Search Results (105)

(1) Background: Hemorrhagic fever with renal syndrome (HFRS) remains a prevalent zoonosis in Eurasia. Orthohantavirus puumalaense (PUUV), carried by bank voles (Myodes glareolus), is the principal zoonotic pathogen of HFRS in this region. Despite ongoing efforts to develop effective drugs and vaccines against PUUV, this challenge remains. (2) Aim: In this study, we aimed to express a large quantity of the PUUV recombinant N (rN) protein using E. coli. We also sought to develop a protocol for extracting the rN protein from pellets, solubilizing, and refolding it to restore its native form. This protocol is crucial for producing a large quantity of rN protein to develop vaccines and diagnostic tools for HFRS. (3) Methods; PUUV S segment open reading frame (ORF) coding for N protein was synthesized and cloned into the plasmid vector pET-28 (A+). The ORF was transformed, expressed and induced in BL21(DE3) pLysS E. coli strain. Subsequently, rN protein was purified using immobilized metal affinity and ion chromatography. Immune reactivity of rN protein was tested by employing in house and commercial VektoHanta-IgG kit ELISA methods (both in vitro and in vivo). (4) Results: The best conditions for scaling up the expression of the PUUV rN protein were an incubation temperature of 20 °C during a 20 h incubation period, followed by induction with 0.5 mM IPTG. The most significant protein yield was achieved when the pellets were incubated in denaturing buffer with 8M urea. The highest yield of refolded proteins was attained using non-denaturing buffer (50 mM Tris-HCl) supplemented with arginine. A final 50 μL of PUUV rN protein solution with a concentration of 7 mg/mL was recovered from 1 L of culture. The rN protein elicited an antibody response in vivo and reacted with serum taken from patients with HFRS by ELISA in vitro. (5) Conclusion: Therefore, the orthohantavirus N protein’s ability to elicit immune response in vivo suggests that it can be used to develop vaccines against PUUV after conducting in vitro and in vivo studies to ascertain neutralising antibodies. Full article

Orthohantaviruses are tri-segmented negative-sense RNA viruses that can cause severe pathologies in humans. Currently, limited information exists on the molecular interactions driving orthohantavirus assembly in infected cells. Specifically, it is not clear how its glycoproteins (i.e., Gn and Gc) interact with other viral or host molecules. In this study, we use one- and two-color Number and Brightness fluorescence microscopy approaches to quantitatively characterize the interactions between orthohantavirus glycoproteins and the nucleoprotein in transfected cells. Our results indicate that orthohantavirus nucleoprotein homo-interactions are strongly affected by the host environment. Furthermore, we report evidence of Gc–nucleoprotein interactions, based on (i) the high fluorescence cross-correlation between these two proteins and (ii) the increased Gc-Gc interactions observed in the presence of nucleoprotein. Finally, experiments on a Gc deletion mutant suggest that the observed protein–protein interactions are mediated by the cytoplasmic tail of Gc. In conclusion, this study provides new insights into the role of the interactions between orthohantavirus glycoproteins and nucleoprotein in the context of viral assembly. Full article

Orthohantavirus infection is a zoonotic disease transmitted to humans through contact with infected rodents. In Eurasia, Old World Orthohantaviruses can cause haemorrhagic fever with renal syndrome (HFRS), while in the Americas, New World Orthohantaviruses are responsible for hantavirus cardiopulmonary syndrome (HCPS). In Kazakhstan, the first recorded cases of HFRS appeared in the West Kazakhstan region in 2000, which has since then been established as an endemic area due to the presence of stable rodent reservoirs and recurring human infections. Routine diagnosis of HFRS in this region relies primarily on immunoassays. To enhance diagnostic precision, we aimed to implement both serological and molecular methods on samples from suspected HFRS cases in the endemic West Kazakhstan region and non-endemic Almaty City. A total of 139 paired serum, saliva, and urine samples were analysed using IgM/IgG ELISA, immunoblot assays, and qPCR. Our findings confirm that suspected HFRS cases in West Kazakhstan are associated with the Puumala virus serotype. Full article

