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.

Lipofuscin in brains of patients and mice with epidemic hemorrhagic fever

We have previously shown that the lipofuscin in the brain seems to have in-creased in amount in autopsy cases of epidemic hemorrhagic fever.The purpose of thisstudy was to testify if there is really such an increase.Lipfuscin in 10 sections from everybrain of 10 autopsy cases,stained with Sudan Ⅳ,Sudan black and H.E.,was carefully es-timated and found to be greatly increased as compared with the controls of the same agewithout brain disease.Animal experiment was also conducted on 15 sucking BALB/c miceby I.P.inoculation of 100 LD50（0.05ml）of strain Chen of hemorrhagic fever virus,andon 15 mice without inoculation as controls.No lipofuscin was detected in the controls.However,in the brains of experimental mice,lipofuscin was found to be markedly in-creased,especially in the necrotic cells.The findings suggest that the over-productionand deposition of lipofuscin may be a mild change caused by the virus and its related fac-tors,which might be enhanced by hypotension and shock.

Abnormalities of Cellular Immunoregulation in Patients with Epidemic Hemorrhagic Fever

In this report,a comparative study is made of the function test of spontaneousT suppressor cell（STs）and T Lymphocyte subsets in patients with epidemic hemorrha-gic fever（EHF）.It was found that in the early stages of the disease the activity of STs wasmarkedly lower than that of the controls,while the percentage of CD?+ cells increasedsignificantly,which led to the decrease and reciprocation of CD4/CD8 ratio,and that theactivity of STs was reversely related to the proportion of CD8+ cells on linear regressionanalysis,indicating that the CD8+ cells increased may mainly belong to cytotoxic T cells.It was also shown that the changes of STs function and CD4/CD8 ratio were related tothe abnormalities of serum C3 content and circulating immune complex.The results sug-gest that the disturbance of host cellular immunoregulation may play an important rolein the pathogenesis of EHF.

Pathological changes and viral antigen distribution in the lymphatic tissue of Patients with epidemic hemorrhagic fever

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Epidemic Hemorrhagic Fever in Hubei Frovince,The Peoples Repulic of China: A Clinical and Serological Study

Between July 1975 and April 1980, 71 patients were admitted to the 2ndAttached Hospital of Hubei Provincial Medical College in Wuchang withthe diagnosis of Epidemic Hemorrhagic Fever (EHF). The clinical courseamong these patients was similar to that described for patients with KoreanHemorrhagic Fever, Nephropathica Epidemica of Scandanavia and Hemor-rhagic Fever with Renal Syndrome of the U.S.S.R. The overall mortalitywas 11.2％. Sera obtaincd from some of these patients as well as frompatients admitted to the First Attached Hospital of Hubei Provincial Me-dical College were tested against an antigen associated with Korean He-morrhagic Fever and showed exceedingly high antibody titers. We concludethat EHF in Centfal China represents the same disease process as KoreanHemorrhagic Fever.

A Universal Real-Time Fluorescence qPCR Method for Identifying Epidemic Strains of African Swine Fever Virus

Objective Establishing a highly sensitive real-time fluorescence quantitative PCR (qPCR) method for universal testing of epidemic African swine fever virus (ASFV) strains. Methods The ASFV p72 gene was targeted to design primer probes covering 24 p72 genotypes. The optimal amount of dimethylsulphoxide (DMSO) for qPCR amplification was determined, Various sensitivity and limit of detection (LOD) tests were performed, and clinical samples from China and imported goods were tested. Results The optimal primer-probe combination could specifically detect ASFV, 1.5% DMSO was optimal for qPCR, and LOD reached 3.2 copies/μL with good reproducibility (n = 20, p = 0.369). The method was employed to test 142 clinically suspected samples, of which 30 pig blood and 37 pig tissue samples were ASFV-positive. Moreover, the positive testing rate for ASFV was higher than for the standard qPCR method recommended by the Office International Des Epizooties (OIE), and for the commercially available kit. Thus, our method is superior for testing weakly positive samples with low virus titre, and epidemic strains present in imported goods. Conclusion Our method could be employed for universal testing of epidemic ASFV strains worldwide, ensuring wider coverage of hosts and ASFV strains/endemic strains, reducing false negatives, and benefitting early diagnosis.

