Hantavirus Species Research Articles

Development of a seroepidemiological tool for bat-borne and shrew-borne hantaviruses and its application using samples from Zambia.

Rodent-borne orthohantaviruses are the causative agents of hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Apart from the classic rodent-borne hantaviruses, numerous species of hantaviruses have been identified in shrews and bats; however, their antigenicity and pathogenicity are unknown. This study focused on developing a serological method to detect antibodies against bat- and shrew-borne hantaviruses. Five bat-borne (Brno, Dakrong, Quezon, Robina, and Xuan Song) and 6 shrew-borne (Asama, Altai, Cao Bang, Nova, Seewis, and Thottapalayam) viruses were selected based on the phylogenetic differences in their N proteins. The recombinant N (rN) proteins of these viruses were expressed as antigens in Vero E6 and 293T cell lines using the pCAGGS/MCS vector. Antisera against the Nus-tagged rN fusion proteins of these viruses (mouse anti-Brno, Dakrong, Quezon, Robina, Xuan Song, Asama, Cao Bang, and Nova, while rabbit anti-Altai, Seewis and Thottapalayam) were also generated. Antigenic cross-reactivity was examined in antisera and rN-expressing Vero E6 cells. The rN proteins of almost all the tested viruses, except for the Quezon and Robina viruses, showed independent antigenicity. For serological screening of bat samples, 5 rNs of the bat-borne viruses were expressed together in a single transfection protocol. Similarly, 6 rNs of shrew-borne viruses were expressed. Reactivities of the mixed antigen system were also examined across the singly transfected Vero cell lines to ensure that all antigens were expressed. Using these antigens, bat serum samples collected from Zambia were screened using the indirect immunofluorescence antibody test (IFAT). Selected positive samples were individually tested for the respective antigens by IFAT and western blot assays using rN-expressing 293T cell lysates. Of the 1,764 bat serum samples tested, 11.4% and 17.4% were positive for bat and shrew mixed antigens, respectively. These samples showed positive reactions to the Brno, Dakrong, Quezon, Xuan Son, Robina, Asama, Altai, Cao Bang, or Thottapalayam virus antigens. These observations suggest that the mixed-antigen screening system is useful for serological screening For Orthohantavirus infections and that bats in Zambia are likely exposed to not only bat-borne hantaviruses but also to shrew-borne hantaviruses.

Genome-wide support for incipient Tula hantavirus species within a single rodent host lineage.

Evolutionary divergence of viruses is most commonly driven by co-divergence with their hosts or through isolation of transmission after host shifts. It remains mostly unknown, however, whether divergent phylogenetic clades within named virus species represent functionally equivalent byproducts of high evolutionary rates or rather incipient virus species. Here, we test these alternatives with genomic data from two widespread phylogenetic clades in Tula orthohantavirus (TULV) within a single evolutionary lineage of their natural rodent host, the common vole Microtus arvalis. We examined voles from forty-two locations in the contact region between clades for TULV infection by reverse transcription (RT)-PCR. Sequencing yielded twenty-three TULV Central North and twenty-one TULV Central South genomes, which differed by 14.9-18.5 per cent at the nucleotide and 2.2-3.7 per cent at the amino acid (AA) level without evidence of recombination or reassortment between clades. Geographic cline analyses demonstrated an abrupt (<1 km wide) transition between the parapatric TULV clades in continuous landscape. This transition was located within the Central mitochondrial lineage of M. arvalis, and genomic single nucleotide polymorphisms showed gradual mixing of host populations across it. Genomic differentiation of hosts was much weaker across the TULV Central North to South transition than across the nearby hybrid zone between two evolutionary lineages in the host. We suggest that these parapatric TULV clades represent functionally distinct, incipient species, which are likely differently affected by genetic polymorphisms in the host. This highlights the potential of natural viral contact zones as systems for investigating the genetic and evolutionary factors enabling or restricting the transmission of RNA viruses.

