| 1. |
- Alm, Erik, et al.
(författare)
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Universal Single-Probe RT-PCR Assay for Diagnosis of Dengue Virus Infections
- 2014
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Ingår i: PLoS Neglected Tropical Diseases. - : Public Library of Science (PLoS). - 1935-2727 .- 1935-2735. ; 8:12, s. e3416-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Dengue is a mosquito-borne viral disease that has become more prevalent in the last few decades. Most patients are viremic when they present with symptoms, and early diagnosis of dengue is important in preventing severe clinical complications associated with this disease and also represents a key factor in differential diagnosis. Here, we designed and validated a hydrolysis-probe-based one-step real-time RT-PCR assay that targets the genomes of dengue virus serotypes 1-4. Methodology/Principal Findings: The primers and probe used in our RT-PCR assay were designed to target the 39 untranslated region of all complete genome sequences of dengue virus available in GenBank (n=3,305). Performance of the assay was evaluated using in vitro transcribed RNA, laboratory-adapted virus strains, external control panels, and clinical specimens. The linear dynamic range was found to be 10(4)-10(11) GCE/mL, and the detection limit was between 6.0x10(2) and 1.1x10(3) GCE/mL depending on target sequence. The assay did not cross-react with human RNA, nor did it produce false-positive results for other human pathogenic flaviviruses or clinically important etiological agents of febrile illnesses. We used clinical serum samples obtained from returning travelers with dengue-compatible symptomatology (n = 163) to evaluate the diagnostic relevance of our assay, and laboratory diagnosis performed by the RT-PCR assay had 100% positive agreement with diagnosis performed by NS1 antigen detection. In a retrospective evaluation including 60 archived serum samples collected from confirmed dengue cases 1-9 days after disease onset, the RT-PCR assay detected viral RNA up to 9 days after appearance of symptoms. Conclusions/Significance: The validation of the RT-PCR assay presented here indicates that this technique can be a reliable diagnostic tool, and hence we suggest that it be introduced as the method of choice during the first 5 days of dengue symptoms.
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| 2. |
- Bergqvist, Cecilia, et al.
(författare)
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Multiplex Nucleic Acid Suspension Bead Arrays for Detection and Subtyping of Filoviruses
- 2015
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 53:4, s. 1368-1370
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Tidskriftsartikel (refereegranskat)abstract
- Here we describe multiplex suspension bead array systems that allow fast and reliable detection of reverse transcriptase (RT) PCR amplified filovirus genomes and also enable subtyping of Ebola virus species and Marburg virus strains. These systems have an analytical sensitivity equivalent to that of RT-PCR.
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| 3. |
- Hinkula, Jorma, et al.
(författare)
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Immunization with DNA Plasmids Coding for Crimean-Congo Hemorrhagic Fever Virus Capsid and Envelope Proteins and/or Virus-Like Particles Induces Protection and Survival in Challenged Mice
- 2017
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Ingår i: Journal of Virology. - : AMER SOC MICROBIOLOGY. - 0022-538X .- 1098-5514. ; 91:10
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Tidskriftsartikel (refereegranskat)abstract
- Crimean-Congo hemorrhagic fever virus (CCHFV) is a bunyavirus causing severe hemorrhagic fever disease in humans, with high mortality rates. The requirement of a high-containment laboratory and the lack of an animal model hampered the study of the immune response and protection of vaccine candidates. Using the recently developed interferon alpha receptor knockout (IFNAR(- / -)) mouse model, which replicates human disease, we investigated the immunogenicity and protection of two novel CCHFV vaccine candidates: a DNA vaccine encoding a ubiquitin-linked version of CCHFV Gc, Gn, and N and one using transcriptionally competent virus-like particles (tc-VLPs). In contrast to most studies that focus on neutralizing antibodies, we measured both humoral and cellular immune responses. We demonstrated a clear and 100% efficient preventive immunity against lethal CCHFV challenge with the DNA vaccine. Interestingly, there was no correlation with the neutralizing antibody titers alone, which were higher in the tc-VLP-vaccinated mice. However, the animals with a lower neutralizing titer, but a dominant cell-mediated Th1 response and a balanced Th2 response, resisted the CCHFV challenge. Moreover, we found that in challenged mice with a Th1 response (immunized by DNA/DNA and boosted by tc-VLPs), the immune response changed to Th2 at day 9 postchallenge. In addition, we were able to identify new linear B-cell epitope regions that are highly conserved between CCHFV strains. Altogether, our results suggest that a predominantly Th1-type immune response provides the most efficient protective immunity against CCHFV challenge. However, we cannot exclude the importance of the neutralizing antibodies as the surviving immunized mice exhibited substantial amounts of them. IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is responsible for hemorrhagic diseases in humans, with a high mortality rate. There is no FDAapproved vaccine, and there are still gaps in our knowledge of the immune responses to infection. The recently developed mouse models mimic human CCHF disease and are useful to study the immunogenicity and the protection by vaccine candidates. Our study shows that mice vaccinated with a specific DNA vaccine were fully protected. Importantly, we show that neutralizing antibodies are not sufficient for protection against CCHFV challenge but that an extra Th1-specific cellular response is required. Moreover, we describe the identification of five conserved B-cell epitopes, of which only one was previously known, that could be of great importance for the development of diagnostics tools and the improvement of vaccine candidates.
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| 4. |
- Karlberg, Helen, et al.
