Transcriptome profiling highlights regulated biological processes and type III interferon antiviral responses upon Crimean-Congo hemorrhagic fever virus infection

Crimean-Congo hemorrhagic fever virus(CCHFV)is a biosafety level-4(BSL-4)pathogen that causes Crimean-Congo hemorrhagic fever(CCHF)characterized by hemorrhagic manifestation,multiple organ failure and high mortality rate,posing great threat to public health.Despite the recently increasing research efforts on CCHFV,host cell responses associated with CCHFV infection remain to be further characterized.Here,to better understand the cellular response to CCHFV infection,we performed a transcriptomic analysis in human kidney HEK293 cells by high-throughput RNA sequencing(RNA-seq)technology.In total,496 differentially expressed genes(DEGs),including 361 up-regulated and 135 down-regulated genes,were identified in CCHFV-infected cells.These regulated genes were mainly involved in host processes including defense response to virus,response to stress,regulation of viral process,immune response,metabolism,stimulus,apoptosis and protein catabolic process.Therein,a significant up-regulation of type III interferon(IFN)signaling pathway as well as endoplasmic reticulum(ER)stress response was especially remarkable.Subsequently,representative DEGs from these processes were well validated by RT-qPCR,confirming the RNA-seq results and the typical regulation of IFN responses and ER stress by CCHFV.Furthermore,we demonstrate that not only type I but also type III IFNs(even at low dosages)have substantial anti-CCHFV activities.Collectively,the data may provide new and comprehensive insights into the virus-host interactions and particularly highlights the potential role of type III IFNs in restricting CCHFV,which may help inform further mechanistic delineation of the viral infection and development of anti-CCHFV strategies.

Crimean-Congo hemorrhagic fever virus in Central,Eastern,and South-eastern Asia

Crimean-Congo hemorrhagic fever(CCHF),caused by Crimean-Congo hemorrhagic fever virus(CCHFV),is endemic in Africa,Asia,and Europe,but CCHF epidemiology and epizootiology is only rudimentarily defined for most regions.Here we summarize what is known about CCHF in Central,Eastern,and South-eastern Asia.Searching multiple international and country-specific databases using a One Health approach,we defined disease risk and burden through identification of CCHF cases,anti-CCHFV antibody prevalence,and CCHFV isolation from vector ticks.We identified 2313 CCHF cases that occurred in 1944–2021 in the three examined regions.Central Asian countries reported the majority of cases(2,026).In Eastern Asia,China was the only country that reported CCHF cases(287).In South-eastern Asia,no cases were reported.Next,we leveraged our previously established classification scheme to assign countries to five CCHF evidence levels.Six countries(China,Kazakhstan,Kyrgyzstan,Tajikistan,Turkmenistan,and Uzbekistan)were assigned to level 1 or level 2 based on CCHF case reports and the maturity of the countries’surveillance systems.Two countries(Mongolia and Myanmar)were assigned to level 3 due to evidence of CCHFV circulation in the absence of reported CCHF cases.Thirteen countries in Eastern and South-eastern Asia were categorized in levels 4 and 5 based on prevalence of CCHFV vector ticks.Collectively,this paper describes the past and present status of CCHF reporting to inform international and local public-health agencies to strengthen or establish CCHFV surveillance systems and address shortcomings.

Isolation,Characterization,and Phylogenetic Analysis of Two New Crimean-Congo Hemorrhagic Fever Virus Strains from the Northern Region of Xinjiang Province,China

