Pathophysiology of hantavirus pulmonary syndrome in rhesus macaques.

The pathophysiology of hantavirus pulmonary syndrome (HPS) remains unclear because of a lack of surrogate disease models with which to perform pathogenesis studies. Nonhuman primates (NHP) are considered the gold standard model for studying the underlying immune activation/suppression associated with immunopathogenic viruses such as hantaviruses; however, to date an NHP model for HPS has not been described. Here we show that rhesus macaques infected with Sin Nombre virus (SNV), the primary etiological agent of HPS in North America, propagated in deer mice develop HPS, which is characterized by thrombocytopenia, leukocytosis, and rapid onset of respiratory distress caused by severe interstitial pneumonia. Despite establishing a systemic infection, SNV differentially activated host responses exclusively in the pulmonary endothelium, potentially the mechanism leading to acute severe respiratory distress. This study presents a unique chronological characterization of SNV infection and provides mechanistic data into the pathophysiology of HPS in a closely related surrogate animal model. We anticipate this model will advance our understanding of HPS pathogenesis and will greatly facilitate research toward the development of effective therapeutics and vaccines against hantaviral diseases.

Characterization of a Cdc42 protein inhibitor and its use as a molecular probe.

Cdc42 plays important roles in cytoskeleton organization, cell cycle progression, signal transduction, and vesicle trafficking. Overactive Cdc42 has been implicated in the pathology of cancers, immune diseases, and neuronal disorders. Therefore, Cdc42 inhibitors would be useful in probing molecular pathways and could have therapeutic potential. Previous inhibitors have lacked selectivity and trended toward toxicity. We report here the characterization of a Cdc42-selective guanine nucleotide binding lead inhibitor that was identified by high throughput screening. A second active analog was identified via structure-activity relationship studies. The compounds demonstrated excellent selectivity with no inhibition toward Rho and Rac in the same GTPase family. Biochemical characterization showed that the compounds act as noncompetitive allosteric inhibitors. When tested in cellular assays, the lead compound inhibited Cdc42-related filopodia formation and cell migration. The lead compound was also used to clarify the involvement of Cdc42 in the Sin Nombre virus internalization and the signaling pathway of integrin VLA-4. Together, these data present the characterization of a novel Cdc42-selective allosteric inhibitor and a related analog, the use of which will facilitate drug development targeting Cdc42-related diseases and molecular pathway studies that involve GTPases.

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.

Highly differentiated, resting gn-specific memory CD8+ T cells persist years after infection by andes hantavirus.

In man, infection with South American Andes virus (ANDV) causes hantavirus cardiopulmonary syndrome (HCPS). HCPS due to ANDV is endemic in Southern Chile and much of Argentina and increasing numbers of cases are reported all over South America. A case-fatality rate of about 36% together with the absence of successful antiviral therapies urge the development of a vaccine. Although T-cell responses were shown to be critically involved in immunity to hantaviruses in mouse models, no data are available on the magnitude, specificity and longevity of ANDV-specific memory T-cell responses in patients. Using sets of overlapping peptides in IFN-gamma ELISPOT assays, we herein show in 78 Chilean convalescent patients that Gn-derived epitopes were immunodominant as compared to those from the N- and Gc-proteins. Furthermore, while the relative contribution of the N-specific response significantly declined over time, Gn-specific responses remained readily detectable ex vivo up to 13 years after the acute infection. Tetramer analysis further showed that up to 16.8% of all circulating CD3(+)CD8(+) T cells were specific for the single HLA-B*3501-restricted epitope Gn(465-473) years after the acute infection. Remarkably, Gn(465-473)-specific cells readily secreted IFN-gamma, granzyme B and TNF-alpha but not IL-2 upon stimulation and showed a 'revertant' CD45RA(+)CD27(-)CD28(-)CCR7(-)CD127(-) effector memory phenotype, thereby resembling a phenotype seen in other latent virus infections. Most intriguingly, titers of neutralizing antibodies increased over time in 10/17 individuals months to years after the acute infection and independently of whether they were residents of endemic areas or not. Thus, our data suggest intrinsic, latent antigenic stimulation of Gn-specific T-cells. However, it remains a major task for future studies to proof this hypothesis by determination of viral antigen in convalescent patients. Furthermore, it remains to be seen whether Gn-specific T cells are critical for viral control and protective immunity. If so, Gn-derived immunodominant epitopes could be of high value for future ANDV vaccines.

Incidence rate for hantavirus infections without pulmonary syndrome, Panama.

During 2001-2007, to determine incidence of all hantavirus infections, including those without pulmonary syndrome, in western Panama, we conducted 11 communitywide surveys. Among 1,129 persons, antibody prevalence was 16.5%-60.4%. Repeat surveys of 476 found that patients who seroconverted outnumbered patients with hantavirus pulmonary syndrome by 14 to 1.

