The Role of Long Noncoding RNA Negative Regulator of Interferon Response in the Regulation of Hantaan Virus Infection.

Hantaan virus (HTNV) is prevalent in Eurasia. It causes hemorrhagic fever with renal syndrome (HFRS). Long noncoding RNAs (lncRNAs) play key roles in regulating innate immunity. Among these, lncRNA negative regulator of interferon response (NRIR) was reported as an inhibitor of several interferon (IFN)-stimulated genes. Our results showed that: NRIR expression was upregulated by HTNV infection in a type I IFN-dependent manner. The expression of NRIR in CD14+ monocytes from HFRS patients in acute phase was significantly higher than that in convalescent phase and healthy controls. HTNV infection in some HTNV-compatible cells was promoted by NRIR. NRIR negatively regulated innate immunity, especially IFITM3 expression. Localized in the nucleus, NRIR bound with HNRNPC, and knockdown of HNRNPC significantly weakened the effect of NRIR in promoting HTNV infection and restored IFITM3 expression. These results indicated that NRIR regulates the innate immune response against HTNV infection possibly through its interaction with HNRNPC and its influence on IFITM3.

High-resolution phylogeographical surveillance of Hantaan orthohantavirus using rapid amplicon-based Flongle sequencing, Republic of Korea.

Orthohantaviruses, etiological agents of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome, pose a critical public health threat worldwide. Hantaan orthohantavirus (HTNV) outbreaks are particularly endemic in Gyeonggi Province in northern area of the Republic of Korea (ROK). Small mammals were collected from three regions in the Gyeonggi Province during 2017 and 2018. Serological and molecular prevalence of HTNV was 25/201 (12.4%) and 10/25 (40%), respectively. A novel nanopore-based diagnostic assay using a cost-efficient Flongle chip was developed to rapidly and sensitively detect HTNV infection in rodent specimens within 3 h. A rapid phylogeographical surveillance of HTNV at high-resolution phylogeny was established using the amplicon-based Flongle sequencing. In total, seven whole-genome sequences of HTNV were newly obtained from wild rodents collected in Paju-si (Gaekhyeon-ri) and Yeoncheon-gun (Hyeonga-ri and Wangnim-ri), Gyeonggi Province. Phylogenetic analyses revealed well-supported evolutionary divergence and genetic diversity, enhancing the resolution of the phylogeographic map of orthohantaviruses in the ROK. Incongruences in phylogenetic patterns were identified among HTNV tripartite genomes, suggesting differential evolution for each segment. These findings provide crucial insights into on-site diagnostics, genome-based surveillance, and the evolutionary dynamics of orthohantaviruses to mitigate hantaviral outbreaks in HFRS-endemic areas in the ROK.

Hantaan virus inhibits type I interferon response by targeting RLR signaling pathways through TRIM25.

Hantaan virus (HTNV) is responsible for hemorrhagic fever with renal syndrome (HFRS), primarily due to its ability to inhibit host innate immune responses, such as type I interferon (IFN-I). In this study, we conducted a transcriptome analysis to identify host factors regulated by HTNV nucleocapsid protein (NP) and glycoprotein. Our findings demonstrate that NP and Gc proteins inhibit host IFN-I production by manipulating the retinoic acid-induced gene I (RIG-I)-like receptor (RLR) pathways. Further analysis reveals that HTNV NP and Gc proteins target upstream molecules of MAVS, such as RIG-I and MDA-5, with Gc exhibiting stronger inhibition of IFN-I responses than NP. Mechanistically, NP and Gc proteins interact with tripartite motif protein 25 (TRIM25) to competitively inhibit its interaction with RIG-I/MDA5, suppressing RLR signaling pathways. Our study unveils a cross-talk between HTNV NP/Gc proteins and host immune response, providing valuable insights into the pathogenic mechanism of HTNV.

Investigation of a subunit protein vaccine for HFRS based on a consensus sequence between envelope glycoproteins of HTNV and SEOV.

