Andes Virus Genome Mutations That Are Likely Associated with Animal Model Attenuation and Human Person-to-Person Transmission.

We performed whole-genome sequencing with bait enrichment techniques to analyze Andes virus (ANDV), a cause of human hantavirus pulmonary syndrome. We used cryopreserved lung tissues from a naturally infected long-tailed colilargo, including early, intermediate, and late cell culture, passages of an ANDV isolate from that animal, and lung tissues from golden hamsters experimentally exposed to that ANDV isolate. The resulting complete genome sequences were subjected to detailed comparative genomic analysis against American orthohantaviruses. We identified four amino acid substitutions related to cell culture adaptation that resulted in attenuation of ANDV in the typically lethal golden hamster animal model of hantavirus pulmonary syndrome. Changes in the ANDV nucleocapsid protein, glycoprotein, and small nonstructural protein open reading frames correlated with mutations typical for ANDV strains associated with increased virulence in the small-animal model. Finally, we identified three amino acid substitutions, two in the small nonstructural protein and one in the glycoprotein, that were only present in the clade of viruses associated with efficient person-to-person transmission. Our results indicate that there are single-nucleotide polymorphisms that could be used to predict strain-specific ANDV virulence and/or transmissibility. IMPORTANCE Several orthohantaviruses cause the zoonotic disease hantavirus pulmonary syndrome (HPS) in the Americas. Among them, HPS caused by Andes virus (ANDV) is of great public health concern because it is associated with the highest case fatality rate (up to 50%). ANDV is also the only orthohantavirus associated with relatively robust evidence of person-to-person transmission. This work reveals nucleotide changes in the ANDV genome that are associated with virulence attenuation in an animal model and increased transmissibility in humans. These findings may pave the way to early severity predictions in future ANDV-caused HPS outbreaks.

Polymorphisms in human leukocyte antigens, human platelet antigens, and cytokine genes in hantavirus cardiopulmonary syndrome patients from Ribeirão Preto, Brazil.

Hantavirus cardiopulmonary syndrome is a severe human disease associated with hantavirus infection. The clinical course of illness varies greatly among individuals, possibly due to viral and immunological elements and the influence of host genetic factors on clinical outcome. As the magnitude of immune activation has been associated with disease severity, polymorphisms in genes involved in the immune response that may affect the development of this syndrome were investigated. Polymorphisms in the TGF-ß, IL-10, IL-6, and IFN-γ genes, human leukocyte antigens (HLA), and human platelet alloantigens (HPA) genes were investigated in 122 patients with Araraquara virus infection from Ribeirão Preto, Brazil. Patients were divided into two groups: hantavirus cardiopulmonary syndrome (HCPS group; n = 26) and hantavirus seropositive only (n = 96). The frequencies of HLA alleles, cytokines and platelet antigen genotypes were evaluated in both groups and compared to a control group. The data demonstrated no significant influence of the HLA alleles, HPA, IL-6, and IL-10 genotypes on susceptibility to hantavirus infection. However, the hantavirus seropositive group presented a significantly higher frequency of a polymorphism associated with a high IFN-γ production than the HCPS group. In addition, a genotype associated with high TGF-ß production was found more frequently in individuals infected with hantavirus than in the control group. This phenotype was associated with a less intense thrombocytopenia in the HCPS group and may be protective against the most severe form of hantavirus disease. More studies are required to quantify further the influence of the high TGF-ß producer phenotype on the outcome of hantavirus infection.

Hantavirus infection prevalence in wild rodents and human anti-hantavirus serological profiles from different geographic areas of South Brazil.

Paraná state presents the fourth highest number of accumulated cases of hantavirus pulmonary syndrome in Brazil. To map the risk areas for hantavirus transmission we carried out a study based on rodent trapping and determined the anti-hantavirus seroprevalence in these animals and in the inhabitants of these localities. Overall seroprevalence in rodents and humans were 2.5% and 2.4%, respectively. Eighty-two percent of the seropositive rodents were genetically analyzed. Phylogenetic analyses revealed that hantaviruses from rodent samples cluster with Araucária (Juquitiba-like) or Jaborá hantavirus genotypes. The Jaborá strain was identified in Akodon serrensis and Akodon montensis, whereas the Araucária strain was detected in Oligoryzomys nigripes, Oxymycterus judex, A. montensis, and Akodon paranaensis, with the latter species being identified for the first time as a natural host. These findings expose the complex relationships between virus and reservoirs in Brazil, which could have an impact on hantavirus transmission dynamics in nature and human epidemiology.

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.

T cells and pathogenesis of hantavirus cardiopulmonary syndrome and hemorrhagic fever with renal syndrome.

We previously hypothesized that increased capillary permeability observed in both hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS) may be caused by hantavirus-specific cytotoxic T cells attacking endothelial cells presenting viral antigens on their surface based on clinical observations and in vitro experiments. In HCPS, hantavirus-specific T cell responses positively correlated with disease severity. In HFRS, in one report, contrary to HCPS, T cell responses negatively correlated with disease severity, but in another report the number of regulatory T cells, which are thought to suppress T cell responses, negatively correlated with disease severity. In rat experiments, in which hantavirus causes persistent infection, depletion of regulatory T cells helped infected rats clear virus without inducing immunopathology. These seemingly contradictory findings may suggest delicate balance in T cell responses between protection and immunopathogenesis. Both too strong and too weak T cell responses may lead to severe disease. It is important to clarify the role of T cells in these diseases for better treatment (whether to suppress T cell functions) and protection (vaccine design) which may need to take into account viral factors and the influence of HLA on T cell responses.

