Localization of Dengue Virus in Naturally Infected Human Tissues, by Immunohistochemistry and In Situ Hybridization
15 Apr 2004-The Journal of Infectious Diseases (Oxford University Press)-Vol. 189, Iss: 8, pp 1411-1418
TL;DR: Tissue specimens from patients with serologically or virologically confirmed dengue infections are studied by immunohistochemistry (IHC) and in situ hybridization (ISH), to localize viral antigen and RNA, respectively.
Abstract: Dengue viral antigens have been demonstrated in several types of naturally infected human tissues, but little is known of whether these same tissues have detectable viral RNA. We studied tissue specimens from patients with serologically or virologically confirmed dengue infections by immunohistochemistry (IHC) and in situ hybridization (ISH), to localize viral antigen and RNA, respectively. IHC was performed on specimens obtained from 5 autopsies and 24 biopsies and on 20 blood-clot samples. For ISH, antisense riboprobes to the dengue E gene were applied to tissue specimens in which IHC was positive. Viral antigens were demonstrated in Kupffer and sinusoidal endothelial cells of the liver; macrophages, multinucleated cells, and reactive lymphoid cells in the spleen; macrophages and vascular endothelium in the lung; kidney tubules; and monocytes and lymphocytes in blood-clot samples. Positive-strand viral RNA was detected in the same IHC-positive cells found in the spleen and blood-clot samples. The strong, positive ISH signal in these cells indicated a high copy number of viral RNA, suggesting replication.
Citations
More filters
TL;DR: A global strategy aimed at increasing the capacity for surveillance and outbreak response, changing behaviours and reducing the disease burden using integrated vector management in conjunction with early and accurate diagnosis has been advocated.
Abstract: Dengue fever and dengue haemorrhagic fever are important arthropod-borne viral diseases. Each year, there are ∼50 million dengue infections and ∼500,000 individuals are hospitalized with dengue haemorrhagic fever, mainly in Southeast Asia, the Pacific and the Americas. Illness is produced by any of the four dengue virus serotypes. A global strategy aimed at increasing the capacity for surveillance and outbreak response, changing behaviours and reducing the disease burden using integrated vector management in conjunction with early and accurate diagnosis has been advocated. Antiviral drugs and vaccines that are currently under development could also make an important contribution to dengue control in the future.
1,732 citations
TL;DR: From the Oxford University Clinical Research Unit and Wellcome Trust Major Overseas Programme, Ho Chi Minh City, Vietnam, and the Centre for Tropical Medicine, University of Oxford, Churchill Hospital, Oxford, United Kingdom.
Abstract: From the Oxford University Clinical Research Unit and Wellcome Trust Major Overseas Programme (C.P.S., J.J.F., B.W.), Hospital for Tropical Diseases (N.V.C.), Ho Chi Minh City, Vietnam; and the Centre for Tropical Medicine, University of Oxford, Churchill Hospital, Oxford, United Kingdom (C.P.S., J.J.F., B.W.). Address reprint requests to Dr. Farrar at the Hospital for Tropical Diseases, Oxford University Clinical Research Unit, 190 Ben Ham Tu, Quan 5, Ho Chi Minh City, Vietnam, or at jfarrar@oucru.org.
1,174 citations
TL;DR: A personalized approach to the study of pathogenesis will elucidate the basis of individual risk for development of DHF and DSS as well as identify the genetic and environmental bases for differences in risk forDevelopment of severe disease.
Abstract: Summary: Much remains to be learned about the pathogenesis of the different manifestations of dengue virus (DENV) infections in humans. They may range from subclinical infection to dengue fever, dengue hemorrhagic fever (DHF), and eventually dengue shock syndrome (DSS). As both cell tropism and tissue tropism of DENV are considered major determinants in the pathogenesis of dengue, there is a critical need for adequate tropism assays, animal models, and human autopsy data. More than 50 years of research on dengue has resulted in a host of literature, which strongly suggests that the pathogenesis of DHF and DSS involves viral virulence factors and detrimental host responses, collectively resulting in abnormal hemostasis and increased vascular permeability. Differential targeting of specific vascular beds is likely to trigger the localized vascular hyperpermeability underlying DSS. A personalized approach to the study of pathogenesis will elucidate the basis of individual risk for development of DHF and DSS as well as identify the genetic and environmental bases for differences in risk for development of severe disease.
