Andreas Rang

Hantavirus-induced immunity in rodent reservoirs and humans.

Summary: Hantaviruses are predominantly rodent‐borne pathogens, although recently novel shrew‐associated hantaviruses were found. Within natural reservoir hosts, hantairuses do not cause obvious pathogenetic effects; transmission to humans, however, can lead to hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome, depending on the virus species involved. This review is focussed on the recent knowledge on hantavirus‐induced immune responses in rodent reservoirs and humans and their impact on susceptibility, transmission, and outcome of hantavirus infections. In addition, this review incorporates a discussion on the potential role of direct cell‐virus interactions in the pathogenesis of hantavirus infections in humans. Finally, questions for further research efforts on the immune responses in potential hantavirus reservoir hosts and humans are summarized.

Hantaan Virus Triggers TLR3-Dependent Innate Immune Responses

Immediately after viral infection, innate responses including expression of IFN-α/β and IFN-stimulated genes (ISGs) are elicited ubiquitously by recruitment of specific pathogen recognition receptors. The velocity to induce IFN-α/β and ISGs in response to an infection is often decisive for virulence. Interestingly, in primary endothelial cells ISGs are induced later by hantaviruses pathogenic to humans than those considered to be nonpathogenic or of low virulence. Here we demonstrate that pathogenic Hantaan (HTNV) and putatively nonpathogenic Prospect Hill hantavirus (PHV) differentially activate innate responses in the established cell lines A549 and HuH7. STAT1α phosphorylation was detectable 3 h after PHV inoculation but not within the first 2 days after HTNV inoculation. The velocity to induce the ISGs MxA and ISG15 correlated inversely with amounts of virus produced. Moreover, expression of the inflammatory chemokine CCL5 was also induced differentially. Both hantaviruses induced innate responses via TRAF3 (TNF receptor-associated factor 3), and TLR3 was required for HTNV-induced expression of MxA, but not for the MxA induction triggered by PHV. Infection of RIG-I-deficient HuH7.5 cells revealed that RIG-I (retinoic acid receptor I) was not necessary for induction of innate responses by PHV. Taken together, these data suggest that HTNV and PHV elicit different signaling cascades that converge via TRAF3. Early induction of antiviral responses might contribute to efficient elimination of PHV. Subsequent to clearance of the infection, innate responses most likely cease; vice versa, retarded induction of antiviral responses could lead to increased HTNV replication and dissemination, which might cause a prolonged inflammatory response and might contribute to the in vivo virulence.

Genetic reassortment between high-virulent and low-virulent Dobrava-Belgrade virus strains

The tri-segmented RNA genome of hantaviruses facilitates genetic reassortment by segment swapping when cells are co-infected with different virus strains. We found efficient in vitro reassortment between members of two different genetic lineages of the Dobrava-Belgrade virus species, the weakly virulent DOBV-Aa and highly virulent DOBV-Af. In all reassortants, S and L segments originated from the same parental strain, and only the M segment was exchanged. To identify functional differences between the parental strains DOBV-Aa and DOBV-Af in cell culture and to compare them with the reassortants, we studied elements of the innate immunity in virus-infected cells. The contrasting phenotypes of the parental viruses were maintained by the reassortants carrying the respective S and L segments of the parental virus and were not influenced by the origin of the M segment.

Infection of in vivo differentiated human mast cells with hantaviruses.

Increased vascular permeability is a key feature of the pathological symptoms caused by hantaviruses. Here, we analysed the interaction between hantaviruses and mast cells, which regulate vascular homeostasis. In highly purified human skin mast cells increasing amounts of Hantaan (HTNV) and, to a lower extent, Prospect Hill (PHV) virions were produced. Replication was confirmed by the production of viral plus-strand RNA as determined by a virus strand-specific RT-PCR. PHV but not HTNV elicited early expression of beta interferon, MxA, ISG15 and CCL5 consistent to studies with other cell types. The data demonstrate that mature mast cells are permissive to infection with hantaviruses. This interaction might contribute to the development of vascular leakage syndrome.

Dobrava-Belgrade hantavirus from Germany shows receptor usage and innate immunity induction consistent with the pathogenicity of the virus in humans.