Due to the global resurgence of hemorrhagic fever with renal syndrome (HFRS), more attention is being focused on this dangerous illness. In China and Korea, the only vaccines available are the virus-inactivated vaccine against Hantaan virus (HTNV) or Seoul virus (SEOV), but their efficacy and safety are inadequate. Therefore, it is important to develop new vaccines that are safer and more efficient to neutralize and regulate areas with a high prevalence of HFRS. We employed bioinformatics methods to design a recombinant protein vaccine based on conserved regions of protein consensus sequences in HTNV and SEOV membranes. The S2 Drosophila expression system was utilized to enhance protein expression, solubility and immunogenicity. After the Gn and Gc proteins of HTNV and SEOV were successfully expressed, mice were immunized, and the humoral immunity, cellular immunity, and in vivo protection of the HFRS universal subunit vaccine were systematically evaluated in mouse models. These results indicated that the HFRS subunit vaccine generated elevated levels of binding and neutralizing antibodies, particularly IgG1, compared to that of the traditional inactivated HFRS vaccine. Additionally, the spleen cells of immunized mice secreted IFN-r and IL-4 cytokines effectively. Moreover, the HTNV-Gc protein vaccine successfully protected suckling mice from HTNV infection and stimulated GC responses. In this research, a new scientific approach is investigated to develop a universal HFRS subunit protein vaccine that is capable of producing effective humoral and cellular immunity in mice. The results suggest that this vaccine could be a promising candidate for preventing HFRS in humans.

Construction of a Hantaan Virus Phage Antibody Library and Screening for Potential Neutralizing Activity.

China is one of the main epidemic areas for hemorrhagic fever with renal syndrome (HFRS). Currently, there is no human antibody specific to Hantaan virus (HTNV) for the emergency prevention and treatment of HFRS. To prepare human antibodies with neutralizing activity, we established an anti-HTNV phage antibody library using phage display technology by transforming peripheral blood mononuclear cells (PBMCs) of patients with HFRS into B lymphoblastoid cell lines (BLCLs) and extracting cDNA from BLCLs that secreted neutralizing antibodies. Based on the phage antibody library, we screened HTNV-specific Fab antibodies with neutralizing activities. Our study provides a potential way forward for the emergency prevention of HTNV and specific treatment of HFRS.

Insight into the Hantaan virus RNA-dependent RNA polymerase inhibition using in-silico approaches.

The Hantaan virus (HTN) is a member of the hantaviridae family. It is a segmented type, negative-strand virus (sNSVs). It causes hemorrhagic fever with renal syndrome, which includes fever, vascular hemorrhage, and renal failure. This illness is one of the most serious hemorrhagic diseases in the world, and it is a major public health concern due to its high mortality rate. The Hantaan virus RNA-dependent RNA polymerase complex (RdRp) is involved in viral RNA transcription and replication for the survival and transmission of this virus. Therefore, it is a primary target for antiviral drug development. Interference with the endonucleolytic "cap-snatching" reaction by the HTN virus RdRp endonuclease domain is a particularly appealing approach for drug discovery against this virus. This RdRp endonuclease domain of the HTN virus has a metal-dependent catalytic activity. We targeted this metal-dependent enzymatic activity to identify inhibitors that can bind and disrupt this endonuclease enzyme activity using in-silico approaches i.e., molecular docking, molecular dynamics simulation, predicted absorption, distribution, metabolism, excretion, toxicity (ADMET) and drug-likeness studies. The docking studies showed that peramivir, and ingavirin compounds can effectively bind with the manganese ions and engage with other active site residues of this protein. Molecular simulations also showed stable binding of these ligands with the active site of HTN RdRp. Simulation analysis showed that they were in constant contact with the active site manganese ions and amino acid residues of the HTN virus endonuclease domain. This study will help in better understanding the HTN and related viruses.