Crimean-Congo hemorrhagic fever(CCHF)caused by the CCHF virus(CCHFV)is a tick-borne natural focal disease with a mortality rate of approximately 50%.CCHFV is widely prevalent in Africa,southern Asia,the Middle East,and southeast Europe.CCHF outbreaks have been reported previously in Xinjiang province,China,especially in its southern region.Epidemiological surveys conducted on ticks and animals have revealed the presence of CCHFV strains in ticks,rodents,and infected individuals from cities and counties in southern Xinjiang.Phylogenetic analyses revealed that the Chinese CCHFV strains belong to one genotype,based on complete sequences of the S segments of its negative-stranded RNA genome.The present study reports two new CCHFV strains isolated from Hyalomma asiaticum asiaticum ticks collected from Fukang City and Wujiaqu City in the northern region of Xinjiang.Viral characteristics and their evolutionary relationships were analyzed through metagenomic and reverse-transcription PCR analyses;these analyses indicated that the genotype of both strains was different from that of other Chinese strains.Furthermore,previous reports of CCHFV in Xinjiang were reviewed and phylogenetic analyses were performed.CCHFV was found to prevail in Fukang City in Junggar Basin for more than 20 years,and that Fukang City and Wujiaqu City are considered natural reservoirs of different genotypes of CCHFV strains.Our findings facilitate the understanding of CCHFV distribution in Xinjiang province and provide insights into the evolutionary relationships among Chinese CCHFV strains.

Prevalence and Phylogenetic Analysis of Crimean-Congo Hemorrhagic Fever Virus in Ticks from Different Ecosystems in Xinjiang,China

The Crimean-Congo hemorrhagic fever virus(CCHFV),a member of the genus Orthonairovirus and family Nairoviridae,is transmitted by ticks and causes severe hemorrhagic disease in humans.To study the epidemiology of CCHFV in different ecosystems in Xinjiang,China,a total of 58,932 ticks were collected from Tarim Basin,Junggar Basin,Tianshan Mountain,and Altai Mountain from 2014 to 2017.Hyalomma asiaticum asiaticum was the dominant tick species in Tarim and Junggar basins,whereas Dermacentor nuttalli and Hyalomma detritum were found in Tianshan Mountain and Altai Mountain,respectively.Reverse transcription-polymerase chain reaction of the CCHFV small(S)genome segment was used for the molecular detection.The CCHFV-positive percentage was 5.26%,6.85%,1.94%,and 5.56% in Tarim Basin,Junggar Basin,Tianshan Mountain,and Altai Mountain,respectively.Sequences of the S segment were used for phylogenetic analysis and the results showed that the newly identified CCHFV strains belonged to two clades.Our study confirms that H.asiaticum asiaticum is the major vector of CCHFV in desert habitats which is consistent with previous studies,and also suggests that H.detritum and D.nuttalli are emerging vectors for CCHFV in Xinjiang.Moreover,this study reports the presence of CCHFV in the mountain habitat of Xinjiang for the first time,suggesting that future surveillance of CCHFV should also include mountainous areas.

A new strain of Crimean-Congo hemorrhagic fever virus isolated from Xinjiang, China

Crimean-Congo hemorrhagic fever virus(CCHFV) is a highly pathogenic tick-borne virus with a fatality rate of up to 50% in humans. CCHFV is widely distributed in countries around the world.Outbreaks of CCHFV infection in humans have occurred in prior years in Xinjiang Province, China.Epidemiological surveys have detected CCHFV RNA in ticks and animals; however, few isolates were identified. In this study, we identified and isolated a new CCHFV strain from Hyalomma asiaticum asiaticum ticks collected from north of Tarim Basin in Xinjiang, China. A preliminary investigation of infection and antigens expression of CCHFV was performed in newborn mice. The target tissues for CCHFV replication in newborn mice were identified. The analysis of the phylogenetic relationships with other Chinese strains suggested that diverse genotypes of CCHFV have circulated in Xinjiang for years. These findings provide important insights into our understanding of CCHFV infection and evolution as well as disease prevention and control for local residents.