Hantaviruses, including the Sin Nombre virus (SNV) and Andes virus (ANDV), are associated with severe global health risks, causing high mortality rates in hantavirus pulmonary syndrome (HPS) patients. Neutralizing antibodies are essential for virus clearance and survival, making neutralization assays critical for understanding immunity and evaluating therapeutic strategies. In this study, we developed a recombinant vesicular stomatitis virus (VSV)-based surrogate system expressing SNV and ANDV glycoproteins (GPCs), enabling neutralization studies under biosafety level 2 conditions. The neutralization titers obtained with the VSV-based system closely matched the findings from authentic hantavirus assays performed under biosafety level 3 conditions, confirming its potential as a useful tool for determining immune responses and advancing hantavirus research. Full article

In this work, we performed the genetic characterization of a new variant of orthohantavirus associated with a fatal case of hantavirus pulmonary syndrome, outside the known endemic region, in northwestern Argentina. We first confirmed an orthohantavirus infection by ELISA, testing for the detection of IgM and IgG antibodies. Then, we extracted RNA from 100 microliters of serum, the only sample available, followed by RT-PCR. The amplicons were sequenced using Sanger and next-generation sequencing technology. We obtained partial sequences of 1253 bp, 799 bp and 1675 bp from the S-, M- and L-segments, respectively, showing low sequence identities with all the previously characterized hantaviruses (10.9%, 13.5% and 15.1% of the divergence, respectively). The phylogenetic analysis showed that this virus belongs to the Orthohantavirus andesense species (ANDV), and among the ANDV-like variants, it is more closely related to the Lechiguanas clade. Similar percentages of divergence were considered sufficient to distinguish AND-like variants in previous works. As the patient had no travel history before the onset of disease was reported, we conducted rodent surveys to confirm the presence of reservoirs. The rodent assemblage was compatible with the transitional zone among different ecoregions (Yungas, Chaco and Monte). Moreover, one of the species captured, Oligoryzomys flavescens, was previously described as a reservoir of hantavirus. This species may either host several variants across its range or encompass a species complex, as proposed by some authors. Full article

Orthohantavirus infection is a rodent-to-human zoonotic disease with a worldwide distribution, resulting in more than 200,000 cases per year. Human infection leads to two diseases, haemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome, with mortality rates ranging from 1% to 38%. Apart from the data on cases presenting obvious clinical symptoms, the true prevalence is poorly understood, especially in the occupational groups considered to be at risk of exposure. As there is currently no approved therapy or vaccine, surveillance is essential to locate the presumed site of infection following orthohantavirus outbreaks in order to control the spread of infection. To this end, the use of rapid diagnostic tools is essential to rapidly provide data on viral circulation. This review focuses mainly on the available diagnostic methods, both serological and biomolecular, and the surveillance systems used for orthohantaviruses. The information gathered could provide a valid basis for the implementation of further surveillance systems in a country lacking up-to-date data. Full article

Background: Old World orthohantaviruses are the aetiological agent of Haemorrhagic Fever with Renal Syndrome (HFRS) disease. Worldwide, the two most prominent pathogens of HFRS are Seoul orthohantavirus (SEOV) and Hantaan orthohantavirus (HTNV). There is currently no specific treatment nor widely licensed vaccine form hantaviruses. Methods: This study developed a virus-vectored vaccine approach using modified vaccinia Ankara (MVA) incorporating a SEOV-HTNV chimeric nucleoprotein antigen. Results: The vaccine demonstrated the induction of humoral and cellular immunity. In the absence of a disease model, a reduction in the viral load of a susceptible mouse strain with type-I interferon receptor deficiency (A129) was used to ascertain protective effects after challenge with SEOV. Results demonstrated a significant reduction in and/or clearance of viral RNA in immunised animals. Conclusions: An MVA viral vector vaccine incorporating the nucleoprotein as antigen offers a promising approach for Hantavirus vaccine development. Full article

Orthohantaviruses (also known as hantaviruses) are pathogens that cause two distinct, yet related forms of severe human disease: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). These diseases pose a significant threat to global public health due to their high case fatality rates, which can range from 1% to 50%. In recent years, an increasing number of countries and regions have reported human cases, underscoring the urgent need for improved understanding, prevention, and treatment strategies. Given the severity of these diseases and the lack of specific post-exposure antiviral treatments, preventive measures are critical. For several decades, substantial efforts have been dedicated to developing orthohantavirus vaccines, leading to significant advancements. The first large-scale deployment involved inactivated vaccines, which played a crucial role in reducing HFRS incidence in South Korea and China. Subunit vaccines, viral vector vaccines, and virus-like particle (VLP) vaccines have also been extensively researched. Nucleic acid vaccines, including both mRNA and DNA vaccines, hold the greatest potential for future development due to their rapid design and production cycles, ability to elicit robust immune responses, ease of storage and transportation, and adaptable production platforms. Ongoing advancements in computer technology and artificial intelligence promise to further enhance the development of more effective orthohantavirus vaccines. Full article