816 citations
Cites background from "Localization of Dengue Virus in Nat..."
...In contrast, there is evidence for the presence of DENV antigen in the pulmonary vascular endothelium (101)....
[...]
...A review of the literature describing findings on autopsy samples from a total of 160 fatal cases, mostly children or young adolescents (4 to 18 years old) who died within 36 h of developing shock, revealed, in order of frequency, the presence of DENV in cells in the skin (104), liver (13, 14, 53, 54d, 69, 96, 101, 104, 137, 164, 173, 199, 208), spleen (13, 14, 101, 164, 199, 208), lymph node (13, 14, 101, 104, 173, 199, 208), kidney (14, 78, 101), bone marrow (13, 78, 101, 173), lung (13, 78, 101, 137, 164, 173), thymus (106), and brain (164)....
[...]
...RNA in these cells would more likely be explained by a mechanism of pinocytosis (101)....
[...]
TL;DR: An increased understanding of the adaptive immune response to natural d Dengue virus infection and candidate dengue vaccines has helped to define the specific antibody and T cell responses that are associated with either protective or pathological immunity during dengued infection.
Abstract: Dengue is a mosquito-borne viral disease of expanding geographical range and incidence. The existence of four viral serotypes and the association of prior dengue virus infection with an increased risk for more severe disease have presented significant obstacles to vaccine development. An increased understanding of the adaptive immune response to natural dengue virus infection and candidate dengue vaccines has helped to define the specific antibody and T cell responses that are associated with either protective or pathological immunity during dengue infection. Further characterization of immunological correlates of disease outcome and the validation of these findings in vaccine trials will be invaluable for developing effective dengue vaccines.
650 citations
TL;DR: The unique immunological concerns in dengue virus vaccine development are discussed and the current prospects for the development of an acceptable vaccine are discussed, with a goal that is likely to be reached in the near future.
Abstract: The number of cases of severe dengue disease continues to grow in endemic areas of southeast Asia, Central and South America, and other subtropical regions. Children bear the greatest burden of disease, and the development of an effective vaccine remains a global public health priority. A tetravalent vaccine is urgently needed and must be effective against all four dengue virus serotypes, be cost-effective and provide long-term protection. In this Review we discuss the unique immunological concerns in dengue virus vaccine development and the current prospects for the development of an acceptable vaccine, a goal that is likely to be reached in the near future.
604 citations
References
More filters
TL;DR: In this paper, a procedure for extracting plasmid DNA from bacterial cells is described, which is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day, yet yields DNA which is pure enough to be digestible by restriction enzymes.
Abstract: A procedure for extracting plasmid DNA from bacterial cells is described. The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.
13,805 citations
01 Jan 1979
TL;DR: The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes, and achievesequate pH control without using a pH meter.
Abstract: Aprocedure forextracting plasmid DNAfrombacterial cellsis described. Themethodissimpleenough topermit theanalysis bygel electrophoresis of100ormoreclonesperdayyetyieldsplasmid DNAwhichis pureenough tobedigestible byrestriction enzymes.Theprinciple ofthe methodisselective alkaline denaturation ofhighmolecular weightchromosomal DNAwhilecovalently closedcircular DNAremains double-stranded. Adequate pHcontrol isaccomplished without usinga pHmeter.Uponneutralization, chromosomal DNArenatures toformaninsoluble clot,leaving plasmid DNAin thesupernatant. Largeandsmallplasmid DNAshavebeenextracted bythis method.
13,233 citations
"Localization of Dengue Virus in Nat..." refers methods in this paper
...Plasmids with inserts in 2 orientations were isolated by use of a modified alkaline lysis method [24]....
[...]
TL;DR: This work has identified a severe syndrome, dengue hemorrhagic fever/dengue shock syndrome, in Southeast Asian children, which recently has also been identified in children infected with the virus in Puerto Rico.
