiple organs by days 6 and 8, respectively. Levels of infectious virus in the blood increased 4 to 5 logs between days 6 and 8. Pulmonary edema was first detected ultrastructurally on day 6. Ultrastructural analysis of lung tissues revealed the presence of large inclusion bodies and substantial numbers of vacuoles within infected endothelial cells. Paraendothelial gaps were not observed, suggesting that fluid leakage was transcellular and directly attributable to infecting virus. Taken together, these data imply that HPS treatment strategies aimed at preventing virus replication and dissemination will have the greatest probability of success if administered before the viremic phase; however, because vascular leakage is associated with infected endothelial cells, a therapeutic strategy targeting viral replication might be effective even at later times (e.g., after disease onset).     

Deer mouse movements in peridomestic and sylvan settings in relation to Sin Nombre virus antibody prevalence

Prevalence of antibody to Sin Nombre virus (SNV) has been found to be nearly twice as high in deer mice (Peromyscus maniculatus) in peridomestic settings as in sylvan settings in two studies in Montana and one in New Mexico. We investigated whether this difference may be related to a difference in deer mouse movements in the two settings. We used radiotelemetry to determine home range size and length of movement for 22 sylvan (1991-1992) and 40 peridomestic deer mice (1995-1999). We also determined the percentage of locations inside versus outside of buildings for peridomestic mice. Though variable, average home range size for female deer mice was significantly smaller for peridomestic deer mice than for sylvan deer mice. The smaller home range in peridomestic settings may concentrate shed SNV, and protection from solar ultraviolet radiation inside buildings may increase environmental persistence of SNV. Both these factors could lead to increased SNV exposure of deer mice within peridomestic populations and result in higher antibody prevalence. Peridomestic deer mice moved between buildings and outside areas, which is evidence that SNV can be transmitted between peridomestic and sylvan populations.     

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.     

Long-term dynamics of Sin Nombre viral RNA and antibody in deer mice in Montana

Infections with hantaviruses in the natural host rodent may result in persistent, asymptomatic infections involving shedding of virus into the environment. Laboratory studies have partially characterized the acute and persistent infection by Sin Nombre virus (SNV) in its natural host, the deer mouse (Peromyscus maniculatus). However, these studies have posed questions that may best be addressed using longitudinal studies involving sequential sampling of individual wild-caught, naturally infected mice. Using enzyme immunoassay and polymerase chain reaction (PCR) analysis of monthly blood samples, we followed the infection status of deer mice in a mark-recapture study in Montana for 2 yr. Only six of 907 samples without IgG antibody to SNV contained detectable SNV RNA, suggesting that there is a very brief period of viremia before the host develops detectable antibody. The simultaneous presence of both antibody and viral RNA in blood was detected in consecutive monthly samples for as long as 3 mo. However, chronic infection was typified by alternating characteristics of PCR positivity and PCR negativity. Two possible interpretations of these results are that 1) viral RNA may be consistently present in the blood of chronically infected deer mouse, but that viral RNA is near the limits of PCR detectability or 2) SNV RNA sporadically appears in blood as a consequence of unknown physiological events. The occurrence of seasonal patterns in the proportion of samples that contains antibody and that also contained SNV RNA demonstrated a temporal association between recent infection (antibody acquisition) and presence of viral RNA in blood.     

Purification and characterization of the Sin Nombre virus nucleocapsid protein expressed in Escherichia coli

Sin Nombre virus is a member of the Hantavirus genus, family Bunyaviridae, and is an etiologic agent of hantavirus pulmonary syndrome. The hantavirus nucleocapsid (N) protein plays an important role in the encapsidation and assembly of the viral negative-sense genomic RNA. The Sin Nombre N protein was expressed as a C-terminal hexahistidine fusion in Escherichia coli and initially purified by nickel-affinity chromatography. We developed methods to extract the soluble fraction and to solubilize the remainder of the N protein using denaturants. Maximal expression of protein from native purification was observed after a 1.5-h induction with IPTG (2.4 mg/L). The zwitterionic detergent Chaps did not enhance the yield of native purifications, but increased the yield of protein obtained from insoluble purifications. Both soluble and insoluble materials, purified by nickel-affinity chromatography, were also subjected to Hi Trap SP Sepharose fast-flow (FF) chromatography. Both soluble and insoluble proteins had a similar A(280) profile on the Sepharose FF column, and both suggested the presence of a nucleic acid contaminant. The apparent dissociation constant of the N protein, purified by nickel-affinity and SP Sepharose FF chromatography, and the 5' end of the viral S-segment genome were measured using a filter binding assay. The N protein-vRNA complex had an apparent dissociation constant of 140 nM.     

Rapid and simultaneous screening and quantitation of antitetanus and anti-HBs antibodies using Laurell's method]

Every transfusion Center which must hunt out anti-tetanus and anti-HBs antibodies to obtain specific corresponding immunoglobulins, is submitted to rapidity, precision and low cost necessity. In order to satisfy these obligations, the authors describe a simple and rapid method, allowing use of the same serum, through a single stage, and simultaneously, screening and quantitation of anti-tetanus antibodies (according to Laurells' method) and of fractionation suitable anti-HBs antibodies (through electro immunodiffusion). Technical conditions exposed in this paper allow direct quantitation of anti-tetanus antibodies from 1 to 30-40 IU; dilutions can be made for upper titers. False negative reactions due to zone effect, as observed in EID and passive haemagglutination do not occur while using this method. Its routine use contributes to the increase of the percentage of fractionation suitable plasmas, without overloading the screening laboratory's tash.     

A simple and rapid method for screening amylolytic bacteria

A laboratory class was designed for the study of the ecology of amylolytic bacteria in soil, although other sources may be equally suitable for this purpose. Groups of three students carried out the following: (a) preparation and sterilization of medium and plates, (b) collection and preparation of soil samples, spreading the samples on the plates, (c) incubation of the plates at 37 degrees C overnight, a further 1 h incubation at 60 degrees C to observe amylolytic activity due to thermophilic bacteria, and (d) interpretation and discussion of the results. These tasks are accomplished in two periods of 4h on consecutive days. No sophisticated instruments are required for these experiments, which can be carried out in three classes of 4h each. On the first day the students prepare culture media, buffers and reagents, as well as collect and grow soil samples. The second day is spent for both taxonomic identification of colonies and the HAI determination.