Cryo-EM structure of glycoprotein C from Crimean-Congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever virus(CCHFV)is a causative agent of serious hemorrhagic diseases in humans with high mortality rates.CCHFV glycoprotein Gc plays critical roles in mediating virus-host membrane fusion and has been studied extensively as an immunogen.However,the molecular mechanisms involved in membrane fusion and Gc-specific antibody-antigen interactions remain unresolved largely because structural information of this glycoprotein is missing.We designed a trimeric protein including most of the ectodomain region of Gc from the prototype CCHFV strain,Ib Ar10200,which enabled the cryo-electron microscopy structure to be solved at a resolution of 2.8Å.The structure confirms that CCHFV Gc is a class Ⅱ fusion protein.Unexpectedly,structural comparisons with other solved Gc trimers in the postfusion conformation revealed that CCHFV Gc adopted hybrid architectural features of the fusion loops from hantaviruses and domain Ⅲ from phenuiviruses,suggesting a complex evolutionary pathway among these bunyaviruses.Antigenic sites on CCHFV Gc that protective neutralizing antibodies target were mapped onto the CCHFV Gc structure,providing valuable information that improved our understanding of potential neutralization mechanisms of various antibodies.

Hemorrhagic fever with renal syndrome virus infection in livers studied by in situ hybridization and immunohistochemistry

To clarify whether hemorrhagic fever with renal syndrome (HFRS) virus is cytopathic or not for the liver, HFRS virus RNA in liver tissue from 19 patients with HFRS and the livers of naturally infected rats with virus were examined by in situ hybridization

Tracing in vivo of the vascular and cellular membranous permeabilities in the experimentally infected suckling mice with Chen strain hemorrhagic fever with renal syndrome virus

the vascular and cellular membranous permeabilities in the experimentally infected suckling BALB/c mice with Chen strain hemorrhagic fever with renal syndrome virus (HFRSV) were studied by employing horseradish peroxidase (HRP), colloidal lanthanum and colloidal gold labelled antibodies as tracers and applying the tracers to the mice in vivo via tail veins, and the tissues were observed under light mcroscope and electron microscope. The vascular and cellular membranous permeabilities in the infected increased as the tracers appeared in the perivascular and interstitial tissues as well as in the cytoplasms of some parenchymal cells of the organs ,while the permeabilities remained normal in the control. The rasults suggest that in the infected mice, it might be the virus infection that was mainly responsible for the abnormal permeabilities.

In situ hybridization detection of persistent infection and viral RNA distribution in laboratory rats with hemorrhagic fever with renal syndrome virus

Digoxigenin-labelled Hantaan specific cDNA probes were used in the present study for in situ hybridization detection of hemorrhagic fever with renal syndrome virus(HFRSV) persistent infection and viral RNA distribution in naturally infected laboratory rat

Tracing of the viral antigens by gold-labelled antibodies in the experimentally infected suckling mice with Chen strain hemorrhagic fever with renal syndrome virus and ultrastructural observation

Our previous works were done on the abnormal permeability of vasculature and cellular membrane after hemorrhagic fever with renal syndrome virus (HFRSV) infection. In this study, the internalization of viral antigens in the experimentally infected suckling BALB/c mice with Chen strain HFRSV were further studied by the application of the colloidal gold-labelled polyclonal and monoclonal antibodies against HFRSV (GLAb) to the infected animals via tail veins. The normal mice with GLAb and the infected mice with colloidal gold-labelled indifferent antibodies (GLIg) were also employed as experimental controls. At certain time intervals after the injection,the animals were sacrificed and tissues were observed under light and electron microscopes. In the normal mice,the conjugates were confined to the vasculature and reliculo-endothelial system and localized in the lysosomes of phagocytes. In the infected animals,the GLIg conjugates could be used to demonstrate the abnormal permeability,but could not show the antigen localization,while the GLAb could be internalized into the cytoplasm of the parenchymal cells and localized in the free ribosome,Golgi apparatus,granules within vesicles,and inclusion body-like structures Compared to the infected animals with GLIg,obvious tissue structure alternations under LM were observed in the infected mice with GLAb. The ultrastructural changes of destruction and abnormal structures frequently occurred in the cells of the infected animals. The typical virion,immature virion and inclusion body could be found but only in a few cells. The results indicated that the free ribosomes,Golgi apparatus and vesices may be related to viral infection and positive viral antigen could not represent the virus structure only. It is suggested that the antibodies produced in the bodies after HFRSV infection can bind not only with the extracel lular viral antigens to form immunocomplexes to induce tissue lesions but also the cytoplasmic viral antigens of the infected cells through the damaged cellular membranes.