Frequent Import and Multiple Sources of Dengue Fever have Changed the Epidemic Situation of the Disease in Fujian Province,China

Objective The aim of this study was to update the epidemic situation of dengue fever(DF) and provide new insights for the consideration of disease control in Fujian province,China.Methods Details about DF cases in Fujian reported during 2004–2017 were collected and analyzed.The envelope(E) genes of isolates of dengue virus(DENV) were sequenced for phylogenetic analysis.Results The number of imported DF cases had increased dramatically since 2013,and the source regions expanded from Southeast Asia to South Asia,America,Oceania,and Africa,as well as the surrounding provinces.This resulted in local outbreaks and indigenous cases of DF that occurred more frequently,with 10 of 13 local outbreaks and 85.9%(1,252/1,458) of indigenous cases reported in2013–2017.Compared with only two coastal cities before 2013,four coastal and one inland city in 2013–2017 experienced the local DF outbreaks.The phylogenetic analysis of E genes confirmed that the import of DENV,not only from abroad but also from the surrounding provinces,played an important role in dissemination and local outbreaks of DF in Fujian.Conclusions The frequent import of DF cases from not only abroad but also the surrounding provinces resulted in increased incidence,frequent local outbreaks,and expansion of distribution in Fujian in recent years.There is a need for urgent measures to improve disease control in this province.

Epidemic Characteristics and Spatio-Temporal Patterns of HFRS in Qingdao City,China,2010-2022

Objective This study investigated the epidemic characteristics and spatio-temporal dynamics of hemorrhagic fever with renal syndrome(HFRS)in Qingdao City,China.Methods Information was collected on HFRS cases in Qingdao City from 2010 to 2022.Descriptive epidemiologic,seasonal decomposition,spatial autocorrelation,and spatio-temporal cluster analyses were performed.Results A total of 2,220 patients with HFRS were reported over the study period,with an average annual incidence of 1.89/100,000 and a case fatality rate of 2.52%.The male:female ratio was 2.8:1.75.3%of patients were aged between 16 and 60 years old,75.3%of patients were farmers,and 11.6%had both“three red”and“three pain”symptoms.The HFRS epidemic showed two-peak seasonality:the primary fall-winter peak and the minor spring peak.The HFRS epidemic presented highly spatially heterogeneous,street/township-level hot spots that were mostly distributed in Huangdao,Pingdu,and Jiaozhou.The spatio-temporal cluster analysis revealed three cluster areas in Qingdao City that were located in the south of Huangdao District during the fall-winter peak.Conclusion The distribution of HFRS in Qingdao exhibited periodic,seasonal,and regional characteristics,with high spatial clustering heterogeneity.The typical symptoms of“three red”and“three pain”in patients with HFRS were not obvious.

The Lassa Virus Epidemic in Mango, Togo: Therapeutic Route of the First 4 Cases

Introduction: Lassa viral hemorrhagic fever is caused by the Lassa virus. The aim of our study is to describe the therapeutic itinerary of the 4 cases of Lassa virus hemorrhagic of February-March 2017 epidemic that occurred in Mango. Methodology: Our study is a transverse retro-prospective and descriptive study from February, 1st to March, 31st 2017 that dealt with 4 confirmed Lassa fever cases declared positive on the PCR basis;hospitalized or deceased at the hospital “Esperance” of Mango;support center of Lassa viral hemorrhagic fever. Results: we reported 4 clinical observations of Lassa viral hemorrhagic fever diagnosed on the PCR basis during the Lassa fever epidemic. Patients came from Benin (2 cases) or from Burkina-Faso (1 case) and were 25, 60, 52 years old and a premature baby of 13 days. The reasons for admission were external hemorrhage, a pultated tonsillitis and abdominal pains. Fever was observed for all the cases. Complications were marked by hemorrhages and shocks. Only two patients benefitted from Antiviral therapy with Ribavirin and were declared healed. The other two patients did not benefit from the treatment due to diagnostic and therapeutic delays. Lethality was 75% (3 cases) with a highly secured burial. Effective management of contacts was established. Conclusion: Diagnostic and therapeutic delays of patients are responsible of the Dark Prognosis of Lassa fever during the epidemic.