Modeling the Immune Response for Pathogenic and Nonpathogenic Orthohantavirus Infections in Human Lung Microvasculature Endothelial Cells.

Hantaviruses, genus Orthohantavirus, family Hantaviridae, order Bunyavirales, are negative-sense, single-stranded, tri-segmented RNA viruses that persistently infect rodents, shrews, and moles. Of these, only certain virus species harbored by rodents are pathogenic to humans. Infection begins with inhalation of virus particles into the lung and trafficking to the lung microvascular endothelial cells (LMVEC). The reason why certain rodent-borne hantavirus species are pathogenic has long been hypothesized to be related to their ability to downregulate and dysregulate the immune response as well as increase vascular permeability of infected endothelial cells. We set out to study the temporal dynamics of host immune response modulation in primary human LMVECs following infection by Prospect Hill (nonpathogenic), Andes (pathogenic), and Hantaan (pathogenic) viruses. We measured the level of RNA transcripts for genes representing antiviral, proinflammatory, anti-inflammatory, and metabolic pathways from 12 to 72 h with time points every 12 h. Gene expression analysis in conjunction with mathematical modeling revealed a similar profile for all three viruses in terms of upregulated genes that partake in interferon signaling (TLR3, IRF7, IFNB1), host immune cell recruitment (CXCL10, CXCL11, and CCL5), and host immune response modulation (IDO1). We examined secreted protein levels of IFN-β, CXCL10, CXCL11, CCL5, and IDO in two male and two female primary HLMVEC donors at 48 and 60 h post infection. All three viruses induced similar levels of CCL5, CXCL10, and CXCL11 within a particular donor, and the levels were similar in three of the four donors. All three viruses induced different protein secretion levels for both IFN-β and IDO and secretion levels differed between donors. In conclusion, we show that there was no difference in the transcriptional profiles of key genes in primary HLMVECs following infection by pathogenic and nonpathogenic hantaviruses, with protein secretion levels being more donor-specific than virus-specific.

Multiple Lineages of Hantaviruses Harbored by the Iberian Mole (Talpa occidentalis) in Spain.

The recent detection of both Nova virus (NVAV) and Bruges virus (BRGV) in European moles (Talpa europaea) in Belgium and Germany prompted a search for related hantaviruses in the Iberian mole (Talpa occidentalis). RNAlater®-preserved lung tissue from 106 Iberian moles, collected during January 2011 to June 2014 in Asturias, Spain, were analyzed for hantavirus RNA by nested/hemi-nested RT-PCR. Pairwise alignment and comparison of partial L-segment sequences, detected in 11 Iberian moles from four parishes, indicated the circulation of genetically distinct hantaviruses. Phylogenetic analyses, using maximum-likelihood and Bayesian methods, demonstrated three distinct hantaviruses in Iberian moles: NVAV, BRGV, and a new hantavirus, designated Asturias virus (ASTV). Of the cDNA from seven infected moles processed for next generation sequencing using Illumina HiSeq1500, one produced viable contigs, spanning the S, M and L segments of ASTV. The original view that each hantavirus species is harbored by a single small-mammal host species is now known to be invalid. Host-switching or cross-species transmission events, as well as reassortment, have shaped the complex evolutionary history and phylogeography of hantaviruses such that some hantavirus species are hosted by multiple reservoir species, and conversely, some host species harbor more than one hantavirus species.

Antigenic mapping and functional characterization of human New World hantavirus neutralizing antibodies.