Study on Biologic Activity for Membrane of Normal Bone Marrow Cells with Infection of Epidemic Hemorrhagic Fever Virus

Using DPH fluorescence probe, the membrane of normal bone marrow cells with infection of epidemic hemorrhagic fever virus (EHFV) was labeled. The membrane lipid fluidity was obviously decreased from the membrane lipid fluorescence polarization. The membrane lipid fluidity of lympho- cyte, monocyte and neutrophilic granulocyte was dynamically observed. After culturing the cells for 1, 6, 24 and 72 h, it was found that all the membrane lipid fluidity of the infected cells was de- creased obviously with the longer the culturing time, the more obvious it. Compared with the normal control groups, there was a significant difference statistically (P<0. 05-0. 01). It was suggested that the decrease of the membrane lipid fluidity of normal bone marrow cell with infection of EHFV had correlation with the degree of virus invading and cellfunction injury.

Sequencing, Expression and Diagnostic Application of the Nucleoprotein Gene of Xinjiang Hemorrhagic Fever Virus

In order to analyze the nucleoprotein (NP) gene of Crimean-Congo hemorrhagic fever virus (CCHFV), viral RNA was amplified by RT-PCR by using the proof-reading DNA polymerase to produce the complete NP gene. The PCR product was sequenced, analyzed for phylogenesis and cloned into the expression vector pET32a and the recombinant plasmid expressed in E.coli BL-21 with high yield. The primarily purified fused protein was used to coat ELISA plates for the detect antibodies. It was found the similarities between NP gene of BA88166 and other XHFVs in nucleotide level and amino acid contents were very significant, and the NP gene of BA88166 encoded a nucleoprotein with 482 amino acid and a deduced molecular weight (MW) of 54?kDa. Western blot assay showed that the fusion protein expressed in bacteria possessed good antigenicity. The results with ELISA for the detection of the human and animal sera collected in endemic areas were found to be in good accordance to the clinical diagnosis. It concluded that the relations of NP genes of XHFV BA88166 and other XHFVs appeared to be evolutionally close. The methodologies established in this study were accurate, specific, rapid and reproducible for the clinical examinations and epidemiological survey.

Study on the Cytotoxic T Lymphocytes Clone Specific for the Nucleocapsid Protein of Hantaan Virus from Peripheral Blood in Patients with Hemorrhagic Fever with Renal Syndrome

In order to elucidate the molecular and immunological mechanisms as well as the pathogenesis of hemorrhagic fever with renal syndrome (HFRS), the CD8 + cytotoxic T lymphocytes (CTL) clone was established directly from peripheral blood mononuclear cells (PBMC) of patients with HFRS. The activities of CTL were detected as usual with EBV-transformed lymphoblastoid cell line (BLCL) as target cells. The results showed that the CTL clone could recognized and killed the target cells with specificity of nucleocapsid protein of Hantaan virus (HTNVNP) with the cytotoxicity percentages of 50.2%, 25.4% and 39.0% respectively. These results demonstrated that the antigenic epitopes of HTNVNP mainly located on the C-terminal of the viral nucleocapsid protein.