Prognosis and Evolution of Lassa Virus Infected Persons during the 2017 Epidemic in Togo

Introduction: Lassa viral hemorrhagic fever is common in West Africa. Almost 300,000 persons are affected each year with 5000 deaths. The mice of the genus mastomys is the wild tank. Objective: The aim of our study is to describe clinic, therapeutic and evolution of the affected patients during February-March 2017 epidemic that occurred in the north of Togo. Methodology: Our study is a record review study from patients’ record, who were hospitalized from February, 1st to March, 31st 2017 at Mango hospital. Lassa diagnosis was performed by PCR. They patients have received Ribavirin and blood transfusion when necessary. Results: We have reported 5 clinical observations of Lassa viral hemorrhagic fever. Patients came from Benin (03 cases), from Burkina-Faso (1 case), from Togo (1 case) and were 25, 34, 60, 52 years old and a premature baby of 13 days. External hemorrhage and abdominal pains were the main symptoms. Fever was observed for all the cases. Complications were marked by hemorrhages and shocks. Only 3 patients had benefitted of Antiviral therapy with Ribavirin. The other 2 patients did not benefit from the treatment because the diagnosis of Lassa fever was done the day they dead before the treatment started. Lethality was 80% (4 cases) with a highly secured burial. Effective management of contacts was done. Conclusion: Diagnostic and therapeutic delays of patients are responsible of the bad prognosis of the disease.

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.

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.

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.

STUDIES ON THE ENVIRONMENT STRUCTURE OF NATURAL NIDI AND EPIDEMIC AREAS OF HEMORRHAGIC FEVER WITH RENAL SYNDROME IN CHINA

The environment structure of natural nidi and epidemic areas of hemorrhagic fever with renal syndrome (HFRS) possesses characteristics of topography, hydrology, climate, soil, vegetation, and animals. The natural nidi and epidemic areas of HFRS are distributed mainly in plain and hilly regions under 500 meters above the sea level; in plentiful-water zones and transitional zones; in temperate zone and subtropical zone of the eastern monsoon region; in the abundant-aluminum soil region and the sial soil region; in the eastern China damp forest region (agricultural districts and northeast forest districts); in the eastern Asia dampness-prefering animal geographic region. Apodemus agrarius and Rattus norvegicus are the thriving animal population, predominant species or common species as well as the main reservoir hosts and sources of infection in the natural nidi and epidemic areas of HFRS.

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.

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.

Application of Conglutinin-ELISA for the detection of Circulating Immune Complexes in Sera of Patients with Epidemic Hemorrhagic Fever.

Conglutinin was extracted and purified from bovine’s sera and was used in ELISA for the detection of circulating immune complexes in the sera of patients suffering from epidemic hemorrhagic fever (EHF). The detected rates of circu-

Trans Complementation of Replication-defective Omsk Hemorrhagic Fever Virus for Antiviral Study

Omsk hemorrhagic fever virus(OHFV) is a tick-borne flavivirus classified as a biosafety level-4(BSL4) pathogen. Studies of OHFV are restricted to be conducted within BSL4 laboratories. Currently, no commercial vaccines or antiviral drugs are available against OHFV infection. In this study, we recovered a replication-deficient OHFV with an NS1 deletion(OHFVDNS1) and reporter virus replacing NS1 with the Gaussia luciferase(Gluc)(OHFV-ΔNS1-Gluc). Both the defective OHFVDNS1 and OHFV-ΔNS1-Gluc virus could only replicate efficiently in the BHK21 cell line expressing NS1(BHK21NS1) but not in na?ve BHK21 cells. The Gluc reporter gene of OHFV-ΔNS1-Gluc virus was maintained stably after serial passaging of BHK21NS1 cells and was used to surrogate the replication of OHFV. Using NITD008, OHFV-ΔNS1-Gluc virus was validated for antiviral screening, and high-throughput screening parameters were optimized in a 96-well plate format with a calculated Z0 value above 0.5. The OHFV-ΔNS1-Gluc reporter virus is a powerful tool for antiviral screening as well as viral replication and pathogenesis studies in BSL2 laboratories.

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