VEGFR2 and Src kinase inhibitors suppress Andes virus-induced endothelial cell permeability.

Hantaviruses predominantly infect human endothelial cells and, in the absence of cell lysis, cause two diseases resulting from increased vascular permeability. Andes virus (ANDV) causes a highly lethal acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). ANDV infection enhances the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF) by increasing signaling responses directed by the VEGFR2-Src-VE-cadherin pathway, which directs adherens junction (AJ) disassembly. Here we demonstrate that inhibiting pathway-specific VEGFR2 and Src family kinases (SFKs) blocks ANDV-induced endothelial cell permeability. Small interfering RNA (siRNA) knockdown of Src within ANDV-infected endothelial cells resulted in an ∼70% decrease in endothelial cell permeability compared to that for siRNA controls. This finding suggested that existing FDA-approved small-molecule kinase inhibitors might similarly block ANDV-induced permeability. The VEGFR2 kinase inhibitor pazopanib as well as SFK inhibitors dasatinib, PP1, bosutinib, and Src inhibitor 1 dramatically inhibited ANDV-induced endothelial cell permeability. Consistent with their kinase-inhibitory concentrations, dasatinib, PP1, and pazopanib inhibited ANDV-induced permeability at 1, 10, and 100 nanomolar 50% inhibitory concentrations (IC(50)s), respectively. We further demonstrated that dasatinib and pazopanib blocked VE-cadherin dissociation from the AJs of ANDV-infected endothelial cells by >90%. These findings indicate that VEGFR2 and Src kinases are potential targets for therapeutically reducing ANDV-induced endothelial cell permeability and, as a result, capillary permeability during HPS. Since the functions of VEGFR2 and SFK inhibitors are already well defined and FDA approved for clinical use, these findings rationalize their therapeutic evaluation for efficacy in reducing HPS disease. Endothelial cell barrier functions are disrupted by a number of viruses that cause hemorrhagic, edematous, or neurologic disease, and as a result, our findings suggest that VEGFR2 and SFK inhibitors should be considered for regulating endothelial cell barrier functions altered by additional viral pathogens.

Association of -308G/A polymorphism in the tumor necrosis factor-alpha gene promoter with susceptibility to development of hantavirus cardiopulmonary syndrome in the Ribeirão Preto region, Brazil.

Activation of the immune response in hantavirus cardiopulmonary syndrome (HCPS) leads to a high TNF production, probably contributing to the disease. The polymorphic TNF2 allele (TNF -308G/A) has been associated with increased cytokine production. We investigated the association of the TNF2 allele with the outcome of hantavirus infection in Brazilian patients. A total of 122 hantavirus-exposed individuals (26 presenting HCPS and 96 only hantavirus seroconversion) were studied. The TNF2 allele was more frequently found in HCPS patients than in individuals with positive serology for hantavirus but without a history of HCPS illness, suggesting that the TNF2 allele could represent a risk factor for developing HCPS.

Hantavirus cardiopulmonary syndrome: immune response and pathogenesis.

Hantaviruses are emerging viruses in the Americas that cause cardiopulmonary syndrome with high lethality. The intense cellular immune response to hantavirus alters normal endothelial cell barrier functions and seems to be harmful to the host. On the other hand, the humoral immune response seems to be essential for recovery from infection.

Pathogenic and nonpathogenic hantaviruses differentially regulate endothelial cell responses.

Hantaviruses cause two human diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Hantaviruses infect human endothelial cells but cause little or no damage to the infected endothelium. We analyzed with Affymetrix DNA Arrays (Santa Clara, CA) the endothelial cell transcriptional responses directed by hantaviruses associated with HPS [New York-1 virus (NY-1V)], HFRS [Hantaan virus (HTNV)], or by a hantavirus not associated with human disease [Prospect Hill virus (PHV)]. Hantavirus infections induced 117 cellular genes and repressed 25 genes by >3-fold, 4 days postinfection (p.i.). Although >80% of cells were infected by each virus 1 day p.i., PHV induced or repressed 67 genes at this early time compared with three genes altered by HTNV or NY-1V. The early high-level induction of 24 IFN-stimulated genes by PHV (4- to 229-fold) represents a fundamental difference in the temporal regulation of cellular responses by pathogenic and nonpathogenic hantaviruses. Because all hantaviruses induced >23 IFN-stimulated genes at late times p.i., pathogenic hantaviruses appear to suppress early cellular IFN responses that are activated by nonpathogenic hantaviruses. At late times p.i., 13 genes were commonly induced by HTNV and NY-1V that were not induced by PHV. In contrast to NY-1V, HTNV uniquely induced a variety of chemokines and cell adhesion molecules (i.e., IL-8, IL-6, GRO-beta, ICAM), as well as two complement cascade-associated factors that may contribute to immune components of HFRS disease. NY-1V failed to induce most cellular chemokines directed by HTNV (3/14) or genes primarily activated by NF-kappaB. However, NY-1V uniquely induced beta3 integrin-linked potassium channels, which could play a role in HPS-associated vascular permeability. These studies provide a basic understanding of hantavirus-directed cellular responses that are likely to differentiate pathogenic and nonpathogenic hantaviruses, contribute to HFRS and HPS pathogenesis, and provide insight into disease mechanisms and potential therapeutic interventions.