Spatial but not temporal co-divergence of a virus and its mammalian host.

Co-divergence between host and parasites suggests that evolutionary processes act across similar spatial and temporal scales. Although there has been considerable work on the extent and correlates of co-divergence of RNA viruses and their mammalian hosts, relatively little is known about the extent to which virus evolution is determined by the phylogeographic history of host species. To test hypotheses related to co-divergence across a variety of spatial and temporal scales, we explored phylogenetic signatures in Andes virus (ANDV) sampled from Chile and its host rodent, Oligoryzomys longicaudatus. ANDV showed strong spatial subdivision, a phylogeographic pattern also recovered in the host using both spatial and genealogical approaches, and despite incomplete lineage sorting. Lineage structure in the virus seemed to be a response to current population dynamics in the host at the spatial scale of ecoregions. However, finer scale analyses revealed contrasting patterns of genetic structure across a latitudinal gradient. As predicted by their higher substitution rates, ANDV showed greater genealogical resolution than the rodent, with topological congruence influenced by the degree of lineage sorting within the host. However, despite these major differences in evolutionary dynamics, the geographic structure of host and virus converged across large spatial scales.

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.

Drug Repurposing from an Academic Perspective.

Academia and small business research units are poised to play an increasing role in drug discovery, with drug repurposing as one of the major areas of activity. Here we summarize project status for a number of drugs or classes of drugs: raltegravir, cyclobenzaprine, benzbromarone, mometasone furoate, astemizole, R-naproxen, ketorolac, tolfenamic acid, phenothiazines, methylergonovine maleate and beta-adrenergic receptor drugs, respectively. Based on this multi-year, multi-project experience we discuss strengths and weaknesses of academic-based drug repurposing research. Translational, target and disease foci are strategic advantages fostered by close proximity and frequent interactions between basic and clinical scientists, which often result in discovering new modes of action for approved drugs. On the other hand, lack of integration with pharmaceutical sciences and toxicology, lack of appropriate intellectual coverage and issues related to dosing and safety may lead to significant drawbacks. The development of a more streamlined regulatory process world-wide, and the development of pre-competitive knowledge transfer systems such as a global healthcare database focused on regulatory and scientific information for drugs world-wide, are among the ideas proposed to improve the process of academic drug discovery and repurposing, and to overcome the "valley of death" by bridging basic to clinical sciences.

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.

Molecular epidemiology of Powassan virus in North America.

Powassan virus (POW) is a tick-borne flavivirus distributed in Canada, the northern USA and the Primorsky region of Russia. POW is the only tick-borne flavivirus endemic to the western hemisphere, where it is transmitted mainly between Ixodes cookei and groundhogs (Marmota monax). Deer tick virus (DTV), a genotype of POW that has been frequently isolated from deer ticks (Ixodes scapularis), appears to be maintained in an enzootic cycle between these ticks and white-footed mice (Peromyscus leucopus). DTV has been isolated from ticks in several regions of North America, including the upper Midwest and the eastern seaboard. The incidence of human disease due to POW is apparently increasing. Previous analysis of tick-borne flaviviruses endemic to North America have been limited to relatively short genome fragments. We therefore assessed the evolutionary dynamics of POW using newly generated complete and partial genome sequences. Maximum-likelihood and Bayesian phylogenetic inferences showed two well-supported, reciprocally monophyletic lineages corresponding to POW and DTV. Bayesian skyline plots based on year-of-sampling data indicated no significant population size change for either virus lineage. Statistical model-based selection analyses showed evidence of purifying selection in both lineages. Positive selection was detected in NS-5 sequences for both lineages and envelope sequences for POW. Our findings confirm that POW and DTV sequences are relatively stable over time, which suggests strong evolutionary constraint, and support field observations that suggest that tick-borne flavivirus populations are extremely stable in enzootic foci.

Phage display selection of cyclic peptides that inhibit Andes virus infection.

Specific therapy is not available for hantavirus cardiopulmonary syndrome caused by Andes virus (ANDV). Peptides capable of blocking ANDV infection in vitro were identified using antibodies against ANDV surface glycoproteins Gn and Gc to competitively elute a cyclic nonapeptide-bearing phage display library from purified ANDV particles. Phage was examined for ANDV infection inhibition in vitro, and nonapeptides were synthesized based on the most-potent phage sequences. Three peptides showed levels of viral inhibition which were significantly increased by combination treatment with anti-Gn- and anti-Gc-targeting peptides. These peptides will be valuable tools for further development of both peptide and nonpeptide therapeutic agents.