Increased CD4+CD8+ Double Positive T Cells during Hantaan Virus Infection.

Hantaan virus (HTNV) infection causes an epidemic of hemorrhagic fever with renal syndrome (HFRS) mainly in Asia. It is well known that T cells mediated anti-viral immune response. Although previous studies showed that double positive T (DP T) cells, a little portion of T lymphocytes, were involved in adaptive immune response during virus infection, their kinetic changes and roles in HTNV infection have not yet been explored. In this study, we characterized DP T cells from HFRS patients based on flow cytometry data combined with scRNA-seq data. We showed that HTNV infection caused the upregulation of DP T cells in the peripheral blood, which were correlated with disease stage. The scRNA-seq data clustered DP T cells, unraveled their gene expression profile, and estimated the ordering of these cells. The production of granzyme B and CD107a from DP T cells and the abundant TCR distribution indicated the anti-viral property of DP T cells. In conclusion, this study identified, for the first time, an accumulation of DP T cells in the peripheral blood of HFRS patients and suggested these DP T cells belonging to CD8+T cells lineage. The DP T cells shared the similar characteristics with cytotoxic T cells (CTL) and exerted an anti-viral role in HFRS.

Emerging hantavirus infection in wild rodents captured in suburbs of Gwangju Metropolitan City, South Korea.

BACKGROUND: Hemorrhagic fever with renal syndrome (HFRS) caused by hantaviruses is a frequently reported acute hemorrhagic fever in South Korea. These viruses are transmitted by various rodent species such as Apodemus agrarius. METHODOLOGY/PRINCIPAL FINDINGS: To investigate hantavirus infection and seroprevalence in rodents, wild rodents were captured from two districts in the suburbs of Gwangju Metropolitan City from January 2016 to December 2018. Nested reverse-transcription polymerase chain reaction (RT-PCR) targeting the hantavirus-specific L segment and indirect immunofluorescence antibody (IFA) assay using Hantaan virus antigen slides were performed. A total of 585 wild rodents were captured-512 A. agrarius, 49 Crocidura lasiura, and 24 Myodes regulus. Nested RT-PCR was performed to examine the rate of hantavirus infection in wild rodents, and 1.88% (11/585) of all rodents, 1.17% (6/512) of A. agrarius, 6.12% (3/49) of C. lasiura, and 8.33% (2/24) of M. regulus tested positive. The nucleotide sequence analysis of the eleven PCR-positive products revealed that six PCR products showed over 85% sequence similarity with the Jeju virus, four showed over 99.7% similarity with the Hantaan virus, and one showed over 95.3% homology with the Imjin virus. Moreover, IgG antibodies against the Hantaan virus were detected in 6.15% (36/585) of all rodents, 6.8% (35/512) of A. agrarius, and 4.17% (1/24) of M. regulus. IgG antibodies were not detected in C. lasiura. CONCLUSIONS/SIGNIFICANCE: Hantaviruses were detected in all three wild rodent species of A. agrarius, C. lasiura, and M. regulus captured in the suburbs of Gwangju Metropolitan City, South Korea, and it was demonstrated that they were various strains of hantaviruses such as the Hantaan, Jeju, and Imjin viruses.

Urinary genome detection and tracking of Hantaan virus from hemorrhagic fever with renal syndrome patients using multiplex PCR-based next-generation sequencing.