Background Dobrava-Belgrade virus (DOBV) is a European hantavirus causing hemorrhagic fever with renal syndrome (HFRS) in humans with fatality rates of up to 12%. DOBV-associated clinical cases typically occur also in the northern part of Germany where the virus is carried by the striped field mouse (Apodemus agrarius). However, the causative agent responsible for human illness has not been previously isolated. Methodology/Principal Findings Here we report on characterization of a novel cell culture isolate from Germany obtained from a lung tissue of “spillover” infected yellow necked mouse (A. flavicollis) trapped near the city of Greifswald. Phylogenetic analyses demonstrated close clustering of the new strain, designated Greifswald/Aa (GRW/Aa) with the nucleotide sequence obtained from a northern German HFRS patient. The virus was effectively blocked by specific antibodies directed against β3 integrins and Decay Accelerating Factor (DAF) indicating that the virus uses same receptors as the highly pathogenic Hantaan virus (HTNV). In addition, activation of selected innate immunity markers as interferon β and λ and antiviral protein MxA after viral infection of A549 cells was investigated and showed that the virus modulates the first-line antiviral response in a similar way as HTNV. Conclusions/Significance In summary, our study reveals novel data on DOBV receptor usage and innate immunity induction in relationship to virus pathogenicity and underlines the potency of German DOBV strains to act as human pathogen.

Generation and characterization of genetic reassortants between Puumala and Prospect Hill hantavirus in vitro.

Hantaviruses belong to the family Bunyaviridae characterized by tri-segmented RNA genomes. Depending on the hantavirus species, infection can lead to hantavirus cardiopulmonary or haemorrhagic fever with renal syndrome. In vitro studies suggest that pathogenic hantaviruses evade induction of innate antiviral responses, and this ability might determine the virulence in humans. Since reverse genetic systems are not available, in vitro reassortment is currently the only way to culture defined hantavirus variants. Here, we demonstrate for the first time the generation of a reassortant between a pathogenic Old World and a non-pathogenic New World hantavirus in vitro. The reassortant contained the glycoprotein coding M-segment derived from the pathogenic Puumala virus (PUUV) and the other genomic segments coding for the nucleocapsid protein and RNA-dependent RNA-polymerase from Prospect Hill virus (PHV), which is taken as non-pathogenic in humans. Exchange of the M-segment was confirmed by sequencing and virus neutralization test with PUUV-specific sera. Functional analysis of the reassortant and parental viruses revealed characteristic growth kinetics and innate immune responses as determined by expression analyses for lambda interferon and MxA, and by interferon-stimulated response element reporter gene studies. Consistent with previous studies with other pathogenic hantaviruses, PUUV elicited reduced innate responses if compared with PHV. In all these functional assays the reassortant revealed PHV-like phenotypes. Thus, neither the PUUV M-segment nor entry via specific M-segment directed pathways modulated the virus type-specific innate responses. Moreover, the data imply that this approach might be an option for production of attenuated viruses that could be used as vaccines against pathogenic hantaviruses.

Hantavirus Emergence in Rodents, Insectivores and Bats: What Comes Next?

Hantaviruses have attracted increased public interest in recent years and are a clear example of an emerging virus. The enveloped virions of hantaviruses contain a tri-segmented single-stranded RNA genome of negative polarity. The initial discovery of hantaviruses dates back to investigations initiated after a large number of United Nations military personnel were affected by a severe disease during the Korean conflict in the 1950s. These investigations resulted in the discovery of Hantaan virus, the prototype species within the genus, in the striped field mouse (Apodemus agrarius). In the following years a large number of additional hantaviruses in other rodents were identified. The congruence in the phylogenetic relationship of the viruses and their rodent reservoirs was explained by a virus–host co-divergence. Further phylogenetic investigations and the discovery of novel hantaviruses indicated host-switch and subsequent host adaptation as an alternative evolution mechanism. In humans hantaviruses cause two disease syndromes, hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome. Due to similarities in the disease symptoms it is currently suggested to unify both as “hantavirus disease.” For detection of hantavirus infections a broad spectrum of molecular and serological diagnostic methods exists. Problems for hantavirus diagnostics in patients arise from the short-termed viremia and the strong cross-reactivity of sera against the nucleocapsid protein, frequently used in serological assays. The geographical distribution of the hantaviruses follows the range of their reservoir rodents. Human pathogenic hantaviruses are most likely distributed world-wide, but so far with little evidence for their presence in Africa, and Australia in particular. The recent discoveries of novel hantaviruses in shrews, moles and even bats raise questions about the origin and molecular evolution of hantaviruses. Importantly, the consequences of these novel viruses for public health are still unknown. The emergence of hantaviruses might be driven by various factors. Changes in small mammal reservoir population dynamics represent one of the major drivers for the occurrence of disease clusters and outbreaks. Long-term monitoring studies of small mammal reservoirs are needed to develop and validate prediction models and to implement them in early warning tools. Future hantavirus studies will profit from the application of Next Generation Sequencing technique, and the development of a reverse-genetics system.

