Molecular epidemiology of dengue virus strains from finnish travelers.We characterized 11 dengue virus dengue virus n. A virus of the genus Flavivirus that is the cause of dengue. (DENV DENV Department of Environment (Canada) ) isolates obtained from Finnish travelers during 2000-2005 using monoclonal antibodies This is a list of monoclonal antibodies, antibodies which are clones of a single parent cell. When used as medications, the generic names end in -mab (see "Nomenclature of monoclonal antibodies"). and phylogenetic phy·lo·ge·net·ic adj. 1. Of or relating to phylogeny or phylogenetics. 2. Relating to or based on evolutionary development or history. analysis. The analysis of DENV isolated from travelers contributes to the global picture of strain distribution and circulation. The isolates included all serotypes, including a DENV-2 isolate from Ghana. ********** Dengue viruses (DENV 1-4) are mosquito-borne members of the family Flaviviridae, genus Flavivirus. Dengue dengue or breakbone fever or dandy fever Infectious, disabling mosquito-borne fever. Other symptoms include extreme joint pain and stiffness, intense pain behind the eyes, a return of fever after brief pause, and a characteristic rash. is regarded as the most significant arboviral disease in the world. Disease incidence and prevalence are rising in dengue-endemic areas, and travelers are increasingly affected. The disease can vary from asymptomatic to febrile febrile /feb·rile/ (feb´ril) pertaining to or characterized by fever. feb·rile adj. Of, relating to, or characterized by fever; feverish. disease, classic dengue fever dengue fever (dĕng`gē, –gā), acute infectious disease caused by four closely related viruses and transmitted by the bite of the Aedes mosquito; it is also known as breakbone fever and bone-crusher disease. , or complications such as dengue hemorrhagic fever hemorrhagic fever (hĕm'ərăj`ĭk), any of a group of viral diseases characterized by sudden onset, muscle and joint pain, fever, bleeding, and shock from loss of blood. or dengue shock syndrome. Several virus- and host-specific factors have been suggested to correlate with severe disease outcomes, which are mostly associated with secondary infections (1). These outcomes are not common in European travelers, and deaths are rare (2). In recent years, the Years, The the seven decades of Eleanor Pargiter’s life. [Br. Lit.: Benét, 1109] See : Time number of annually diagnosed cases has increased in Finland from an average of 10 to >20 in 2006 (Huhtamo et al., unpub, data). In the present study, samples collected during 1999-2005 were studied by virus isolation. Virus isolates were not obtained from year 1999 samples; all isolates obtained from these samples were from the years 2000-2005. The Study Patients returning from dengue-endemic areas with fever and other symptoms compatible with dengue were treated mainly at university hospitals in Finland This is a list of hospitals in Finland.
A technique that uses a fluorochrome to indicate the occurrence of a specific antigen-antibody reaction. The fluorochrome labels either an antigen or an antibody. assay (IFA Immunofluorescent assay (IFA) A blood test sometimes used to confirm ELISA results instead of using the Western blotting. In an IFA test, HIV antigen is mixed with a fluorescent compound and then with a sample of the patient's blood. ), and IgM-enzyme immunoassay Immunoassay An assay that quantifies antigen or antibody by immunochemical means. The antigen can be a relatively simple substance such as a drug, or a complex one such as a protein or a virus. (Focus Technologies, Cypress, CA, USA). For this study, serum specimens from all patients were aliquoted and stored at -70[degrees]C. From patients with dengue diagnosis, acute-phase serum specimens with IgG titers [less than or equal to] 320 (IFA) were chosen for virus isolation (n = 40). Virus isolations were done simultaneously in 2 cell lines: in Vero E6 cells (ATCC ATCC American Type Culture Collection, see there CRL-1586) grown in minimal essential medium at 37[degrees]C and 5% C[O.sub.2], and in C6/36 Aedes albopictus Noun 1. Aedes albopictus - striped native of Japan thriving in southwestern and