BACKGROUND: Hantavirus infection occurs through the inhalation of aerosolized excreta, including urine, feces, and saliva of infected rodents. The presence of Hantaan virus (HTNV) RNA or infectious particles in urine specimens of patient with hemorrhagic fever with renal syndrome (HFRS) remains to be investigated. METHODOLOGY/PRINCIPAL FINDINGS: We collected four urine and serum specimens of Republic of Korea Army (ROKA) patients with HFRS. We performed multiplex PCR-based next-generation sequencing (NGS) to obtain the genome sequences of clinical HTNV in urine specimens containing ultra-low amounts of viral genomes. The epidemiological and phylogenetic analyses of HTNV demonstrated geographically homogenous clustering with those in Apodemus agrarius captured in highly endemic areas, indicating that phylogeographic tracing of HTNV genomes reveals the potential infection sites of patients with HFRS. Genetic exchange analyses showed a genetic configuration compatible with HTNV L segment exchange in nature. CONCLUSION/SIGNIFICANCE: Our results suggest that whole or partial genome sequences of HTNV from the urine enabled to track the putative infection sites of patients with HFRS by phylogeographically linking to the zoonotic HTNV from the reservoir host captured at endemic regions. This report raises awareness among physicians for the presence of HTNV in the urine of patients with HFRS.

The changing epidemiology of hemorrhagic fever with renal syndrome in Southeastern China during 1963-2020: A retrospective analysis of surveillance data.

BACKGROUND: Hemorrhagic fever with renal syndrome (HFRS) is a rodent-borne disease caused by hantavirus which was endemic Zhejiang Province, China. In this study, we aim to explore the changing epidemiology of HFRS in Zhejiang, identify high-risk areas and populations, and evaluate relevant policies and interventions to better improve HFRS control and prevention. METHODS: Surveillance data on HFRS during 1963-2020 in Zhejiang Province were extracted from Zhejiang Provincial Center for Disease Control and Prevention archives and the Chinese Notifiable Disease Reporting System. The changing epidemiological characteristics of HFRS including seasonal distribution, geographical distribution, and demographic features, were analyzed using joinpoint regression, autoregressive integrated moving average model, descriptive statistical methods, and Spatio-temporal cluster analysis. RESULTS: From 1963 to 2020, 114 071 HFRS cases and 1269 deaths were reported in Zhejiang Province. The incidence increased sharply from 1973 and peaked in 1986, then decreased steadily and maintained a stable incidence from 2004. HFRS cases were reported in all 11 prefecture-level cities of Zhejiang Province from 1963 to 2020. The joint region (Shengzhou, Xinchang, Tiantai, and surrounding areas), and Kaihua County are the most seriously affected regions throughout time. After 1990, the first HFRS incidence peak was in May-June, with another one from November to January. Most HFRS cases occurred in 21- (26.48%) and 30- years group (24.25%) from 1991 to 2004, but 41- (25.75%) and 51-years (23.30%) had the highest proportion from 2005 to 2020. Farmers accounted for most cases (78.10%), and cases are predominantly males with a male-to-female ratio of 2.6:1. It was found that the median time from onset to diagnosis was 6.5 days (IQR 3.75-10.42), and the time from diagnosis to disease report was significantly shortened after 2011. CONCLUSIONS: We observed dynamic changes in the seasonal distribution, geographical distribution, and demographic features of HFRS, which should be well considered in the development of control and prevention strategies in future. Additional researches are warranted to elucidate the environmental, meteorological, and social factors associated with HFRS incidence in different decades.

Acute Appendicitis Associated with Hantaan Virus Infection.

Hantaviruses are Bunyaviridae viruses that cause hemorrhagic fever with renal syndrome. Appendicitis caused by Hantaan virus has not been reported previously. An 81-year-old man who underwent laparoscopic appendectomy for suspected appendicitis based on abdominal pain, fever, hypotension, and computed tomography findings. Based on a suspicion of hemorrhagic fever with renal syndrome, the patient's plasma was simultaneously analyzed using an indirect immunofluorescent antibody assay and nested reverse transcription-polymerase chain reaction (RT-PCR). The appendix tissue was also analyzed using nested RT-PCR and immunohistochemical (IHC) staining to identify the presence of Hantaan virus. Nested RT-PCR detected the presence of Hantaan virus, and indirect immunofluorescent antibody assay results revealed the presence of elevated antibody levels. Furthermore, IHC staining of the appendix tissue confirmed Hantaan virus antigens in the peripheral nerve bundle. Based on these findings, we confirmed the nerve tropism of the Hantaan virus. Hantaan virus in plasma and appendix tissue samples was confirmed using PCR and phylogenetic tree analysis. Moreover, we detected hypertrophy of the submucosa and periappendiceal adipose tissue nerve bundle along with Hantaan virus antigens in peripheral nerve bundles using IHC staining. Hence, we report that Hantaan virus infection may be accompanied by appendicitis.

