Validation of an antigenic site targeted by monoclonal antibodies against Puumala virus.

Identification of B-cell epitopes facilitates the development of vaccines, therapeutic antibodies and diagnostic tools. Previously, the binding site of the bank vole monoclonal antibody (mAb) 4G2 against Puumala virus (PUUV, an orthohantavirus in the Hantaviridae family of the Bunyavirales order) was predicted using a combination of methods, including pepscan, phage-display, and site-directed mutagenesis of vesicular stomatitis virus (VSV) particles pseudotyped with Gn and Gc glycoproteins from PUUV. These techniques led to the identification of the neutralization escape mutation F915A. To our surprise, a recent crystal structure of PUUV Gc in complex with Fab 4G2 revealed that residue F915 is distal from epitope of mAb 4G2. To clarify this issue and explore potential explanations for the inconsistency, we designed a mutagenesis experiment to probe the 4G2 epitope, with three PUUV pseudoviruses carrying amino acid changes E725A, S944F, and S946F, located within the structure-based 4G2 epitope on the Gc. These amino acid changes were able to convey neutralization escape from 4G2, and S944F and S946F also conveyed escape from neutralization by human mAb 1C9. Furthermore, our mapping of all the known neutralization evasion sites from hantaviral Gcs onto PUUV Gc revealed that over 60 % of these sites reside within or close to the epitope of mAb 4G2, indicating that this region may represent a crucial area targeted by neutralizing antibodies against PUUV, and to a lesser extent, other hantaviruses. The identification of this site of vulnerability could guide the creation of subunit vaccines against PUUV and other hantaviruses in the future.

Neutralizing Antibody Titers in Hospitalized Patients with Acute Puumala Orthohantavirus Infection Do Not Associate with Disease Severity.

Nephropathia epidemica (NE), a mild form of haemorrhagic fever with renal syndrome (HFRS), is an acute febrile illness caused by Puumala orthohantavirus (PUUV). NE manifests typically with acute kidney injury (AKI), with a case fatality rate of about 0.1%. The treatment and management of hantavirus infections are mainly supportive, although neutralizing monoclonal antibodies and immune sera therapeutics are under investigation. In order to assess the potential use of antibody therapeutics in NE, we sought to determine the relationship between circulating PUUV neutralizing antibodies, PUUV nucleocapsid protein (N) IgG antibodies, and viral loads with markers of disease severity. The study included serum samples of extensively characterized patient cohorts (n = 116) from Tampere University Hospital, Finland. The results showed that upon hospitalization, most patients already had considerable neutralizing and anti-PUUV-N IgG antibody levels. However, contrary to expectations, neutralizing antibody titers from the first day of hospitalization did not appear to protect from AKI or correlate with more favorable disease outcomes. This indicates that further studies are needed to investigate the applicability of neutralizing antibodies as a therapy for hospitalized NE patients.

Identification of Norovirus and Human Parechovirus in Patients With Hand, Foot and Mouth Disease Syndrome.

BACKGROUND: Hand, foot and mouth disease (HFMD) is caused mostly by enteroviruses. However, other viral agents also can cause similar syndromes, and hence, the infections they cause are often misdiagnosed clinically. To determine non-enterovirus etiologic agents in HFMD-like cases, we screened enterovirus-negative samples collected from the patients who were clinically diagnosed as HFMD in China. METHODS: Two hundred enterovirus-negative samples were collected previously in Wenzhou city of Zhejiang province, China. Both high throughput sequencing and RT-PCR were used to screen viral agents. In addition, their clinical features were analyzed. RESULTS: Norovirus (NoV) and human parechovirus (HPeV) were identified from 22 (11.00%) and 9 (4.50%) samples, respectively. In addition, the complete genome sequences were recovered from 4 NoV-positive samples, and the VP1/3Dpol gene sequences were recovered from 5 HPeV-positive samples. Phylogenetic analyses of the NoV sequences revealed that they were closely related to those circulated in other regions of China. Notably, 4 genotypes of HPeVs, including HPeV-1, HPeV-4, HPeV-5 and HPeV-14, were found, indicating high genetic diversity of the virus. Frequent recombination between various genotypes was also observed in the HPeVs. Although most of the patients presented with the clinical features of HFMD, 4 patients infected with NoV GII.4 and 3 patients infected with HPeV-1 (1) and HPeV-4 (2) were characterized with diarrhea. Finally, tonsillitis, convulsion and granulocytopenia were observed in 1 NoV GII.4 patient, while liver dysfunction was found in 1 NoV GII.17 patient. CONCLUSIONS: These data reveal the variety of agents in the cases clinically diagnosed as HFMD.