Crimean-Congo hemorrhagic fever from the immunopathogenesis, clinical, diagnostic, and therapeutic perspective: A scoping review

Crimean-Congo hemorrhagic fever virus(CCHFV) is responsible for widespread tick-borne zoonotic viral disease CCHF in African, Middle Eastern, Asian, and European countries. CCHFV can be spread to humans through tick bites or contact with infected animals or humans, and it often progresses from asymptomatic to severe/lethal illness, with fatality rates ranging from 10% to 40% in humans. Today, CCHF is growing into a significant public health concern due to its very high prevalence, severity of the condition, and lack of available vaccines and specific treatments. Recent research has been drawn towards a more accurate study of CCHFV characteristics, including the structure, genetic diversity, mechanisms involved in pathogenesis and immunopathogenesis, and clinical features. In addition, the use of animal models(mouse and non-human primates) and advanced diagnostic tools in recent years has resulted in a significant advance in CCHF related studies. In this context, we summarized the latest findings about CCHF research, its health complications, animal models, current diagnosis, vaccination, and CCHF treatments, and therapeutic strategies. Furthermore, we discussed existing deficiencies and problems in CCHFV analysis, as well as areas that still need to yield conclusive answers.

Epidemiology and Mutational Analysis of Global Strains of Crimean-Congo Haemorrhagic Fever Virus

Crimean-Congo hemorrhagic fever (CCHF) is a severe illness with high fatality.Cases are reported in several countries in Africa,Europe,the Middle East,and Asia.Phylogenetic analyses based on the virus S (nucleocapsid),M (glycoprotein),and L (polymerase) genome segments sequences indicate distinct geographic lineages exist but their specific genetic characteristics require elucidation.In this work we collected all full length S segment sequences and generated a phylogenetic tree based on the alignment of these 62 samples.We then analyzed the alignment using entries from AAIndex,the Amino Acid Index database,to identify amino acid mutations that performed significant changes in charge,pka,hydropathy and side chain volume.Finally,we mapped these changes back to the tree and alignment to identify correlated mutations or sites that characterized a specific lineage.Based on this analysis we are able to propose a number of sites that appear to be important for virus function and which would be good candidates for experimental mutational analysis studies.

CLINICAL SIGNIFICANCE OF DETECTING SERUM ANTIBODIES TO NUCLEOPROTEIN AND GLYCOPROTEIN G_2 OF HANTAAN VIRUS IN PATIENTS WITH HEMORRHAGIC FEVER WITH RENAL SYNDROME

Antibody blocking enzyme linked immunosorbent assays, respectively detecting antibodies to Hantaan virus nucleoprotein (NPAb) and glycoprotein GZ (G,Ab), were developed using monoclonal antibody L133, L13r3, LV48A and LVZB28B NPAb and GZAb in 291 serum samples from 65 patientswith kemorrkagic fever with renal syudrome (HFRS) were detfrmlned by these methods. The positive rates or NPAb were 90N on day 2-3 and 100 % on day 8-9 arter onset of disease, respectively.NPAb titers Increased during fever period and reached Peak levels during kypotensive and oliguric periods of HFRS. It was suggested that NPAb might be an important component Involved in the immunopathogenlc lin'alrmeut of HFRS and the detection of NPAb might be useful for the early diagnosis or HFRS. The I,osltlve rates and titers of GZAh were very low during the rirst three periods,namely rever, hypoteuslve and ollgurlc periods, and reached high levels during the convalescent period. GRAb titers were negatively related to the I,rotelnurla levels during the course of HFRS. It wasIndicated that GZAb might be the main component or neutralizing autlhodles to Hantaan virus Infection and the efrlclent production or GZAb was a good marker ror predicting the recovery and betterprognosis of HFRS.

Crimean-Congo Hemorrhagic Fever: An Overview

Crimean-Congo hemorrhagic fever (CCHF) is a zoonotic viral infection that is a serious threat to humans. The disease is widely distributed in Africa, Asia, and Europe and has developed into a serious public health concern. Humans become infected through the bites of ticks, by contact with a patient with CCHF, or by contact with blood or tissues from viremic livestock. Microvascular instability and impaired hemostasis are the hallmarks of the infection. Infection in human begins with nonspecific febrile symptoms, but may progress to a serious hemorrhagic syndrome with high mortality rates. Enzyme-linked immunoassay (ELISA) and polymerase chain reaction (PCR) are the most used and specific tests for the diagnosis. The mainstay of treatment is supportive. Although definitive studies are not available, ribavirin is suggested to be effective especially at the earlier phase of the infection. Uses of universal protective measures are the best way to avoid the infection. In this review, all aspects of CCHF are overviewed in light of the current literature.