Structural Basis for a Neutralizing Antibody Response Elicited by a Recombinant Hantaan Virus Gn Immunogen.

Hantaviruses are a group of emerging pathogens capable of causing severe disease upon zoonotic transmission to humans. The mature hantavirus surface presents higher-order tetrameric assemblies of two glycoproteins, Gn and Gc, which are responsible for negotiating host cell entry and constitute key therapeutic targets. Here, we demonstrate that recombinantly derived Gn from Hantaan virus (HTNV) elicits a neutralizing antibody response (serum dilution that inhibits 50% infection [ID50], 1:200 to 1:850) in an animal model. Using antigen-specific B cell sorting, we isolated monoclonal antibodies (mAbs) exhibiting neutralizing and non-neutralizing activity, termed mAb HTN-Gn1 and mAb nnHTN-Gn2, respectively. Crystallographic analysis reveals that these mAbs target spatially distinct epitopes at disparate sites of the N-terminal region of the HTNV Gn ectodomain. Epitope mapping onto a model of the higher order (Gn-Gc)4 spike supports the immune accessibility of the mAb HTN-Gn1 epitope, a hypothesis confirmed by electron cryo-tomography of the antibody with virus-like particles. These data define natively exposed regions of the hantaviral Gn that can be targeted in immunogen design. IMPORTANCE The spillover of pathogenic hantaviruses from rodent reservoirs into the human population poses a continued threat to human health. Here, we show that a recombinant form of the Hantaan virus (HTNV) surface-displayed glycoprotein, Gn, elicits a neutralizing antibody response in rabbits. We isolated a neutralizing (HTN-Gn1) and a non-neutralizing (nnHTN-Gn2) monoclonal antibody and provide the first molecular-level insights into how the Gn glycoprotein may be targeted by the antibody-mediated immune response. These findings may guide rational vaccine design approaches focused on targeting the hantavirus glycoprotein envelope.

Multiplex PCR-Based Nanopore Sequencing and Epidemiological Surveillance of Hantaan orthohantavirus in Apodemus agrarius, Republic of Korea.

Whole-genome sequencing of infectious agents enables the identification and characterization of emerging viruses. The MinION device is a portable sequencer that allows real-time sequencing in fields or hospitals. Hantaan orthohantavirus (Hantaan virus, HTNV), harbored by Apodemus agrarius, causes hemorrhagic fever with renal syndrome (HFRS) and poses a critical public health threat worldwide. In this study, we aimed to evaluate the feasibility of using nanopore sequencing for whole-genome sequencing of HTNV from samples having different viral copy numbers. Amplicon-based next-generation sequencing was performed in A. agrarius lung tissues collected from the Republic of Korea. Genomic sequences of HTNV were analyzed based on the viral RNA copy numbers. Amplicon-based nanopore sequencing provided nearly full-length genomic sequences of HTNV and showed sufficient read depth for phylogenetic analysis after 8 h of sequencing. The average identity of the HTNV genome sequences for the nanopore sequencer compared to those of generated from Illumina MiSeq revealed 99.8% (L and M segments) and 99.7% (S segment) identities, respectively. This study highlights the potential of the portable nanopore sequencer for rapid generation of accurate genomic sequences of HTNV for quicker decision making in point-of-care testing of HFRS patients during a hantavirus outbreak.

A novel genotype of Hantaan orthohantavirus harbored by Apodemus agrarius chejuensis as a potential etiologic agent of hemorrhagic fever with renal syndrome in Republic of Korea.