midwestern United States and spreading to the Caribbean; potential carrier of serious diseases Asian tiger mosquito cells (ATCC CRL-1660) grown in Leibowitch L-15 medium at room temperature. Cells in 25-[cm.sup.2] flasks were incubated with 50 [micro]L of patient serum for 1 hour and observed for 24 days for cytopathic effects (CPEs). When CPEs were evident, cells were harvested for IFA, and RNA RNA: see nucleic acid. RNA in full ribonucleic acid One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic was extracted from supernatants for reverse transcriptase-PCR (RT-PCR RT-PCR reverse transcriptase-polymerase chain reaction. See PCR1. ). In the absence of CPEs, cells were subcultured after 7 days into 75-[cm.sup.2] culture flasks and studied by IFA on days 7 and 24. In IFA, the cells were stained with a DENV-positive serum and DENV-type-specific monoclonal antibodies (MAbs) (3). RNA was extracted from IFA- or CPE-positive culture supernatants with a Viral RNA Mini Kit (QIAGEN, Valencia, CA, USA) according to according to prep. 1. As stated or indicated by; on the authority of: according to historians. 2. In keeping with: according to instructions. 3. the manufacturer's instructions. RT-PCR targeting the capsid-premembrane (C-preM) region was performed using DENV-specific primers (4), Expand reverse transcriptase Reverse transcriptase Any of the deoxyribonucleic acid (DNA) polymerases present in particles of retroviruses which are able to carry out DNA synthesis using an RNA template. (Roche, Basel, Switzerland) and Taq DNA polymerase DNA polymerase /DNA po·lym·er·ase/ (pah-lim´er-as) any of various enzymes catalyzing the template-directed incorporation of deoxyribonucleotides into a DNA chain, particularly one using a DNA template. (Fermentas, Glen Burnie, MD, USA). A total of 11 DENV strains were isolated from different geographic locations, including the 4 serotypes (DENV1, n = 4; DENV-2, n = 2; DENV-3, n = 3; DENV-4, n = 2; Table). The serum samples yielding virus isolates were drawn within 1 week after onset of symptoms, which included fever, headache, muscular pain, rash, and nausea. Most of these samples were positive for antibodies to DENV (IgM positive, n = 8; IgG positive, n = 5). Isolates were either strains that grew in both of the tested cell lines (n = 6) or strains that grew only in C6/36 cells (n = 5). Two of the DENV-3 isolates (2 and 7) were detectable considerably earlier in Vero E6 than in C6/36 cells. DENV-1 isolates showed 2 distinct growth patterns; isolates 4 and 8 grew only in C6/36 cells, and isolates 3 and 11 grew in both tested cell lines (Table). All isolates were successfully serotyped with the RTPCR RTPCR Reverse Transcriptase Polymerase Chain Reaction of Lanciotti et al. (4), in agreement with results of the MAb IFA. However, isolate 3 (DENV- 1) had particular properties in type-specific MAb IFA, depending on the cell type because it showed positive results in infected C6/36 cells and negative results in infected VE6 cells. First-round RT-PCR amplicons were purified by using ExoSAP-IT (US Biochemicals, Cleveland, OH, USA), and directly sequenced. When necessary, the envelope gene was amplified using previously described primers (5) and sequenced. Nucleotide sequences of the isolates were aligned with published DENV sequences from GenBank (online Appendix Table, available from www.cdc.gov/ EID/content/14/1/80-appT.htm) using ClustalW (www.ebi. ac.uk/tools/clustalw). Phylogenetic analysis was performed by the neighbor-joining method with a Kimura 2-parameter model using MEGA3 software version 3.1 (6). Phylogenetic analyses (Figure 1) showed that isolates 3, 4, and 8 (DENV-1) clustered with Asiatic DENV-1 strains of genotype I (7), which corresponded with the patients' travel history. Isolate 11 (DENV-1) from India clustered with a genotype III strain isolated a year earlier from the Seychelles. Isolate 6 (DENV-2), obtained from Sri Lanka Sri Lanka (srē läng`kə) [Sinhalese,=resplendent land], formerly Ceylon, ancient Taprobane, officially Democratic Socialist Republic of Sri Lanka, island republic (2005 est. pop. in 2003, clustered with a strain isolated in the same year from India. Unlike the other isolates, isolate 