Identification and characterization of a novel subtype of Tula virus in Microtus arvalis obscurus voles sampled from Xinjiang, China.

Although most of Arvicolinae associated hantaviruses can not cause disease in humans, hemorrhagic fever with renal syndrome (HFRS) cases caused by Tula virus (TULV) have been described in Europe since 2002. In addition to Europe, TULV was also identified in the Microtus arvalis obscurus voles sampled from Kazakhstan, which shares borders with China. To gain more insight into the molecular epidemiology of TULV, a total of 365 rodents representing 7 species of 4 subfamily (Arvicolinae, Murinae, Gerbillinae, and Cricetinae) were captured in Qapqal county, Xinjiang, northwest China. Hantavirus RNA was recovered from 40 lung tissue samples of M. arvalis obscurus, with the prevalence of 10.96%. Genetic analysis revealed that all recovered viral sequences were most closely related to those of TULV, but exhibited >11% nucleotide differences from all currently known TULV, suggesting that they may represent a new subtype of TULV. In the S tree, the newly identified viruses formed a distinct lineage and showed a close evolutionary relationship with those sampled from Southwestern Siberia and Kazakhstan. However, they exhibited a different clustering pattern in both the M and the L trees, suggesting the possibility of genetic reassortment. Finally, the recombination event was also observed in Xinjiang TULV viruses. In sum, all these data reveal a complex evolutionary history of TULV in Central Asia.

Phylogeography of Puumala orthohantavirus in Europe.

Puumala virus is an RNA virus hosted by the bank vole (Myodes glareolus) and is today present in most European countries. Whilst it is generally accepted that hantaviruses have been tightly co-evolving with their hosts, Puumala virus (PUUV) evolutionary history is still controversial and so far has not been studied at the whole European level. This study attempts to reconstruct the phylogeographical spread of modern PUUV throughout Europe during the last postglacial period in the light of an upgraded dataset of complete PUUV small (S) segment sequences and by using most recent computational approaches. Taking advantage of the knowledge on the past migrations of its host, we identified at least three potential independent dispersal routes of PUUV during postglacial recolonization of Europe by the bank vole. From the Alpe-Adrian region (Balkan, Austria, and Hungary) to Western European countries (Germany, France, Belgium, and Netherland), and South Scandinavia. From the vicinity of Carpathian Mountains to the Baltic countries and to Poland, Russia, and Finland. The dissemination towards Denmark and North Scandinavia is more hypothetical and probably involved several independent streams from south and north Fennoscandia.

Genetic analyses of Seoul hantavirus genome recovered from rats (Rattus norvegicus) in the Netherlands unveils diverse routes of spread into Europe.

Seoul virus (SEOV) is the etiologic agent of hemorrhagic fever with renal syndrome. It is carried by brown rats (Rattus norvegicus), a commensal rodent that closely cohabitates with humans in urban environments. SEOV has a worldwide distribution, and in Europe, it has been found in rats in UK, France, Sweden, and Belgium, and human cases of SEOV infection have been reported in Germany, UK, France, and Belgium. In the search of hantaviruses in brown rats from the Netherlands, we found both serological and genetic evidence for the presence of SEOV in the local wild rat population. To further decipher the relationship with other SEOV variants globally, the complete genome of SEOV in the Netherlands was recovered. SEOV sequences obtained from three positive rats (captured at close trapping locations at the same time) were found highly similar. Phylogenetic analyses demonstrated that two lineages of SEOV circulate in Europe. Strains from the Netherlands and UK, together with the Baxter strain from US, constitute one of these two, while the second includes strains from Europe and Asia. Our results support a hypothesis of diverse routes of SEOV spread into Europe. These findings, combined with other indications on the expansion of the spatial European range of SEOV, suggest an increased risk of this virus for the public health, highlighting the need for increased surveillance.

Defining of MAbs-neutralizing sites on the surface glycoproteins Gn and Gc of a hantavirus using vesicular stomatitis virus pseudotypes and site-directed mutagenesis.