BACKGROUND: Orthohantaviruses, causing hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome, pose a significant public health threat worldwide. Despite the significant mortality and morbidity, effective antiviral therapeutics for orthohantavirus infections are currently unavailable. This study aimed to investigate the prevalence of HFRS-associated orthohantaviruses and identify the etiological agent of orthohantavirus outbreaks in southern Republic of Korea (ROK). METHODOLOGY/PRINCIPAL FINDINGS: We collected small mammals on Jeju Island during 2018-2020. We detected the Hantaan virus (HTNV)-specific antibodies and RNA using an indirect immunofluorescence assay test and reverse transcription-polymerase chain reaction on Apodemus agrarius chejuensis (A. chejuensis). The prevalence of anti-HTNV antibodies among rodents was 14.1%. A total of six seropositive mouse harbored HTNV RNA. The amplicon-based next-generation sequencing provided nearly full-length tripartite genomic sequences of six HTNV harbored by A. chejuensis. Phylogenetic and tanglegram analyses were conducted for inferring evolutionary relationships between orthohantaviruses with their reservoir hosts. Phylogenetic analysis showed a novel distinct HTNV genotype. The detected HTNV genomic sequences were phylogenetically related to a viral sequence derived from HFRS patient in southern ROK. Tanglegram analysis demonstrated the segregation of HTNV genotypes corresponding to Apodemus spp. divergence. CONCLUSIONS/SIGNIFICANCE: Our results suggest that A. chejuensis-borne HTNV may be a potential etiological agent of HFRS in southern ROK. Ancestral HTNV may infect A. chejuensis prior to geological isolation between the Korean peninsula and Jeju Island, supporting the co-evolution of orthohantaviruses and rodents. This study arises awareness among physicians for HFRS outbreaks in southern ROK.

Orthohantaviruses infections in humans and rodents in Baoji, China.

In recent years, hemorrhagic fever with renal syndrome (HFRS) incidence has been becoming a severe public health problem again due to its significant increase in Shaanxi Province, China. Baoji, located in the Guanzhong Plain in the central part of Shaanxi Province, has been severely affected by HFRS since its first emergence in 1955. To better understand the epidemiology of orthohantaviruses infection in humans and the causative agents carried by the rodents, the long-term incidence patterns were analyzed and a molecular epidemiological investigation of orthohantaviruses infection in humans and rodents was performed. During 1984-2019, 13,042 HFRS cases were registered in Baoji, including 275 death cases. Except the first high prevalence of HFRS in 1988-1993, another two epidemic peaks were observed in 1998-2003 and 2012, respectively, although vaccination project was started since 1996. During the same period, HFRS cases in Baoji mainly were recorded in winter suggesting they may be caused by Hantaan orthohantavirus (HTNV), while a small peak of HFRS was also found in summer with unknown reason. Nucleotide identity and phylogenetic analyses demonstrated that a novel clade of HTNV sequences recovered from HFRS cases were closely related to those from rodents, including species close contact with humans, suggesting a direct viral transmission from rodents to humans and the important role in the HTNV transmission the nontraditional rodent hosts may play. Moreover, two distant related Dabieshan orthohantavirus (DBSV) lineages were also identified in Niviventer niviventer in this area demonstrating its considerable genetic diversity. Our data indicated that continual spillover of HTNV from rodents to humans, contributing to the high prevalence of HFRS in humans in Baoji.

Phylogeographic diversity and hybrid zone of Hantaan orthohantavirus collected in Gangwon Province, Republic of Korea.