Hantaviruses are high-priority emerging pathogens carried by rodents and transmitted to humans by aerosolized excreta or, in rare cases, person-to-person contact. While infections in humans are relatively rare, mortality rates range from 1 to 40% depending on the hantavirus species. There are currently no FDA-approved vaccines or therapeutics for hantaviruses, and the only treatment for infection is supportive care for respiratory or kidney failure. Additionally, the human humoral immune response to hantavirus infection is incompletely understood, especially the location of major antigenic sites on the viral glycoproteins and conserved neutralizing epitopes. Here, we report antigenic mapping and functional characterization for four neutralizing hantavirus antibodies. The broadly neutralizing antibody SNV-53 targets an interface between Gn/Gc, neutralizes through fusion inhibition and cross-protects against the Old World hantavirus species Hantaan virus when administered pre- or post-exposure. Another broad antibody, SNV-24, also neutralizes through fusion inhibition but targets domain I of Gc and demonstrates weak neutralizing activity to authentic hantaviruses. ANDV-specific, neutralizing antibodies (ANDV-5 and ANDV-34) neutralize through attachment blocking and protect against hantavirus cardiopulmonary syndrome (HCPS) in animals but target two different antigenic faces on the head domain of Gn. Determining the antigenic sites for neutralizing antibodies will contribute to further therapeutic development for hantavirus-related diseases and inform the design of new broadly protective hantavirus vaccines.

Zoonotic pathogens identified in rodents and shrews from four provinces, China, 2015-2022.

Rodents and shrews are major reservoirs of various pathogens that are related to zoonotic infectious diseases. The purpose of this study was to investigate co-infections of zoonotic pathogens in rodents and shrews trapped in four provinces of China. We sampled different rodent and shrew communities within and around human settlements in four provinces of China and characterised several important zoonotic viral, bacterial, and parasitic pathogens by PCR methods and phylogenetic analysis. A total of 864 rodents and shrews belonging to 24 and 13 species from RODENTIA and EULIPOTYPHLA orders were captured, respectively. For viral pathogens, two species of hantavirus (Hantaan orthohantavirus and Caobang orthohantavirus) were identified in 3.47% of rodents and shrews. The overall prevalence of Bartonella spp., Anaplasmataceae, Babesia spp., Leptospira spp., Spotted fever group Rickettsiae, Borrelia spp., and Coxiella burnetii were 31.25%, 8.91%, 4.17%, 3.94%, 3.59%, 3.47%, and 0.58%, respectively. Furthermore, the highest co-infection status of three pathogens was observed among Bartonella spp., Leptospira spp., and Anaplasmataceae with a co-infection rate of 0.46%. Our results suggested that species distribution and co-infections of zoonotic pathogens were prevalent in rodents and shrews, highlighting the necessity of active surveillance for zoonotic pathogens in wild mammals in wider regions.

Phylogeographic analysis of Tula hantavirus highlights a single introduction to central Europe.

Orthohantaviruses are zoonotic pathogens of humans, unique among the bunyaviruses in not being transmitted by an arthropod vector. Tula orthohantavirus (TULV) is an old-world hantavirus, of yet unclear human pathogenicity, with few reported cases of clinically relevant human infection. So far, phylogeographic studies exploring the global pathways of hantaviral migration are scarce and generally do not focus on a specific hantavirus species. The aim of the present study was to reconstruct the dispersal history of TULV lineages across Eurasia based on S segment sequences sampled from different geographic areas. Maximum-likelihood and Bayesian inference methods were used to perform the phylogenetic analysis and phylogeographic reconstructions. Sampling time and trapping localities were obtained for a total of 735 TULV S segment sequences available in public databases at the time of the study. The estimated substitution rate of the analyzed partial S segment alignment was 2.26 × 10-3 substitutions/site/year (95 per cent highest posterior density interval: 1.79 × 10-3 to 2.75 × 10-3). Continuous phylogeography of TULV S segment sequences placed the potential root and origin of TULV spread in the Black Sea region. In our study, we detect a single-lineage introduction of TULV to Europe, followed by local viral circulation further on.

Cluster of human Puumala orthohantavirus infections due to indoor exposure?-An interdisciplinary outbreak investigation.