Hantaviruses are zoonotic pathogens associated with severe human diseases such as hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Despite the extensive study of rodent-borne hantaviruses, research on bat-associated hantaviruses remains limited. This study aimed to investigate the seroprevalence and cross-reactivity of neotropical bat samples with rodent- and bat-associated recombinant hantavirus nucleoproteins (rNPs) to improve hantavirus surveillance in the Brazilian Atlantic Forest. The studied bat population consisted of 336 blood samples collected over nearly a decade in five Brazilian states (Bahia, Rio de Janeiro, Santa Catarina, Paraná, and Minas Gerais). Antibodies were detected using IgG ELISA assays with rNPs from bat-borne Mobatvirus xuansonense (XSV) and Loanvirus brunaense (BRNV) and the rodent-borne hantaviruses Orthohantavirus andesense (ANDV) and Orthohantavirus seoulense (SEOV). Results indicated a higher seroprevalence for the BRNV rNP (36.6%) compared to ANDV (7.4%), SEOV (5.7%), and XSV (0.6%). The high sensitivity of the BRNV rNP and the cross-reactivity observed with the ANDV rNP, the main protein used for serological tests in the Americas, indicates that BRNV rNP is a better antigen for the accurate detection of antibodies against hantaviruses in Brazilian bats. These findings underscore the presence of unknown hantaviruses antigenically similar to BRNV in Brazilian bat populations and highlight the urgent need for identifying better antigens for comprehensive hantavirus monitoring in bats. Full article

Hantavirus pulmonary syndrome (HPS) is an American emerging disease caused by the rodent-borne virus genus Orthohantavirus (Family: Hantaviridae: Order: Elliovirales Class: Bunyaviricetes). In Argentina, almost half of the HPS infections occur in the northwestern endemic region. In this study, we monitored rodent abundance during 2022 and 2023 in three sites with different sampling methods (removal trapping, live trapping and hunted rodents by domestic cats) to evaluate their relationship with human infections. We found a similar pattern of variation in rodent abundance across time, and particularly a synchronous rise of rodent abundance that anticipated an HPS outbreak in 2023. Our dynamic regression models revealed a positive relationship between HPS cases and rodent abundance with a three-month lag, as well as rainfall with an eight-month lag. Our results provide a framework for the planning and implementation of public health prevention campaigns based on climatology and rodent monitoring. Domestic cats bringing rodents into houses can be an overlooked risk factor, particularly if viral shedding of infected rodents is magnified by stress. HPS is a disease of public health concern due to its high mortality rate, the lack of a specific therapeutic treatment and no vaccine. Thus, prevention of infections is of the utmost importance. Full article

Hemorrhagic fever with renal syndrome (HFRS) induced by Eurasian pathogenic orthohantaviruses is characterized by acute kidney injury (AKI) with often massive proteinuria. The mechanisms of the organ-specific manifestation are not completely understood. To analyze the role of glomerular and tubular damage in kidney injury induced by HFRS, we measured specific markers in urine samples of patients with acute Puumala virus (PUUV) infection and determined their correlation with disease severity. Levels of α1-microglobulin (α1-MG) and kidney injury molecule 1 (KIM-1), which is expressed by injured tubular epithelial cells, were measured to detect tubular dysfunction and injury. Immunoglobulin G (IgG) and the podocyte specific protein nephrin served as markers for glomerular injury. All four markers were elevated on admission. Markers of glomerular injury, IgG and nephrin, correlated with markers of disease severity such as length of hospitalization, serum creatinine, and proteinuria. In contrast, tubular injury did not correlate with these severity markers. Our results demonstrate that hantavirus infection induces both glomerular and tubular injury early in the clinical course. However, the glomerular dysfunction and podocyte injury seem to contribute directly to disease severity and to play a more central role in HFRS pathogenicity than direct damage to tubular epithelial cells. Full article