Knowledge, attitude and practice of healthcare workers concerning Crimean-Congo hemorrhagic fever in Western Iran

Objective: To determine the knowledge, attitude and practice of healthcare workers in Kermanshah Province about Crimean-Congo hemorrhagic fever(CCHF).Methods: This study was conducted in 2014 on healthcare personnel in different job categories including physicians, nurses, midwives, laboratory staff and network health staff of Kermanshah Province by direct interview.Results: A total of 367 respondents who had more than 5 years of experience in their jobs were interviewed. Among them 91% of physicians and nurses, 97% of midwives and health workers and 96% of laboratory staff stated that they had not been confronted with CCHF patients so far. Regarding knowledge, 76% of physicians, 78% of nurses, 77% of midwives and 58% of laboratory staff believed that the disease is remediable. Most of the interviewed participants stated that the disease pertains to people who are in close contact with domestic animals, but they did not consider their own occupations as one of the risk factors. More than 70% of the respondents believed that the disease may exist in the province or their work field. Generally, the knowledge about CCHF was inadequate, with nurses having the lowest level of knowledge.Conclusions: Knowledge of Kermanshah healthcare staff about CCHF was poor,especially nurses in a high risk job category. Therefore, it is necessary to conduct specific training programs for the disease identification, transmission, prevention, and treatment as well as the use of personal protection and safety devices.

Significance of IL-1Ra and IL-6 gene variants in Turkish patients with Crimean-Congo hemorrhagic fever

Objective: To investigate the association between IL-1 Ra variable number of tandem repeat(rs2234663), IL-6-597 GA(rs1800797), IL-6-572 GC(rs1800796) and the risk of CrimeanCongo hemorrhagic fever(CCHF) in the Turkish patients. Methods: This study included 50 patients infected with CCHF and 50 healthy controls. These variants were genotyped using polymerase chain reaction and/or restriction fragment length polymorphism method. Results: The distribution of the IL-6-572 GC genotypes and alleles varied significantly between the patients and the controls. The subjects carrying IL-6-572 GC GG genotype and G allele had increased risk of developing CCHF compared to the control group(P=0.006, P=0.014, respectively). IL-6-572 GC GC genotype was higher in the controls than the patients(P=0.006). For the triple genotype combinations, the 1/2-GC-GG genotype combination was detected more frequently in the control group than CCHF patients(P=0.016). IL-6(-572/-597) GG-GG genotype was significantly higher in the patient group(P=0.015), while the GC-GG genotype was significantly lower in the patient group(P=0.005). Additionally, the G-G haplotype was significantly higher in the patient group(P=0.042), whereas C-G was found to be significantly lower in the patients than the control group(P=0.037). Conclusions: The results of this study suggest the IL-6-572 GC variant might be genetic markers of sensitivity to CCHF in the Turkish population and may facilitate greater protection against the disease.

Hemorrhagic Fever with Renal Syndrome Associated with Acute Pancreatitis

Hemorrhagic fever with renal syndrome (HFRS) is a systemic infectious disease caused by Hantaviruses and characterized by fevers, bleeding tendencies, gastrointestinal symptoms and renal failure. It encompasses a broad spectrum of clinical presentations, ranging from unapparent or mild illnesses to fulminant hemorrhagic processes. Among the various complications of HFRS, acute pancreatitis is a rare find. In this report, based on clinical data, laboratory and radiologic examination findings, we describe a clinical case, with HFRS from Dobrava virus, associated with acute pancreatitis. The patient was successfully treated by supportive management. Clinicians should be alert to the possibility of HFRS when examining patients with epidemiological data and symptoms of acute pancreatitis.