Puumala orthohantavirus (PUUV) is the most important hantavirus species in Europe, causing the majority of human hantavirus disease cases. In central and western Europe, the occurrence of human infections is mainly driven by bank vole population dynamics influenced by beech mast. In Germany, hantavirus epidemic years are observed in 2‐ to 5‐year intervals. Many of the human infections are recorded in summer and early autumn, coinciding with peaks in bank vole populations. Here, we describe a molecular epidemiological investigation in a small company with eight employees of whom five contracted hantavirus infections in late 2017. Standardized interviews with employees were conducted to assess the circumstances under which the disease cluster occurred, how the employees were exposed and which counteractive measures were taken. Initially, two employees were admitted to hospital and serologically diagnosed with hantavirus infection. Subsequently, further investigations were conducted. By means of a self‐administered questionnaire, three additional symptomatic cases could be identified. The hospital patients' sera were investigated and revealed in one patient a partial PUUV L segment sequence, which was identical to PUUV sequences from several bank voles collected in close proximity to company buildings. This investigation highlights the importance of a One Health approach that combines efforts from human and veterinary medicine, ecology and public health to reveal the origin of hantavirus disease clusters.

Characterization of Hantavirus N Protein Intracellular Dynamics and Localization.

Hantaviruses are enveloped viruses that possess a tri-segmented, negative-sense RNA genome. The viral S-segment encodes the multifunctional nucleocapsid protein (N), which is involved in genome packaging, intracellular protein transport, immunoregulation, and several other crucial processes during hantavirus infection. In this study, we generated fluorescently tagged N protein constructs derived from Puumalavirus (PUUV), the dominant hantavirus species in Central, Northern, and Eastern Europe. We comprehensively characterized this protein in the rodent cell line CHO-K1, monitoring the dynamics of N protein complex formation and investigating co-localization with host proteins as well as the viral glycoproteins Gc and Gn. We observed formation of large, fibrillar PUUV N protein aggregates, rapidly coalescing from early punctate and spike-like assemblies. Moreover, we found significant spatial correlation of N with vimentin, actin, and P-bodies but not with microtubules. N constructs also co-localized with Gn and Gc albeit not as strongly as the glycoproteins associated with each other. Finally, we assessed oligomerization of N constructs, observing efficient and concentration-dependent multimerization, with complexes comprising more than 10 individual proteins.

Academ Virus, a Novel Hantavirus in the Siberian Mole (Talpa altaica) from Russia.

To date, six hantavirus species have been detected in moles (family Talpidae). In this report, we describe Academ virus (ACDV), a novel hantavirus harbored by the Siberian mole (Talpa altaica) in Western Siberia. Genetic analysis of the complete S-, M-, and partial L-genomic segments showed that ACDV shared a common evolutionary origin with Bruges virus, previously identified in the European mole (Talpa europaea), and is distantly related to other mole-borne hantaviruses. Co-evolution and local adaptation of genetic variants of hantaviruses and their hosts, with possible reassortment events, might have shaped the evolutionary history of ACDV.

Prevalence of Hantaviruses Harbored by Murid Rodents in Northwestern Ukraine and Discovery of a Novel Puumala Virus Strain.

In Europe, two species of hantaviruses, Puumala orthohantavirus (PUUV) and Dobrava orthohantavirus (DOBV), cause hemorrhagic fever with renal syndrome in humans. The rodent reservoirs for these viruses are common throughout Ukraine, and hence, the goal of this study was to identify the species and strains of hantaviruses circulating in this region. We conducted surveillance of small rodent populations in a rural region in northwestern Ukraine approximately 30 km from Poland. From the 424 small mammals captured, we identified nine species, of which the most abundant were Myodes glareolus, the bank vole (45%); Apodemus flavicollis, the yellow-necked mouse (29%); and Apodemus agrarius, the striped field mouse (14.6%) Using an indirect immunofluorescence assay, 15.7%, 20.5%, and 33.9% of the sera from M. glareolus, A. glareolus, and A. flavicollis were positive for hantaviral antibodies, respectively. Additionally, we detected antibodies to the hantaviral antigen in one Microtus arvalis, one Mus musculus, and one Sorex minutus. We screened the lung tissue for hantaviral RNA using next-generation sequencing and identified PUUV sequences in 25 small mammals, including 23 M. glareolus, 1 M. musculus, and 1 A. flavicollis, but we were unable to detect DOBV sequences in any of our A. agrarius specimens. The percent identity matrix and Bayesian phylogenetic analyses of the S-segment of PUUV from 14 M. glareolus lungs suggest the highest similarity (92–95% nucleotide or 99–100% amino acid) with the Latvian lineage. This new genetic information will contribute to future molecular surveillance of human cases in Ukraine.