9 (DENV-2), obtained in Ghana in 2005, did not group with any of the representative strains of the C-preM region, for which no African sequences were available in GenBank. The additionally studied envelope gene sequence grouped with previous African isolates of the cosmopolitan genotype (8) (Figure 2). The DENV-3 isolates represented genotype III (9) (Figure 1). Isolate 2 from Cuba clustered with strains from Martinique in agreement with previous data on Cuban strains (10). Isolate 7 (DENV-3), obtained in Sri Lanka in 2004, clustered with strains from Singapore, Sri Lanka, and Taiwan. Isolate 5 was identical in sequence to a strain isolated 1 year earlier from a patient in Brazil who died (11). DENV-4 isolates represented 2 different genotypes; isolate 1 from Sri Lanka clustered with genotype I strains, and isolate 10 from Indonesia clustered with genotype II (12). Conclusions Studies on imported DENV have provided interesting insights to the global picture of circulating strains (13,14), and also have led to the discovery of novel DENV strains and lineages (15,16). In this study, we characterized 11 strains of DENV isolated from Finnish travelers in 2000-2005 and provided new information about strains circulating in India, Sri Lanka, and Ghana. Previous studies have shown that DENV isolation is possible when antibody levels are low (17). However, in this study, most samples yielding virus isolates were antibody positive. The patients had primary infections, except for 1 patient, who had an IgG titer of 320 in the acute phase, which is suggestive of suggestive of Decision making adjective Referring to a pattern by LM or imaging, that the interpreter associates with a particular–usually malignant lesion. See Aunt Millie approach, Defensive medicine. a secondary infection. This was the only patient with any bleeding symptoms, i.e., prolonged bleeding from the venopuncture site. Virus isolates from Finnish travelers were heterogeneous. All patients had dengue fever, including the patient whose isolate was identical in sequence to a strain isolated from a patient who had died. Since the disease outcomes of the patients were uneventful, no associations could be made between the infective virus serotype serotype /se·ro·type/ (ser´o-tip) the type of a microorganism determined by its constituent antigens; a taxonomic subdivision based thereon. se·ro·type n. See serovar. v. or strain and disease severity. [FIGURE 1 OMITTED] Both mammalian and mosquito cells were used in virus isolation, which enabled the detection of other flaviviruses that may have caused seropositivity Seropositivity is the presence of a certain antibody in a blood sample. A patient with seropositivity for a particular antigen or agent is termed seropositive. through cross-reaction. All DENV isolates grew in C6/36 mosquito cells; however, use of 2 cell lines showed variation in the growth patterns of the isolates in different cell types. We observed that some DENV-3 strains were detectable earlier in mammalian Vero E6 cells than in C6/36 cells, which suggested a different capability to infect these cells. This property could not be associated with pathogenicity in this study; thus, the biologic relevance of this phenomenon is unknown. The DENV type-specific MAb IFA showed that one of the DENV-1 isolates (isolate 3) had distinct antigenic properties when cultured in mammalian or mosquito cells. Whether this strain represents MAb-escape properties requires further studies. The phylogenetic grouping of the isolates was consistent with the travel history of the patients in most cases. However, isolate 11 (DENV-1) from India clustered with a genotype III strain isolated a year earlier from the Seychelles, which suggested strain transfer between these countries. Phylogenetic analysis of isolate 9 (Ghana 2005) showed that it could be grouped with other African isolates of the cosmopolitan genotype (Figure 2). To our knowledge, this is the first DENV-2 strain characterized from Ghana (the geographically nearest isolate is from Burkina Faso Burkina Faso (burkē`nə fä`sō), republic (2005 est. pop. 13,925,000), 105,869 sq mi (274,200 sq km), W Africa. It borders on Mali in the west and north, on