Puumala orthohantavirus (PUUV) is an emerging zoonotic virus endemic to Europe and Russia that causes nephropathia epidemica, a mild form of hemorrhagic fever with renal syndrome (HFRS). There are limited options for treatment and diagnosis of orthohantavirus infection, making the search for potential immunogenic candidates crucial. In the present work, various bioinformatics tools were employed to design conserved immunogenic peptides containing multiple epitopes of PUUV nucleocapsid protein. Eleven conserved peptides (90% conservancy) of the PUUV nucleocapsid protein were identified. Three conserved peptides containing multiple T and B cell epitopes were selected using a consensus epitope prediction algorithm. Molecular docking using the HPEP dock server demonstrated strong binding interactions between the epitopes and HLA molecules (ten alleles for each class I and II HLA). Moreover, an analysis of population coverage using the IEDB database revealed that the identified peptides have over 90% average population coverage across six continents. Molecular docking and simulation analysis reveal a stable interaction with peptide constructs of chosen immunogenic peptides and Toll-like receptor-4. These computational analyses demonstrate selected peptides’ immunogenic potential, which needs to be validated in different experimental systems. Full article

Hemorrhagic fever with renal syndrome (HFRS) is a rodent-borne disease widespread in Europe and Asia. HFRS is caused by negative-sensed single-stranded RNA orthohantaviruses transmitted to humans through inhaling aerosolized excreta of infected rodents. Symptoms of HFRS include acute kidney injury, thrombocytopenia, hemorrhages, and hypotension. The immune response raised against viral antigens plays an important role in the pathogenesis of HFRS. Inhibitory co-receptors are essential in regulating immune responses, mitigating immunopathogenesis, and reducing tissue damage. Our research showed an increased soluble form of inhibitory co-receptors TIM-3, LAG-3, and PD-1 in HFRS patients associated with disease severity. Our study aimed to investigate the impact of HFRS on the concentrations of soluble forms of inhibitory receptors TIM-3, LAG-3, and PD-1 in the patient’s serum and the potential correlation with key clinical parameters. Our study aimed to investigate the impact of HFRS on the concentrations of soluble forms of inhibitory receptors TIM-3, LAG-3, and PD-1 in the patient’s serum and their possible association with relevant clinical parameters. Using multiplex immunoassay, we found elevated levels of TIM-3, LAG-3, and PD-1 proteins in the serum of HFRS patients. Furthermore, increased levels were associated with creatinine, urea, lactate dehydrogenase concentrations, and platelet count. These findings suggest that these proteins play a role in regulating the immune response and disease progression. Full article

The protein-L-utilizing Förster resonance energy transfer (LFRET) assay enables mix-and-read antibody detection, as demonstrated for sera from patients with, e.g., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Zika virus, and orthohantavirus infections. In this study, we compared paired serum and whole blood (WB) samples of COVID-19 patients and SARS-CoV-2 vaccine recipients. We found that LFRET also detects specific antibodies in WB samples. In 44 serum–WB pairs from patients with laboratory-confirmed COVID-19, LFRET showed a strong correlation between the sample materials. By analyzing 89 additional WB samples, totaling 133 WB samples, we found that LFRET results were moderately correlated with enzyme-linked immunosorbent assay results for samples collected 2 to 14 months after receiving COVID-19 diagnosis. However, the correlation decreased for samples >14 months after receiving a diagnosis. When comparing the WB LFRET results to neutralizing antibody titers, a strong correlation emerged for samples collected 1 to 14 months after receiving a diagnosis. This study also highlights the versatility of LFRET in detecting antibodies directly from WB samples and suggests that it could be employed for rapidly assessing antibody responses to infectious agents or vaccines. Full article

Orthohantaviruses may cause hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. Andes virus (ANDV) is the only orthohantavirus associated with human–human transmission. Therefore, emergency vaccination would be a valuable public health measure to combat ANDV-derived infection clusters. Here, we utilized a promising vesicular stomatitis virus (VSV)-based vaccine to advance the approach for emergency applications. We compared monovalent and bivalent VSV vectors containing the Ebola virus (EBOV), glycoprotein (GP), and ANDV glycoprotein precursor (GPC) for protective efficacy in pre-, peri- and post-exposure immunization by the intraperitoneal and intranasal routes. Inclusion of the EBOV GP was based on its favorable immune cell targeting and the strong innate responses elicited by the VSV-EBOV vaccine. Our data indicates no difference of ANDV GPC expressing VSV vectors in pre-exposure immunization independent of route, but a potential benefit of the bivalent VSVs following peri- and post-exposure intraperitoneal vaccination. Full article