BACKGROUND: Hantaan orthohantavirus (Hantaan virus, HTNV), harbored by Apodemus agrarius (the striped field mouse), causes hemorrhagic fever with renal syndrome (HFRS) in humans. Viral genome-based surveillance at new expansion sites to identify HFRS risks plays a critical role in tracking the infection source of orthohantavirus outbreak. In the Republic of Korea (ROK), most studies demonstrated the serological prevalence and genetic diversity of orthohantaviruses collected from HFRS patients or rodents in Gyeonggi Province. Gangwon Province is a HFRS-endemic area with a high incidence of patients and prevalence of infected rodents, ROK. However, the continued epidemiology and surveillance of orthohantavirus remain to be investigated. METHODOLOGY/PRINCIPAL FINDINGS: Whole-genome sequencing of HTNV was accomplished in small mammals collected in Gangwon Province during 2015-2018 by multiplex PCR-based next-generation sequencing. To elucidate the geographic distribution and molecular diversity of viruses, we conducted phylogenetic analyses of HTNV tripartite genomes. We inferred the hybrid zone using cline analysis to estimate the geographic contact between two different HTNV lineages in the ROK. The graph incompatibility based reassortment finder performed reassortment analysis. A total of 12 HTNV genome sequences were completely obtained from A. agrarius newly collected in Gangwon Province. The phylogenetic and cline analyses demonstrated the genetic diversity and hybrid zone of HTNV in the ROK. Genetic exchange analysis suggested the possibility of reassortments in Cheorwon-gun, a highly HFRS-endemic area. CONCLUSIONS/SIGNIFICANCE: The prevalence and distribution of HTNV in HFRS-endemic areas of Gangwon Province enhanced the phylogeographic map for orthohantavirus outbreak monitoring in ROK. This study revealed the hybrid zone reflecting the genetic diversity and evolutionary dynamics of HTNV circulating in Gangwon Province. The results arise awareness of rodent-borne orthohantavirus diseases for physicians in the endemic area of ROK.

Protective CD8+ T-cell response against Hantaan virus infection induced by immunization with designed linear multi-epitope peptides in HLA-A2.1/Kb transgenic mice.

BACKGROUND: An effective vaccine that prevents disease caused by hantaviruses is a global public health priority, but up to now, no vaccine has been approved for worldwide use. Therefore, novel vaccines with high prophylaxis efficacy are urgently needed. METHODS: Herein, we designed and synthesized Hantaan virus (HTNV) linear multi-epitope peptide consisting of HLA-A*02-restricted HTNV cytotoxic T cell (CTL) epitope and pan HLA-DR-binding epitope (PADRE), and evaluated the immunogenicity, as well as effectiveness, of multi-epitope peptides in HLA-A2.1/Kb transgenic mice with interferon (IFN)-γ enzyme-linked immunospot assay, cytotoxic mediator detection, proliferation assay and HTNV-challenge test. RESULTS: The results showed that a much higher frequency of specific IFN-γ-secreting CTLs, high levels of granzyme B production, and a strong proliferation capacity of specific CTLs were observed in splenocytes of mice immunized with multi-epitope peptide than in those of a single CTL epitope. Moreover, pre-immunization of multi-epitope peptide could reduce the levels of HTNV RNA loads in the liver, spleen and kidneys of mice, indicating that specific CTL responses induced by multi-epitope peptide could reduce HTNV RNA loads in vivo. CONCLUSIONS: This study may provide an important foundation for the development of novel peptide vaccines for HTNV prophylaxis.

Genetic diversity and evolution of Hantaan virus in China and its neighbors.