Niger in the northeast, on Benin in the southeast, and on Togo, Ghana, and in 1983). This grouping demonstrates sustained circulation of DENV-2 strains in Africa for decades. [FIGURE 2 OMITTED] The 11 DENV isolates represent a random sample from different geographic locations. Three strains were isolated from travelers returning from Sri Lanka, first in 2000 (DENV-4), followed by isolates in 2003 (DENV-2) and 2004 (DENV-3). These strains demonstrate extensive DENV serotype cocirculation. Acknowledgments We thank Ernest Gould for providing the MAbs used in this study; Jukka Lumio, Jarmo Oksi, Irma Koivula, and Karl Sammalkorpi for providing clinical patient data; and Raija Leveelahti, Johanna Myllynen, Essi Hasu, Minna Ulmanen, and Kirsti Raiha for technical assistance. This study was supported by grants from Hospital District of Helsinki and Uusimaa (TYH4211, TYH6215) and National Technology Agency (TEKES TEKES Teknologian kehittämiskeskus (Finnish Technology Agency) ). Ms Huhtamo is a PhD candidate at Helsinki Biomedical bi·o·med·i·cal adj. 1. Of or relating to biomedicine. 2. Of, relating to, or involving biological, medical, and physical sciences. Graduate School and conducts research at the Department of Virology virology, study of viruses and their role in disease. Many viruses, such as animal RNA viruses and viruses that infect bacteria, or bacteriophages, have become useful laboratory tools in genetic studies and in work on the cellular metabolic control of gene expression , Haartman Institute, University of Helsinki The University of Helsinki is not to be confused with the Helsinki University of Technology. The University of Helsinki (Finnish: Helsingin yliopisto, Swedish: Helsingfors universitet . Her research involves mosquito-borne flaviviruses. References (1.) Guzman MG, Kouri G. Dengue: an update. Lancet Infect Dis. 2002;2:33-42. (2.) Wichmann 0, Gascon Gascon inhabitant of Gascony, France; people noted for their bragging. [Fr. Hist.: NCE, 1049] See : Boastfulness J, Sehunk M, Puente S, Siikamaki H, Gjorup I, et al. European network on surveillance of imported infectious diseases. Severe dengue virus infection in travelers: risk factors and laboratory indicators. J Infect Dis. 2007; 195:108-96. (3.) Henchal EA, McCown JM, Seguin MC, Gentry MK, Brandt WE. Rapid identification of dengue virus isolates by using monoclonal antibodies in an indirect immunofluorescence assay. Am J Trop Med Hyg. 1983;32:164-9. (4.) Lanciotti RS, Calisher CH, Gubler DJ, Chang G J, Vorndam AV. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol. 1992;30:545 51. (5.) Uzcategui NY, Camacho D, Comach G, Cuello de Uzcategui R, Holmes EC, Gould EA. evolution and recombination recombination, process of "shuffling" of genes by which new combinations can be generated. In recombination through sexual reproduction, the offspring's complete set of genes differs from that of either parent, being rather a combination of genes from both parents. . J Gen Virol. 2001 ;82:2945-53. (6.) Kumar S, Tamura K, Nei M. MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform. 2004;5:150-63. (7.) A-Nuegoonpipat A, Berlioz-Arthaud A, Chow V, Endy T, Lowry K, Mai le Q, et al. Sustained transmission of dengue virus type 1 in the Pacific due to repeated introductions of different Asian strains. Virology. 2004;329:505-12. (8.) Twiddy SS, Farrar JJ, Vinh Chau N, Wills B, Gould EA, Gritsun T, et al. Phylogenetic relationships and differential selection pressures among genotypes of dengue-2 virus. Virology. 2002;298:63-72. (9.) Messer WB, Gubler D J, Harris E, Sivananthan K, de Silva AM. Emergence and global spread of a dengue serotype 3, subtype (programming) subtype - If S is a subtype of T then an expression of type S may be used anywhere that one of type T can and an implicit type conversion will be applied to convert it to type T. III virus. Emerg infect Dis. 2003;9:800-9. (10.) Miagostovich MP, dos Santos FB, Fumian TM, Guimaraes FR, da Costa EV, Tavares FN, et al. Complete genetic characterization of a Brazilian dengue virus type 3 strain isolated from a fatal outcome fatal outcome, n a consequence that results in death. The course of a disease that results in the death of the patient. . Mem Inst Oswaldo Cruz. 2006;101:307-13. (11.) Rodriguez-Roche R, Alvarez M, Holmes EC, Beruardo L, Kouri G, Gould EA, et al. Dengue virus type 3, Cuba, 2000-2002. Emerg Infect Dis. 2005; 11:773-4. (12.) Klungthong C, Zhang C, Mammen MP Jr, Ubol S, Holmes EC. The molecular epidemiology molecular epidemiology Molecular medicine An evolving field that combines the tools of standard epidemiology–case studies, questionnaires and monitoring of exposure to external factors with the tools of molecular biology–eg, restriction endonucleases, of dengue virus serotype 4 in Bangkok, Thailand. Virology. 