Hantaviral mechanisms driving HLA class I antigen presentation require both RIG-I and TRIF

Hantaviruses are emerging human pathogens. They induce an unusually strong antiviral response of human HLA class I (HLA‐I) restricted CD8+ T cells that may contribute to tissue damage and hantavirus‐associated disease. In this study, we analyzed possible hantaviral mechanisms that enhance the HLA‐I antigen presentation machinery. Upon hantavirus infection of various human and primate cell lines, we observed transactivation of promoters controlling classical HLA molecules. Hantavirus‐induced HLA‐I upregulation required proteasomal activity and was associated with increased TAP expression. Intriguingly, human DCs acquired the capacity to cross‐present antigen upon hantavirus infection. Furthermore, knockdown of TIR domain containing adaptor inducing IFN‐β or retinoic acid inducible gene I abolished hantavirus‐driven HLA‐I induction. In contrast, MyD88‐dependent viral sensors were not involved in HLA‐I induction. Our results show that hantaviruses strongly boost the HLA‐I antigen presentation machinery by mechanisms that are dependent on both retinoic acid inducible gene I and TIR domain containing adaptor inducing IFN‐β.

Non-human primates in outdoor enclosures: Risk for infection with rodent-borne hantaviruses

Different species of non-human primates have been exploited as animal disease models for human hantavirus infections. To study the potential risk of natural hantavirus infection of non-human primates, we investigated serum samples from non-human primates of three species living in outdoor enclosures of the German Primate Center (GPC), Göttingen, located in a hantavirus endemic region of central Germany. For that purpose we used serological assays based on recombinant antigens of the bank vole (Myodes glareolus) transmitted Puumala virus (PUUV) and the common and field vole (Microtus arvalis, Microtus agrestis) associated Tula virus (TULV) which are both broadly geographically distributed in Germany. In 24 out of 251 (9.6%) monkey sera collected in 2006 PUUV- and/or TULV-reactive immunoglobulin G (IgG) antibodies were detected. Investigation of follow-up sera from 13 animals confirmed for two animals a seroconversion due to hantavirus exposure at the GPC. To prove the origin of the infection, wild rodents from the surrounding regions were analyzed by hantavirus-specific reverse transcriptase-PCR analysis. In 6 of the 73 investigated bank voles and 3 of the 19 investigated Microtus spp. PUUV- and TULV-specific nucleic acid sequences, respectively, were detected. In conclusion, our investigations demonstrate for the first time natural infections of non-human primates in outdoor enclosures in Germany. These findings highlight the importance of hantavirus surveillance in those primate housings and corresponding preventive measures against wild rodents, particularly in hantavirus endemic regions.

Defective Particles Can Lead to Underestimated Antibody Titers in Virus Neutralization Tests

Objective: Stocks of RNA viruses often contain divergent mixtures of infectious and defective particles (DPs). Since the surface of both particles share the same immunoreactivity, differing ratios of these particles in stocks used for virus neutralization tests might alter the results. Here the impact of such off-target effects were measured. Methods: To determine the relative content of DPs in 3 stocks with high, medium and low titers of infectious virions, the amounts of nucleocapsid protein and viral RNA were quantified by Western blot and quantitative RT-PCR. Thereafter, stocks with different amount of DPs were used to determine the titer of neutralizing antibodies in serum from a convalescent Puumala virus-infected patient by a focus reduction neutralization test. Results: The relative amount of DPs was at least 5-fold higher in the stock with the lowest titer compared to the stock with the highest. Stocks with lower levels of DPs led to higher antibody titers compared to the analysis performed with stocks containing higher levels of DPs. Conclusion: These results imply that DPs in virus stocks should be considered when performing virus neutralization tests to quantify neutralizing antibodies.