Earlier four monoclonal antibodies (MAbs) against surface glycoproteins Gn and Gc of puumala virus (PUUV, genus Orthohantavirus, family Hantaviridae, order Bunyavirales) were generated and for three MAbs with neutralizing capacity the localization of binding epitopes was predicted using pepscan and phage-display techniques. In this work, we produced vesicular stomatitis virus (VSV) particles pseudotyped with the Gn and Gc glycoproteins of PUUV and applied site-directed mutagenesis to dissect the structure of neutralizing epitopes. Replacement of cysteine amino acid (aa) residues with alanines resulted in pseudotype particles with diminished (16 to 18 %) neut-titres; double Cys→Ala mutants, as well as mutants with bulky aromatic and charged residues replaced with alanines, have shown even stronger reduction in neut-titres (from 25 % to the escape phenotype). In silico modelling of the neut-epitopes supported the hypothesis that these critical residues are located on the surface of viral glycoprotein molecules and thus can be recognized by the antibodies indeed. A similar pattern was observed in experiments with mutant pseudotypes and sera collected from patients suggesting that these neut-epitopes are utilized in a course of human PUUV infection. These data will help understanding the mechanisms of hantavirus neutralization and assist construction of vaccine candidates.

Dobrava hantavirus variants found in Apodemus flavicollis mice in Kirklareli Province, Turkey.

Hantaviruses infect humans via inhalation of viral particles within secretions of infected rodents or rarely through direct contact with infected rodents. Determining the prevalence of hantavirus infections among rodent populations is of vital importance to obtain information on hantavirus-related cases and to predict possible outbreaks. We hypothesized that DOBV strains circulating in the Thrace Region in Turkey would be related to other Balkan DOBV strains. In this study, hantavirus infections in the rodent population of the Kirklareli-Igneada Region (north-western Turkey, near the Bulgarian border) were investigated. This region is of particular importance, as it is located in the south-eastern margin of the European continent and was used as an entrance point of Asian faunal elements into Europe. DOBV infection was detected in eight of 73 rodents; all were of the Apodemus flavicollis species. Partial sequences of the viral S-, M-, and L-genome segments were recovered and compared with previously reported DOBV sequences. The newly characterized Turkish strains were similar to other DOBV variants. Silent nucleotide mutations were dominant. The hantavirus prevalence in the Igneada region was similar to what has been reported in Greece and Bulgaria. For the first time, the M-segment sequences of DOBV from Turkey were recovered and genetic data of hantaviruses from Thrace region of Turkey were obtained.

Hantavirus maintenance and transmission in reservoir host populations.

Hantaviruses are primarily hosted by mammalian species of the orders Rodentia, Eulipotyphla and Chiroptera. Spillover to humans is common, and understanding hantavirus maintenance and transmission in reservoir host populations is important for efforts to curtail human disease. Recent field research challenges traditional phases of virus shedding kinetics derived from laboratory rodent infection experiments. Organ infection sites in non-rodent hosts suggest similar transmission routes to rodents, but require direct assessment. Further advances have also been made in understanding virus persistence (and fadeouts) in fluctuating host populations, as well as occupational, recreational and environmental risk factors associated with spillover to humans. However, despite relevance for both intra-species and inter-species transmission, our understanding of the longevity of hantaviruses in natural environments remains limited.

Vertebrate Reservoirs of Arboviruses: Myth, Synonym of Amplifier, or Reality?

The rapid succession of the pandemic of arbovirus diseases, such as dengue, West Nile fever, chikungunya, and Zika fever, has intensified research on these and other arbovirus diseases worldwide. Investigating the unique mode of vector-borne transmission requires a clear understanding of the roles of vertebrates. One major obstacle to this understanding is the ambiguity of the arbovirus definition originally established by the World Health Organization. The paucity of pertinent information on arbovirus transmission at the time contributed to the notion that vertebrates played the role of reservoir in the arbovirus transmission cycle. Because this notion is a salient feature of the arbovirus definition, it is important to reexamine its validity. This review addresses controversial issues concerning vertebrate reservoirs and their role in arbovirus persistence in nature, examines the genesis of the problem from a historical perspective, discusses various unresolved issues from multiple points of view, assesses the present status of the notion in light of current knowledge, and provides options for a solution to resolve the issue.

Estimation of main diversification time-points of hantaviruses using phylogenetic analyses of complete genomes.