2004;329:168-79. (13.) Teichmann D, Rogler G, Grobusch MP, Schuler-Maue W, Klein E. Imported dengue virus type 2 infection acquired during an outbreak in India. Eur J Clin Microbiol Infect Dis. 1999;18:310-2. (14.) Ito M, Yamada K, Takasaki T, Pandey B, Nerome R, Tajima S, et al. Phylogenetic analysis of dengue viruses isolated from imported dengue patients: possible aid for determining the countries where infections occurred. J Travel Med. 2007; 14:233-44. (15.) Nukui Y, Tajima S, Kotaki A, Ito M, Takasaki T, Koike K, et al. Novel dengue virus type 1 from travelers to Yap State, Micronesia. Emerg Infect Dis. 2006;12:343-6. (16.) Domingo C, Palacios G, Jabado O, Reyes N, Niedrig M, Gascon J, et al. Use of a short fragment of the C-terminal E gene for detection and characterization of two new lineages of dengue virus 1 in India. J Clin Microbiol. 2006;44:1519-29. (17.) Yamada K, Takasaki T, Nawa M, Kurane I. Virus isolation as one of the diagnostic methods for dengue virus infection. J Clin Virol. 2002;24:203 9. Eili Huhtamo, * Nathalie Y. Uzcategui, * Heli Siikamaki, ([dagger]) Auli Saarinen, * Heli Piiparinen, * Antti Vaheri, * and Olli Vapalahti * ([double dagger]) ([section]) * Haartman Institute, University of Helsinki, Finland; ([dagger]) Helsinki University Central Hospital, Finland; ([double dagger]) HUSLAB Hospital District of Helsinki and Uusimaa, Finland; and ([section]) Faculty of Veterinary Medicine veterinary medicine, diagnosis and treatment of diseases of animals. An early interest in animal diseases is found in ancient Greek writings on medicine. Veterinary medicine began to achieve the stature of a science with the organization of the first school in the , University of Helsinki, Finland Address for correspondence: Eili Huhtamo, Haartman Institute, Department of Virology (Haartmaninkatu 3), PO Box 21, 00014, University of Helsinki, Helsinki, Finland; email: eili.huhtamo@helsinki.fi
Table. Dengue virus isolates from Finnish travelers. 2000-2005 *
Patient Strain designation
Virus travel Isolate/ (GenBank accession
serotype history Year case no. no.)
DENV-1 Thailand 2002 3 F9.D1.02
(EU005250)
DENV-1 Malaysia/ 2002 4 F12.D1.02
Thailand (EU005249)
DENV-1 Thailand 2005 8 F31.D1.05
(EU005248)
DENV-1 India 2005 11 F37.D1.05
(EU005247)
DENV-2 Sri Lanka 2003 6 F18.D2.03
(EU005252)
DENV-2 Ghana 2005 9 F32.D2.05
(C-preM, E0005251;
E, EU005258)
DENV-3 Cuba 2002 2 F7.D3.02
(EU005253)
DENV-3 Brazil 2003 5 F13.D3.03
(EU005254)
DENV-3 Sri Lanka 2004 7 F24.D3.04
(EU005255)
DENV-4 Sri Lanka 2000 1 F2.D4.00
(EU005256)
DENV-4 Indonesia 2005 10 F34.D4.05
(EU005257)
IFA screening of infected
cells ([dagger])
Isolation serum
Vero E6 C6/36 antibody status Patient
Virus sex/
serotype dpi Pos dpi Pos dpo Igm IgG age, y
DENV-1 7 +++ 7 ++ 5 - <10 F/23
DENV-1 - - 24 + 7 + 20 F/43
DENV-1 - - 24 + 7 + <10 F/56
DENV-1 5 +++ 5 +++ 3 NA NA M/31
DENV-2 - - 24 + 5 + 320 M/54
DENV-2 7 +++ 7 + 2 + <10 F/22
DENV-3 7 + 24 + 6 + 20 M/55
DENV-3 - - 24 + 5 + <10 M/26
DENV-3 4 +++ 24 + 2 - 20 F/39
DENV-4 10 +++ 10 + 4 + <10 F/42
DENV-4 - - 24 + 5 + 20 M/37
* IFA, immunofluoresence assay; dpi, day postinfection; Pos, proportion
of positive cells; dpo, day post-onset; Ig, immunoglobulin; DENV,
dengue virus; NA, not available; c-preM, capsid-premembrane; E,
envelope.
([dagger]) +, ++, and +++ indicate the relative amount of positive
cells observed in IFA using fluorescence microscope: individual
positive cells observed <10% (+), 10-50% of the cells positive (++),
50%-100% of the cells positive (+++). -, not detected.
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