Epidemiological Characteristics and Regional Risk Prediction of Hemorrhagic Fever with Renal Syndrome in Shandong Province, China.

Background: Hemorrhagic fever with renal syndrome (HFRS), a rodent-borne disease caused by different species of hantaviruses, is widely endemic in China. Shandong Province is one of the most affected areas. This study aims to analyze the epidemiological characteristics of HFRS, and to predict the regional risk in Shandong Province. Methods: Descriptive statistics were used to elucidate the epidemiological characteristics of HFRS cases in Shandong Province from 2010 to 2018. Based on environmental and socioeconomic data, the boosted regression tree (BRT) model was applied to identify important influencing factors, as well as predict the infection risk zones of HFRS. Results: A total of 11,432 HFRS cases were reported from 2010 to 2018 in Shandong, with groups aged 31–70 years (81.04%), and farmers (84.44%) being the majority. Most cases were from central and southeast Shandong. There were two incidence peak periods in April to June and October to December, respectively. According to the BRT model, we found that population density (a relative contribution of 15.90%), elevation (12.02%), grassland (11.06%), cultivated land (9.98%), rural settlement (9.25%), woodland (8.71%), and water body (8.63%) were relatively important influencing factors for HFRS epidemics, and the predicted high infection risk areas were concentrated in central and eastern areas of Shandong Province. The BRT model provided an overall prediction accuracy, with an area under the receiver operating characteristic curve of 0.91 (range: 0.83–0.95). Conclusions: HFRS in Shandong Province has shown seasonal and spatial clustering characteristics. Middle-aged and elderly farmers are a high-risk population. The BRT model has satisfactory predictive capability in stratifying the regional risk of HFRS at a county level in Shandong Province, which could serve as an important tool for risk assessment of HFRS to deploy prevention and control measures.

Geographic Distribution and Phylogeny of Soricine Shrew-Borne Seewis Virus and Altai Virus in Russia.

The discovery of genetically distinct hantaviruses (family Hantaviridae) in multiple species of shrews, moles and bats has revealed a complex evolutionary history involving cross-species transmission. Seewis virus (SWSV) is widely distributed throughout the geographic ranges of its soricid hosts, including the Eurasian common shrew (Sorex araneus), tundra shrew (Sorex tundrensis) and Siberian large-toothed shrew (Sorex daphaenodon), suggesting host sharing. In addition, genetic variants of SWSV, previously named Artybash virus (ARTV) and Amga virus, have been detected in the Laxmann’s shrew (Sorex caecutiens). Here, we describe the geographic distribution and phylogeny of SWSV and Altai virus (ALTV) in Asian Russia. The complete genomic sequence analysis showed that ALTV, also harbored by the Eurasian common shrew, is a new hantavirus species, distantly related to SWSV. Moreover, Lena River virus (LENV) appears to be a distinct hantavirus species, harbored by Laxmann’s shrews and flat-skulled shrews (Sorex roboratus) in Eastern Siberia and far-eastern Russia. Another ALTV-related virus, which is more closely related to Camp Ripley virus from the United States, has been identified in the Eurasian least shrew (Sorex minutissimus) from far-eastern Russia. Two highly divergent viruses, ALTV and SWSV co-circulate among common shrews in Western Siberia, while LENV and the ARTV variant of SWSV co-circulate among Laxmann’s shrews in Eastern Siberia and far-eastern Russia. ALTV and ALTV-related viruses appear to belong to the Mobatvirus genus, while SWSV is a member of the Orthohantavirus genus. These findings suggest that ALTV and ALTV-related hantaviruses might have emerged from ancient cross-species transmission with subsequent diversification within Sorex shrews in Eurasia.