Because of the great variability of their reservoir hosts, hantaviruses are excellent models to evaluate the dynamics of virus-host co-evolution. Intriguing questions remain about the timescale of the diversification events that influenced this evolution. In this paper we attempted to estimate the first ever timing of hantavirus diversification based on thirty five available complete genomes representing five major groups of hantaviruses and the assumption of co-speciation of hantaviruses with their respective mammal hosts. Phylogenetic analyses were used to estimate the main diversification points during hantavirus evolution in mammals while host diversification was mostly estimated from independent calibrators taken from fossil records. Our results support an earlier developed hypothesis of co-speciation of known hantaviruses with their respective mammal hosts and hence a common ancestor for all hantaviruses carried by placental mammals.

Vaccinia virus-free rescue of fluorescent replication-defective vesicular stomatitis virus and pseudotyping with Puumala virus glycoproteins for use in neutralization tests.

Puumala virus (PUUV) grows slowly in cell culture. To study antigenic properties of PUUV, an amenable method for their expression would be beneficial. To achieve this, a replication-defective recombinant vesicular stomatitis virus, rVSVΔG*EGFP, was rescued using BSRT7/5 and encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES)-enabled rescue plasmids. Using these particles, pseudotypes bearing PUUV Sotkamo strain glycoproteins were produced, with titres in the range 105-108, and were used in pseudotype focus reduction neutralization tests (pFRNTs) with neutralizing monoclonal antibodies and patient sera. The results were compared with those from orthodox focus reduction neutralization tests (oFRNTs) using native PUUV with the same samples and showed a strong positive correlation (rs = 0.82) between the methods. While developing the system we identified three amino acids which were mutated in the Vero E6 cell culture adapted PUUV prototype Sotkamo strain sequence, and changing these residues was critical for expression and neutralizing antibody binding of PUUV glycoproteins.

Complete Genome and Phylogeny of Puumala Hantavirus Isolates Circulating in France.

Puumala virus (PUUV) is the agent of nephropathia epidemica (NE), a mild form of hemorrhagic fever with renal syndrome (HFRS) in Europe. NE incidence presents a high spatial variation throughout France, while the geographical distribution of the wild reservoir of PUUV, the bank vole, is rather continuous. A missing piece of the puzzle is the current distribution and the genetic variation of PUUV in France, which has been overlooked until now and remains poorly understood. During a population survey, from 2008 to 2011, bank voles were trapped in eight different forests of France located in areas known to be endemic for NE or in area from where no NE case has been reported until now. Bank voles were tested for immunoglobulin (Ig)G ELISA serology and two seropositive animals for each of three different areas (Ardennes, Jura and Orleans) were then subjected to laboratory analyses in order to sequence the whole S, M and L segments of PUUV. Phylogenetic analyses revealed that French PUUV isolates globally belong to the central European (CE) lineage although isolates from Ardennes are clearly distinct from those in Jura and Orleans, suggesting a different evolutionary history and origin of PUUV introduction in France. Sequence analyses revealed specific amino acid signatures along the N protein, including in PUUV from the Orleans region from where NE in humans has never been reported. The relevance of these mutations in term of pathophysiology is discussed.

Detection of Dobrava hantavirus RNA in Apodemus mice in Bulgaria.

Several Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) in Europe: Dobrava-Belgrade virus (DOBV), Puumala, Saaremaa, Sochi, and Seoul virus. Although HFRS is endemic in Bulgaria, genome sequences of hantaviruses have never been detected in wild rodents. To identify rodent reservoirs, a total of 691 rodents from three endemic regions were trapped in 2011-2012 and screened by TaqMan RT-PCR for detection of hantaviral genomic RNA. Partial small (S) and/or large (L)-segment sequences were recovered from six Apodemus mice: five of the species A. flavicollis and one A. agrarius. Phylogenetic analysis revealed that all recovered sequences belonged to DOBV. On the phylogenetic trees, the novel Bulgarian hantavirus sequences clustered together with sequences of established previously DOBV variants recovered from Bulgarian HFRS patients and also with variants found in wild rodents trapped in Slovenia, Greece, and Slovakia. One of the novel Bulgarian DOBV S-sequences from A. agrarius was related closely to DOBV sequences recovered from A. flavicollis, suggesting a spillover of DOBV from its natural host to A. agrarius mice. The results of this study confirmed the circulation of DOBV in wild rodents in Bulgaria. The complexity of the epidemiological situation in the Balkans requires further studies of hantaviruses in rodent hosts and human HFRS cases.