Ecology, genetic diversity, and phylogeographic structure of andes virus in humans and rodents in Chile.

Andes virus (ANDV) is the predominant etiologic agent of hantavirus cardiopulmonary syndrome (HCPS) in southern South America. In Chile, serologically confirmed human hantavirus infections have occurred throughout a wide latitudinal distribution extending from the regions of Valparaíso (32 to 33 degrees S) to Aysén (46 degrees S) in southern Patagonia. In this study, we found seropositive rodents further north in the Coquimbo region (30 degrees S) in Chile. Rodent seroprevalence was 1.4%, with Oligoryzomys longicaudatus displaying the highest seroprevalence (5.9%), followed by Abrothrix longipilis (1.9%) and other species exhibiting

Recognition of decay accelerating factor and alpha(v)beta(3) 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 alpha(v)beta(3) integrins expressed on Vero E6 cells. SNV binding to DAF is multivalent and of high affinity (K(d) approximately 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.

Small molecule inhibitors of hantavirus infection.

Hantaviruses use α(v)ß(3) integrins on the surface of human host cells as a gateway to invasion, hence compounds that target this receptor may be used as antiviral agents. To accomplish this aim, new peptidomimetic compounds were selected based on similarity to a cyclic peptide known to bind the α(v)ß(3) receptor. This first round of biological screening identified peptidomimetic molecules which were effective hantavirus inhibitors in the low micromolar range, two thousand times more potent than the original cyclic peptide. Pharmacophore models were built to broaden the structural diversity of the second set of compounds screened. Structure-activity relationships (SAR) were drawn from the entire dataset. Further characterization by dose-response studies revealed that three compounds had potency in the nanomolar range. Selectivity assays with a panel of hantaviruses supported the mechanism of inhibition by targeting the α(v)ß(3) receptor, through the ß(3) integrin.

Multivalent presentation of antihantavirus peptides on nanoparticles enhances infection blockade.

Viral entry into susceptible host cells typically results from multivalent interactions between viral surface proteins and host entry receptors. In the case of Sin Nombre virus (SNV), a New World hantavirus that causes hantavirus cardiopulmonary syndrome, infection involves the interaction between viral membrane surface glycoproteins and the human integrin alpha(v)beta(3). Currently, there are no therapeutic agents available which specifically target SNV. To address this problem, we used phage display selection of cyclic nonapeptides to identify peptides that bound SNV and specifically prevented SNV infection in vitro. We synthesized cyclic nonapeptides based on peptide sequences of phage demonstrating the strongest inhibition of infection, and in all cases, the isolated peptides were less effective at blocking infection (9.0% to 27.6% inhibition) than were the same peptides presented by phage (74.0% to 82.6% inhibition). Since peptides presented by the phage were pentavalent, we determined whether the identified peptides would show greater inhibition if presented in a multivalent format. We used carboxyl linkages to conjugate selected cyclic peptides to multivalent nanoparticles and tested infection inhibition. Two of the peptides, CLVRNLAWC and CQATTARNC, showed inhibition that was improved over that of the free format when presented on nanoparticles at a 4:1 nanoparticle-to-virus ratio (9.0% to 32.5% and 27.6% to 37.6%, respectively), with CQATTARNC inhibition surpassing 50% when nanoparticles were used at a 20:1 ratio versus virus. These data illustrate that multivalent inhibitors may disrupt polyvalent protein-protein interactions, such as those utilized for viral infection of host cells, and may represent a useful therapeutic approach.

Case report: T-cell responses during clearance of Andes virus from blood cells 2 months after severe hantavirus cardiopulmonary syndrome.

Hantavirus Cardiopulmonary Syndrome (HCPS) due to Andes virus (ANDV) is endemic in Chile and Argentina and currently demonstrates a case-fatality rate of 37% in humans. By contrast to the chronically infected rodents, it is believed that ANDV in humans is cleared during the acute phase. Moreover, to date, both magnitude and quality of human T-cell responses during ANDV infection and clearance are unknown. Using IFN-gamma and granzyme B ELISPOT assays as well as flow cytometry, we prospectively studied the ANDV-specific T-cell responses in a 56-year-old convalescing survivor of severe HCPS, whose blood cells remained PCR-positive for ANDV-RNA until day 53 after hospital admission, that is, 67 days after infection and 42 days after discharge. PCR-negativity was closely related to the increase and function of (Gn(46-60))-specific IFN-gamma(+) granzyme B(+) CD8(+) T-cells, but not to neutralizing antibody titers. Concurrently, the phenotype of CD45RA(+)CCR7(-) Gn(46-60)-specific T-cells shifted from a CD28(-)CD27(+) "intermediate" to a CD28(-) CD27(-) "late" effector memory beyond day 53 after hospital admission. This is the first report that shows that ANDV can persist in the human hosts for more than 2 months. Moreover, the kinetics of T-cell responses during ANDV clearance may indicate a major role of T-cells for clearance of ANDV and human immunity to this pathogen.