Chloroquine, an Anti-Malaria Drug as Effective Prevention for Hantavirus Infections

We investigated whether chloroquine can prevent hantavirus infection and disease in vitro and in vivo, using the Hantaan virus newborn C57BL/6 mice model and the Syrian hamster model for Andes virus. In vitro antiviral experiments were performed using Vero E6 cells, and Old World and New World hantavirus species. Hantavirus RNA was detected using quantitative RT-PCR. For all hantavirus species tested, results indicate that the IC50 of chloroquine (mean 10.2 ± 1.43 μM) is significantly lower than the CC50 (mean 260 ± 2.52 μM) yielding an overall selectivity index of 25.5. We also investigated the potential of chloroquine to prevent death in newborn mice after Hantaan virus infection and its antiviral effect in the hantavirus Syrian hamster model. For this purpose, C57Bl/6 mother mice were treated subcutaneously with daily doses of chloroquine. Subsequently, 1-day-old suckling mice were inoculated intracerebrally with 5 x 102 Hantaan virus particles. In litters of untreated mothers, none of the pups survived challenge. The highest survival rate (72.7% of pups) was found when mother mice were administered a concentration of 10 mg/kg chloroquine. Survival rates declined in a dose-dependent manner, with 47.6% survival when treated with 5 mg/kg chloroquine, and 4.2% when treated with 1 mg/kg chloroquine. Assessing the antiviral therapeutic and prophylactic effect of chloroquine in the Syrian hamster model was done using two different administration routes (intraperitoneally and subcutaneously using an osmotic pump system). Evaluating the prophylactic effect, a delay in onset of disease was noted and for the osmotic pump, 60% survival was observed. Our results show that chloroquine can be highly effective against Hantaan virus infection in newborn mice and against Andes virus in Syrian hamsters.

Development and design of the Hantavirus registry - HantaReg - for epidemiological studies, outbreaks and clinical studies on hantavirus disease.

BackgroundFrequent outbreaks around the globe and endemic appearance in different parts of the world emphasize the substantial risk of hantavirus diseases. Increasing incidence rates, trends of changing distribution of hantavirus species and new insights into clinical courses of hantavirus diseases call for multinational surveillance. Furthermore, evidence-based guidelines for the management of hantavirus diseases and scoring systems, which allow stratification of patients into risk categories, are lacking.MethodsHantavirus registry (HantaReg) is a novel registry platform facilitating multinational research of hantavirus-caused diseases, such as haemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). HantaReg provides an electronic case report form and uses the General Data Protection Regulation compliant platform clinicalsurveys.net, which can be accessed from any internet browser in the world. Having a modular structure, the registry platform is designed to display or hide questions and items according to the documented case (e.g. patient with HFRS versus HCPS) to facilitate fast, but standardized, data entry. Information categories documented in HantaReg are demographics, pre-existing diseases, clinical presentation, diagnostic and therapeutic approaches, as well as outcome.ConclusionsHantaReg is a novel, ready-to-use platform for clinical and epidemiological studies on hantavirus diseases and facilitates the documentation of the disease course associated with hantavirus infections. HantaReg is expected to promote international collaboration and contributes to improving patient care through the analysis of diagnostic and treatment pathways for hantavirus diseases, providing evidence for robust treatment recommendations. Moreover, HantaReg enables the development of prognosis-indicating scoring systems for patients with hantavirus disease.

Amplicon-Based, Next-Generation Sequencing Approaches to Characterize Single Nucleotide Polymorphisms of Orthohantavirus Species.