Abstract: Dengue viruses occur as four antigenically related but distinct serotypes transmitted to humans by Aedes aegypti mosquitoes. These viruses generally cause a benign syndrome, dengue fever, in the American and African tropics, and a severe syndrome, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), in Southeast Asian children. This severe syndrome, which recently has also been identified in children infected with the virus in Puerto Rico, is characterized by increased vascular permeability and abnormal hemostasis. It occurs in infants less than 1 year of age born to dengue-immune mothers and in children 1 year and older who are immune to one serotype of dengue virus and are experiencing infection with a second serotype. Dengue viruses replicate in cells of mononuclear phagocyte lineage, and subneutralizing concentrations of dengue antibody enhance dengue virus infection in these cells. This antibody-dependent enhancement of infection regulates dengue disease in human beings, although disease severity may also be controlled genetically, possibly by permitting and restricting the growth of virus in monocytes. Monoclonal antibodies show heterogeneous distribution of antigenic epitopes on dengue viruses. These epitopes serve to regulate disease: when antibodies to shared antigens partially neutralize heterotypic virus, infection and disease are dampened; enhancing antibodies alone result in heightened disease response. Further knowledge of the structure of dengue genomes should permit rapid advances in understanding the pathogenetic mechanisms of dengue.
1,607 citations
"Localization of Dengue Virus in Nat..." refers background in this paper
...Infection with dengue virus causes either a relatively mild disease, known as classic dengue fever (DF), or a more severe form, dengue hemorrhagic fever (DHF), a fulminating illness that is characterized by hemorrhagic manifestations and plasma leakage and that can progress to dengue shock syndrome (DSS) and death [4]....
[...]
TL;DR: In vitro antibody-dependent infection of PBL provides a possible model for study of pathogenetic mechanisms in infants with dengue shock syndrome who passively acquire maternal anti-dengue IgG.
Abstract: Cultured mononuclear peripheral blood leukocytes (PBL) from nonimmune human beings and monkeys are nonpermissive to dengue 2 virus (D2V) infection at multiplicities of infection of 0.001-0.1, but become permissive when non-neutralizing dengue antibody is added to medium. D2V infection occurred in PBL prepared from anti-coagulated but not from defibrinated plasma. Infection enhancement was produced by multiple lots of heterotypic anti-dengue raised in several mammalian species. Homotypic anti-dengue neutralized D2V at high concentrations but enhanced at low concentrations; enhancement end point in one serum was 1:320,000. The infection-enhancing factor was a noncytophilic antibody of the IgG class. D2V infection occurred in the absence of heat-labile complement components but did not occur when complexes were prepared with anti- dengue F(ab)(2). Treatment of PBL with several proteases increased permissiveness to D2V infection by immune complexes but not by virus alone. Two rhesus monkey serums collected 14 days after D2V infection contained an IgG antibody with high-titered enhancing activity but with no hemagglutination-inhibition or neutralizing activity. Virus-antibody complexes are irreversibly attached to PBL within 15 min and completely internalized in 60 min. There was considerable variation in cellular infection in different experiments, however, maximum virus yields usually exceeded 1,000 plaque-forming units per 1 x 10(6) PBL occurring between 2 and 4 days in culture. In vitro antibody-dependent infection of PBL provides a possible model for study of pathogenetic mechanisms in infants with dengue shock syndrome who passively acquire maternal anti-dengue IgG.
760 citations
TL;DR: High serum DEN-2 antibody dependent enhancing activity is a significant (relative risk = 6.2) risk factor for severe illness among children in a dengue hemorrhagic fever endemic region.
Abstract: Serum specimens collected during a prospective study of dengue infections among schoolchildren in Bangkok were tested for their ability to enhance dengue 2 (DEN-2) virus growth in human monocytes in vitro. Two groups of dengue-immune sera were compared: 32 dengue antibody positive serum specimens from children who subsequently developed asymptomatic secondary dengue infections; and 9 dengue antibody positive serum specimens from children who subsequently developed severe symptomatic secondary dengue infections, 8 of which were clinically diagnosed as dengue hemorrhagic fever. Antibody-dependent enhancement of virus growth was quantitated by measurement of virus yields in supernatant fluids of normal human monocyte cultures that were infected with DEN-2 virus in the presence of undiluted test serum. Only 4 of 32 (12%) preinfection sera from asymptomatic children, but 6 of 9 (67%) preinfection sera from symptomatic children, had significant enhancing activity (P < 0.001). High serum DEN-2 antibody dependent enhancing activity is a significant (relative risk = 6.2) risk factor for severe illness among children in a dengue hemorrhagic fever endemic region. Dengue antibodies can be neutralizing and therefore protective, or they can be enhancing and increase the risk of dengue hemorrhagic fever.
507 citations