(författare)
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Molecular and Serological Findings in Suspected Patients With Crimean-Congo Hemorrhagic Fever Virus in Iran
- 2015
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Ingår i: Journal of Medical Virology. - : Wiley: 12 months. - 0146-6615 .- 1096-9071. ; 87:4, s. 686-693
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Tidskriftsartikel (refereegranskat)abstract
- Crimean-Congo hemorrhagic fever (CCHF) is an arthropod-borne disease of humans associated with a severe clinical picture, including hemorrhagic syndrome and a high mortality rate. CCHF virus is widely distributed throughout large areas of the world. To characterize the serological status in CCHF patients, paired clinical samples were collected from suspected CCHF patients and analyzed by microbiological and other laboratory analyses with the aim of: determining the presence of neutralizing antibodies against CCHF virus; investigating the cross-reactivity of these neutralizing antibodies against virus isolated from the same outbreak and against other available laboratory strain; and studying the relationship between the isolated virus with other virus by whole genome sequencing. Patients at Boo-Ali Hospital, Zahedan, Iran, with clinical symptoms ranging from mild to severe hemorrhagic fever were included in the study. Two serum samples were taken from each patient, the first as soon as the patient matched the criteria for CCHF notification and the second when the patient was discharged from hospital (2 weeks later). Commercial and in-house assays revealed a positive IgM signal in acute serum samples from six patients. A novel finding was that CCHF patients develop neutralizing antibodies soon after infection. Interestingly these antibodies were able to neutralize other CCHF virus strains too. The complete sequence of the Zahedan 2007 isolate, including the hitherto unknown first L-segment sequence, was identified using an original clinical sample from one patient with confirmed CCHF infection.
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| 5. |
- Ke, Rongqin, et al.
(författare)
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Colorimetric Nucleic Acid Testing Assay for RNA Virus Detection Based on Circle-to-Circle Amplification of Padlock Probes
- 2011
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 49:12, s. 4279-4285
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Tidskriftsartikel (refereegranskat)abstract
- We developed a molecular diagnostic method for detection of RNA virus based on padlock probes and colorimetric readout. The feasibility of our approach was demonstrated by using detection of Crimean-Congo hemorrhagic fever (CCHF) virus as a model. Compared with conventional PCR-based methods, our approach does not require advanced equipment, involves easier assay design, and has a sensitivity of 103 viral copies/ml. By using a cocktail of padlock probes, synthetic templates representing different viral strain variants could be detected. We analyzed 34 CCHF patient samples, and all patients were correctly diagnosed when the results were compared to those of the current real-time PCR method. This is the first time that highly specific padlock probes have been applied to detection of a highly variable target sequence typical of RNA viruses.
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| 6. |
- Lindegren, Gunnel
(författare)
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Optimisation of dengue diagnostic tools in order to increase the knowledge of the pathogenesis
- 2008
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Dengue fever (DF) is the most common global viral mosquito-borne infection, with 100 million estimated cases annually in tropical and subtropical areas. The dengue viruses (genus Flavivirus, Flaviviridae) occur in 4 serotypes (DENV-1 to DENV-4). Dengue diseases range from a mild febrile disease to severe hemorrhagic fever. Infection with one serotype induces a life-long immunity, but does not elicit cross-protective antibodies to the other serotypes. Re-infection with a second serotype is associated with a more severe disease and is also a risk factor for dengue hemorrhagic fever. Each year the Swedish Institute for Infectious Disease and Control receives serum samples from several hundred Swedish travellers, with a suspected acute or past DF after travelling to dengue endemic areas. The aims of this thesis were to introduce methods suitable for the different phases of viremia and antibody development in the early and the late phases of disease, respectively and to determine the optimal methods in relation to the onset and sampling dates. In paper I, dengue IgG immunofluorescence assay (IFA) negative acute serum samples from 57 previously defined Swedish dengue patients (1997-2002), were investigated by different PCR assays and by dengue IgM ELISA. Only samples collected until day 5 post onset were found positive by PCR: in 73% (35/48) of the samples, dengue RNA of serotypes 1, 2 or 3 was detected. The number of genomes/ml varied between 103 and 108, with a gradual decline over time. Dengue-specific IgM antibodies were found in 35% (20/57) of the samples. By a combination of the PCR assays and the IgM ELISA, a dengue diagnoses could be determined in as many as 84% (48/57) of early single samples. When the analyses were consecutively performed on the samples from 2002, 100% (13/13) of the samples were positive either by PCR or by IgM ELISA. In paper II, a dengue micro-NT (m-NT) for detection and serotyping of neutralising antibodies was developed and evaluated. Early convalescent samples (<6 weeks), complemented by late convalescent samples (>5 years) from 20 patients, previously serotyped by PCR were included in the study. The correct serotype was determined in 80% (16/20) of the late convalescent samples, while the serotype could not be determined in 4 patients. One patient did not produce any neutralising antibodies, another patient had most probably had two dengue infections with equally high titres of neutralising antibodies against both. In two patients a significant difference between the serotypes could not be determined. We found no correlation between dengue IFA IgG titres and m-NT titres in samples collected 5-10 years post onset. We have demonstrated that the m-NT is a reliable diagnostic tool for detection and serotyping of neutralising antibodies in late convalescent serum samples of primary dengue cases.
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| 7. |
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| 8. |
- Waldenström, Jonas, et al.
(författare)
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Migrating birds and tickborne encephalitis virus
- 2007
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Ingår i: Emerging Infectious Diseases. - : Centers for Disease Control and Prevention (CDC). - 1080-6040 .- 1080-6059. ; 13:8, s. 1215-8
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Tidskriftsartikel (refereegranskat)abstract
- During spring and autumn 2001, we screened 13,260 migrating birds at Ottenby Bird Observatory, Sweden, and found 3.4% were infested with ticks. Four birds, each a different passerine species, carried tickborne encephalitis virus (TBEV)-infected ticks (Ixodes ricinus). Migrating birds may play a role in the geographic dispersal of TBEV-infected ticks.
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