BACKGROUND: Hantaan virus (HTNV; family Hantaviridae, order Bunyavirales) causes hemorrhagic fever with renal syndrome (HFRS), which has raised serious concerns in Eurasia, especially in China, Russia, and South Korea. Previous studies reported genetic diversity and phylogenetic features of HTNV in different parts of China, but the analyses from the holistic perspective are rare. METHODOLOGY AND PRINCIPAL FINDINGS: To better understand HTNV genetic diversity and gene evolution, we analyzed all available complete sequences derived from the small (S) and medium (M) segments with bioinformatic tools. Eleven phylogenetic groups were defined and showed geographic clustering; 42 significant amino acid variant sites were found, and 19 of them were located in immune epitopes; nine recombinant events and eight reassortments with highly divergent sequences were found and analyzed. We found that sequences from Guizhou showed high genetic divergence, contributing to multiple lineages of the phylogenetic tree and also to the recombination and reassortment events. Bayesian stochastic search variable selection analysis revealed that Heilongjiang, Shaanxi, and Guizhou played important roles in HTNV evolution and migration; the virus may originate from Zhejiang Province in the eastern part of China; and the virus population size expanded from the 1980s to 1990s. CONCLUSIONS/SIGNIFICANCE: These findings revealed the original and evolutionary features of HTNV, which will help to illustrate hantavirus epidemic trends, thus aiding in disease control and prevention.

CRISPR-based platform for carbapenemases and emerging viruses detection using Cas12a (Cpf1) effector nuclease.

CRISPR-Cas12a (also called Cpf1) has been commonly used for genomic editing, based on its ability to generate precise double-stranded DNA (dsDNA) breaks. Recently, it was demonstrated that Cas12a exhibits unspecific ssDNAse activity upon target recognition. This feature allows CRISPR-Cas to be coupled with a ssDNA reporter and generate a fast, accurate and ultrasensitive molecular detection method. Here, we demonstrate that Cas12a was able to detect DNA target sequences corresponding to carbapenemases resistance genes such as KPC, NDM and OXA. Also, with the addition of a reverse-transcription step, we were able to detect viral RNA sequences from DENV, ZIKV and HANTV genomes. In all cases, assay run time was less than two hours. Additionally, we report attomolar levels of detection. This methodology was validated using clinical samples from patients infected with Dengue virus. Reactions were visualized by detection of a fluorescent signal, as well as by the use of a simple lateral flow strip. These results indicate that Cas12a is able to detect both DNA and RNA targets, making it an appropriate and convenient tool to detect all types of pathogens.

RNA-Seq Revealed a Circular RNA-microRNA-mRNA Regulatory Network in Hantaan Virus Infection.

Hantaan virus (HTNV), a Hantavirus serotype that is prevalent in Asia, causes hemorrhagic fever with renal syndrome (HFRS) with high mortality in human race. However, the pathogenesis of HTNV infection remains elusive. Circular RNAs (circRNAs), a new type of non-coding RNAs, play a crucial role in various pathogenic processes. Nevertheless, circRNA expression profiles and their effects on pathogenesis of HTNV infection are still completely unknown. In the present study, RNA sequencing was performed to analyze the circRNA, microRNA (miRNA), and mRNA expression profiles in HTNV-infected and mock-infected human umbilical vein endothelial cells (HUVECs). A total of 70 circRNAs, 66 miRNAs, and 788 mRNAs were differently expressed. Several differentially expressed RNAs were validated by RT-qPCR. Moreover, we verified that some differentially expressed RNAs, such as circ_0000479, miR-149-5p, miR-330-5p, miR-411-3p, RIG-I, CMPK2, PARP10, and GBP1, promoted or inhibited HTNV replication. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis demonstrated that the host genes of differentially expressed circRNAs were principally involved in the innate immune response, the type I interferon (IFN) signaling pathway, and the cytokine-mediated signaling pathway. Additionally, the circRNA-miRNA-mRNA regulatory network was integrally analyzed. The data showed that there were many circRNA-miRNA-mRNA interactions in HTNV infection. By dual-luciferase reporter assay, we confirmed that circ_0000479 indirectly regulated RIG-I expression by sponging miR-149-5p, hampering viral replication. This study for the first time presents a comprehensive overview of circRNAs induced by HTNV and reveals that a network of enriched circRNAs and circRNA-associated competitive endogenous RNAs (ceRNAs) is involved in the regulation of HTNV infection, thus offering new insight into the mechanisms underlying HTNV-host interaction.