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.

Profiling helper T cell subset gene expression in deer mice.

BACKGROUND: Deer mice (Peromyscus maniculatus) are the most common mammals in North America and are reservoirs for several zoonotic agents, including Sin Nombre virus (SNV), the principal etiologic agent of hantavirus cardiopulmonary syndrome (HCPS) in North America. Unlike human HCPS patients, SNV-infected deer mice show no overt pathological symptoms, despite the presence of virus in the lungs. A neutralizing IgG antibody response occurs, but the virus establishes a persistent infection. Limitations of detailed analysis of deer mouse immune responses to SNV are the lack of reagents and methods for evaluating such responses. RESULTS: We developed real-time PCR-based detection assays for several immune-related transcription factor and cytokine genes from deer mice that permit the profiling of CD4+ helper T cells, including markers of Th1 cells (T-bet, STAT4, IFNgamma, TNF, LT), Th2 cells (GATA-3, STAT6, IL-4, IL-5) and regulatory T cells (Fox-p3, IL-10, TGFbeta1). These assays compare the expression of in vitro antigen-stimulated and unstimulated T cells from individual deer mice. CONCLUSION: We developed molecular methods for profiling immune gene expression in deer mice, including a multiplexed real-time PCR assay for assessing expression of several cytokine and transcription factor genes. These assays should be useful for characterizing the immune responses of experimentally- and naturally-infected deer mice.

Use of IgG avidity to indirectly monitor epizootic transmission of sin nombre virus in deer mice (Peromyscus maniculatus).

An IgG avidity assay was developed to differentiate deer mice that had recently acquired Sin Nombre virus (SNV) from those that were infected in the distant past. Using this procedure, low avidity antibodies were predominantly detected in experimentally infected deer mice (89.5%) within the first 30 days post-inoculation. The assay was then applied to sera from naturally infected deer mice collected during a field investigation associated with a cluster of hantavirus pulmonary syndrome cases. A higher proportion of seropositive mice collected during the outbreak had serum with low avidity antibodies (16.7%) when compared with mice trapped four months later (5.7%). Sin Nombre virus RNA was detectable in blood in a similar fraction of low- (45%) and high- (38.7%) avidity groups. Non-adult mice were more likely to contain low-avidity antibodies (44.4%) than were adults (9.6%). Our results indicate that the IgG avidity assay shows promise as a tool to better characterize epizootic intensity and to identify factors involved in SNV transmission.

[The new-world hantaviruses. Ecology and epidemiology of an emerging virus in Latin America]. / Hantavirus del Nuevo Mundo. Ecología y epidemiología de un virus emergente en Latinoamérica.

The hantaviruses are a group of emerging rodent-borne pathogens (family Bunyaviridae; Genus Hantavirus) that are etiologic agents for hemorrhagic fever with renal syndrome (HFRS) in Europe and Asia and hantavirus cardiopulmonary syndrome (HCPS) in the Americas. HFRS is associated with rodents of the family Muridae, subfamilies Murinae and Arvicolinae; HPS is associated with rodents of the subfamily Sigmodontinae. Since the identification of HCPS in USA in 1993, a large number of cases of HPS and an increasing number of hantaviruses and rodent reservoir hosts have been identified in Central and South America. Epidemiologic studies have demonstrated important differences in frequency of infection with hantaviruses in both human and rodent host populations. Antibody prevalences in rodent and human populations may vary from less than 1% to more than 40%. Currently, more than 1500 cases of HCPS have been reported and more than 15 genetically distinct variants of hantaviruses, all associated with sigmodontine rodents, have been identified throughout the Americas. Several characteristics distinguish Latin American HCPS cases from the classical HCPS described for the first time in the USA. These include a variation in severity of disease from moderate and self-limiting to severe, the demonstration of person-to-person transmission, and a somewhat higher incidence of extrapulmonary clinical manifestations in the South American form of HCPS. Nevertheless, our understanding of hantaviruses in the Americas is still far from complete. The factors involved in the dynamics of these viruses in nature, their establishment and transmission within host populations and from hosts to humans, and the variable pathology of these viruses in humans are complex. It is likely that more hantaviruses will be described in the future, and much more data will be required in order to describe the diversity and evolution of this group of pathogens. Latin America, as the center of diversity for Sigmodontine rodents and their hantaviruses is presented with the unique opportunity as well as the challenge of being center stage for continued studies of the dynamics of hantaviruses in natural host populations and the links of host and virus to human populations.