Whole-genome sequencing (WGS) of viruses from patient or environmental samples can provide tremendous insight into the epidemiology, drug resistance or evolution of a virus. However, we face two common hurdles in obtaining robust sequence information; the low copy number of viral genomes in specimens and the error introduced by WGS techniques. To optimize detection and minimize error in WGS of hantaviruses, we tested four amplification approaches and different amplicon pooling methods for library preparation and examined these preparations using two sequencing platforms, Illumina MiSeq and Oxford Nanopore Technologies MinION. First, we tested and optimized primers used for whole segment PCR or one kilobase amplicon amplification for even coverage using RNA isolated from the supernatant of virus-infected cells. Once optimized we assessed two sources of total RNA, virus-infected cells and supernatant from the virus-infected cells, with four variations of primer pooling for amplicons, and six different amplification approaches. We show that 99–100% genome coverage was obtained using a one-step RT-PCR reaction with one forward and reverse primer. Using a two-step RT-PCR with three distinct tiling approaches for the three genomic segments (vRNAs), we optimized primer pooling approaches for PCR amplification to achieve a greater number of aligned reads, average depth of genome, and genome coverage. The single nucleotide polymorphisms identified from MiSeq and MinION sequencing suggested intrinsic mutation frequencies of ~10−5-10−7 per genome and 10−4-10−5 per genome, respectively. We noted no difference in the coverage or accuracy when comparing WGS results with amplicons amplified from RNA extracted from infected cells or supernatant of these infected cells. Our results show that high-throughput diagnostics requiring the identification of hantavirus species or strains can be performed using MiSeq or MinION using a one-step approach. However, the two-step MiSeq approach outperformed the MinION in coverage depth and accuracy, and hence would be superior for assessment of genomes for epidemiology or evolutionary questions using the methods developed herein.

Macropinocytosis and Clathrin-Dependent Endocytosis Play Pivotal Roles for the Infectious Entry of Puumala Virus.

Viruses from the family Hantaviridae are encountered as emerging pathogens causing two life-threatening human zoonoses: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), with case fatality rates of up to 50%. Here, we comprehensively investigated entry of the Old World hantavirus Puumala virus (PUUV) into mammalian cells, showing that upon treatment with pharmacological inhibitors of macropinocytosis and clathrin-mediated endocytosis, PUUV infections are greatly reduced. We demonstrate that the inhibitors did not interfere with viral replication and that RNA interference, targeting cellular mediators of macropinocytosis, decreases PUUV infection levels significantly. Moreover, we established lipophilic tracer staining of PUUV particles and show colocalization of stained virions and markers of macropinosomes. Finally, we report a significant increase in the fluid-phase uptake of cells infected with PUUV, indicative of a virus-triggered promotion of macropinocytosis.IMPORTANCE The family Hantaviridae comprises a diverse group of virus species and is considered an emerging global public health threat. Individual hantavirus species differ considerably in terms of their pathogenicity but also in their cell biology and host-pathogen interactions. In this study, we focused on the most prevalent pathogenic hantavirus in Europe, Puumala virus (PUUV), and investigated the entry and internalization of PUUV into mammalian cells. We show that both clathrin-mediated endocytosis and macropinocytosis are cellular pathways exploited by the virus to establish productive infections and demonstrate that pharmacological inhibition of macropinocytosis or a targeted knockdown using RNA interference significantly reduced viral infections. We also found indications of an increase of macropinocytic uptake upon PUUV infection, suggesting that the virus triggers specific cellular mechanisms in order to stimulate its own internalization, thus facilitating infection.

Replication in the Mononuclear Phagocyte System (MPS) as a Determinant of Hantavirus Pathogenicity.