A tick-borne segmented RNA virus contains genome segments derived from unsegmented viral ancestors.

Although segmented and unsegmented RNA viruses are commonplace, the evolutionary links between these two very different forms of genome organization are unclear. We report the discovery and characterization of a tick-borne virus--Jingmen tick virus (JMTV)--that reveals an unexpected connection between segmented and unsegmented RNA viruses. The JMTV genome comprises four segments, two of which are related to the nonstructural protein genes of the genus Flavivirus (family Flaviviridae), whereas the remaining segments are unique to this virus, have no known homologs, and contain a number of features indicative of structural protein genes. Remarkably, homology searching revealed that sequences related to JMTV were present in the cDNA library from Toxocara canis (dog roundworm; Nematoda), and that shared strong sequence and structural resemblances. Epidemiological studies showed that JMTV is distributed in tick populations across China, especially Rhipicephalus and Haemaphysalis spp., and experiences frequent host-switching and genomic reassortment. To our knowledge, JMTV is the first example of a segmented RNA virus with a genome derived in part from unsegmented viral ancestors.

Co-circulation of multiple hemorrhagic fever diseases with distinct clinical characteristics in Dandong, China.

Hemorrhagic fevers (HF) caused by viruses and bacteria are a major public health problem in China and characterized by variable clinical manifestations, such that it is often difficult to achieve accurate diagnosis and treatment. The causes of HF in 85 patients admitted to Dandong hospital, China, between 2011-2012 were determined by serological and PCR tests. Of these, 34 patients were diagnosed with Huaiyangshan hemorrhagic fever (HYSHF), 34 with Hemorrhagic Fever with Renal Syndrome (HFRS), one with murine typhus, and one with scrub typhus. Etiologic agents could not be determined in the 15 remaining patients. Phylogenetic analyses of recovered bacterial and viral sequences revealed that the causative infectious agents were closely related to those described in other geographical regions. As these diseases have no distinctive clinical features in their early stage, only 13 patients were initially accurately diagnosed. The distinctive clinical features of HFRS and HYSHF developed during disease progression. Enlarged lymph nodes, cough, sputum, and diarrhea were more common in HYSHF patients, while more HFRS cases presented with headache, sore throat, oliguria, percussion pain kidney area, and petechiae. Additionally, HYSHF patients displayed significantly lower levels of white blood cells (WBC), higher levels of creations kinase (CK) and alanine aminotransferase (ALT), while HFRS patients presented with an elevation of blood urea nitrogen (BUN) and creatinine (CREA). These clinical features will assist in the accurate diagnosis of both HYSHF and HFRS. Overall, our data reveal the complexity of pathogens causing HFs in a single Chinese hospital, and highlight the need for accurate early diagnosis and a better understanding of their distinctive clinical features.

Evolution of hantaviruses: co-speciation with reservoir hosts for more than 100 MYR.

The most recent (9th) Report of the International Committee on Taxonomy of Viruses (ICTV) lists 23 established and 30 provisional species in the genus Hantavirus (family Bunyaviridae) (Plyusnin et al., 2012). These virus species are harbored by altogether 51 species of rodents, shrews and moles and thus in most cases it is a relationship of "one hantavirus-one host". Such a tight bond between the two, in combination with the observed association between whole groups of hantaviruses and (sub)families of rodents, helped to develop the widely accepted view of a long-term co-evolution (co-speciation) of these viruses with their hosts. Accumulating evidence of host-switching events, both recent and ancient, however challenged some of the earlier views on hantavirus evolution. In this paper we discuss the concept of hantavirus-host co-speciation and propose a scenario of hantavirus evolution based on the currently available genetic information. This scenario is based on the hypothesis that hantaviruses are very ancient viruses which already existed at the estimated diversification point of major placental clades, of which one includes the ancestors of the order Rodentia and another the ancestors of both orders Eulipotyphla and Chiroptera; the diversification occurred approximately at 90-100 MYA. We also speculate that the evolutionary history of hantaviruses extents even deeper in the past, beyond this time-point, and included the transmission of a (pre)bunyavirus from an insect host to a mammal host.