Members of different virus families including Hantaviridae cause viral hemorrhagic fevers (VHFs). The decisive determinants of hantavirus-associated pathogenicity are still enigmatic. Pathogenic hantavirus species, such as Puumala virus (PUUV), Hantaan virus (HTNV), Dobrava-Belgrade virus (DOBV), and Sin Nombre virus (SNV), are associated with significant case fatality rates. In contrast, Tula virus (TULV) only sporadically causes mild disease in immunocompetent humans and Prospect Hill virus (PHV) so far has not been associated with any symptoms. They are thus defined here as low pathogenic/apathogenic hantavirus species. We found that productive infection of cells of the mononuclear phagocyte system (MPS), such as monocytes and dendritic cells (DCs), correlated well with the pathogenicity of hantavirus species tested. HTNV (intermediate case fatality rates) replicated more efficiently than PUUV (low case fatality rates) in myeloid cells, whereas low pathogenic/apathogenic hantavirus species did not produce any detectable virus titers. Analysis of PHPUV, a reassortant hantavirus derived from a pathogenic (PUUV) and an apathogenic (PHV) hantavirus species, indicated that the viral glycoproteins are not decisive for replication in MPS cells. Moreover, blocking acidification of endosomes with chloroquine decreased the number of TULV genomes in myeloid cells suggesting a post-entry block for low pathogenic/apathogenic hantavirus species in myeloid cells. Intriguingly, pathogenic but not low pathogenic/apathogenic hantavirus species induced conversion of monocytes into inflammatory DCs. The proinflammatory programming of MPS cells by pathogenic hantavirus species required integrin signaling and viral replication. Our findings indicate that the capacity to replicate in MPS cells is a prominent feature of hantaviral pathogenicity.

Field vole-associated Traemmersee hantavirus from Germany represents a novel hantavirus species.

Vole-associated hantaviruses occur in the Old and New World. Tula orthohantavirus (TULV) is widely distributed throughout the European continent in its reservoir, the common vole (Microtus arvalis), but the virus was also frequently detected in field voles (Microtus agrestis) and other vole species. TULV and common voles are absent from Great Britain. However, field voles there harbor Tatenale and Kielder hantaviruses. Here we screened 126 field voles and 13 common voles from Brandenburg, Germany, for hantavirus infections. One common vole and four field voles were anti-TULV antibody and/or TULV RNA positive. In one additional, seropositive field vole a novel hantavirus sequence was detected. The partial S and L segment nucleotide sequences were only 61.1% and 75.6% identical to sympatrically occurring TULV sequences, but showed highest similarity of approximately 80% to British Tatenale and Kielder hantaviruses. Subsequent determination of the entire nucleocapsid (N), glycoprotein (GPC), and RNA-dependent RNA polymerase encoding sequences and determination of the pairwise evolutionary distance (PED) value for the concatenated N and GPC amino acid sequences confirmed a novel orthohantavirus species, tentatively named Traemmersee orthohantavirus. The identification of this novel hantavirus in a field vole from eastern Germany underlines the necessity of a large-scale, broad geographical hantavirus screening of voles to understand evolutionary processes of virus-host associations and host switches.

Highly Divergent Genetic Variants of Soricid-Borne Altai Virus (Hantaviridae) in Eurasia Suggest Ancient Host-Switching Events.

With the recent discovery of genetically distinct hantaviruses (family Hantaviridae) in shrews (order Eulipotyphla, family Soricidae), the once-conventional view that rodents (order Rodentia) served as the primordial reservoir hosts now appears improbable. The newly identified soricid-borne hantaviruses generally demonstrate well-resolved lineages organized according to host taxa and geographic origin. However, beginning in 2007, we detected sequences that did not conform to the prototypic hantaviruses associated with their soricid host species and/or geographic locations. That is, Eurasian common shrews (Sorex araneus), captured in Hungary and Russia, were found to harbor hantaviruses belonging to two separate and highly divergent lineages. We have since accumulated additional examples of these highly distinctive hantavirus sequences in the Laxmann’s shrew (Sorex caecutiens), flat-skulled shrew (Sorex roboratus) and Eurasian least shrew (Sorex minutissimus), captured at the same time and in the same location in the Sakha Republic in Far Eastern Russia. Pair-wise alignment and phylogenetic analysis of partial and full-length S-, M- and/or L-segment sequences indicate that a distinct hantavirus species related to Altai virus (ALTV), first reported in a Eurasian common shrew from Western Siberia, was being maintained in these closely related syntopic soricine shrew species. These findings suggest that genetic variants of ALTV might have resulted from ancient host-switching events with subsequent diversification within the Soricini tribe in Eurasia.