Rapid bedside inactivation of Ebola virus for safe nucleic acid tests

Rapid bedside inactivation of Ebola virus would be a solution for the safety of medical and technical staff, risk containment, sample transport and high-throughput or rapid diagnostic testing during an outbreak. We show that the commercially available MagNA Pure lysis/binding buffer used for nucleic acid extraction inactivates Ebola virus. A rapid bedside inactivation method for nucleic acid tests is obtained by simply adding MagNA Pure lysis/binding buffer directly into vacuum blood collection EDTA-tubes using a thin needle and syringe prior to sampling. The ready-to-use inactivation vacuum tubes are stable for more than 4 months and Ebola virus RNA is preserved in the MagNA Pure lysis/binding buffer for at least 5 weeks independent of the storage temperature. We also show that Ebola virus RNA can be manually extracted from MagNA Pure lysis/binding buffer-inactivated samples using the QIAamp Viral RNA mini kit. We present an easy and convenient method for bedside inactivation using available blood collection vacuum tubes and reagents. We propose to use this simple method for fast, safe and easy bedside inactivation of Ebola virus for safe transport and routine nucleic acid detection

Migrating Birds and Tickborne Encephalitis Virus

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

Quantitative analysis of particles, genomes and infectious particles in supernatants of haemorrhagic fever virus cell cultures

Information on the replication of viral haemorrhagic fever viruses is not readily available and has never been analysed in a comparative approach. Here, we compared the cell culture growth characteristics of haemorrhagic fever viruses (HFV), of the Arenaviridae, Filoviridae, Bunyaviridae, and Flavivridae virus families by performing quantitative analysis of cell culture supernatants by (i) electron microscopy for the quantification of virus particles, (ii) quantitative real time PCR for the quantification of genomes, and (iii) determination of focus forming units by coating fluorescent antibodies to infected cell monolayers for the quantification of virus infectivity

Optimisation of dengue diagnostic tools in order to increase the knowledge of the pathogenesis

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

Optimized Diagnosis of Acute Dengue Fever in Swedish Travelers by a Combination of Reverse Transcription-PCR and Immunoglobulin M Detection

The dengue viruses (genus Flavivirus, family Flaviviridae) are mosquito borne and cause 100 million cases of dengue fever each year in most tropical and subtropical areas of the world. Increased global travel has been accompanied by an increased import not only of dengue but also of severe fevers of unknown origin to Sweden. Fifty-seven Swedish travelers to dengue epidemic areas, with clinical and serologically diagnosed dengue fever, were included in this study. To find fast and reliable methods to diagnose dengue in the early phase of the disease, patient acute-phase sera were investigated for the presence of dengue-specific immunoglobulin M (IgM) antibodies by enzyme-linked immunosorbent assay (ELISA) and also for dengue serotype (DEN-1 to DEN-4)-specific RNA by different PCR assays. The results showed that 15/20 (75%) of the samples collected 5 days or later post onset of disease, but only 5/37 (14%) of the samples collected on days 0 to 4, contained dengue-specific IgM. Of the samples collected on days 0 to 4 post onset, dengue RNAs of subtypes 1, 2, and 3 were detected by multiplex and/or by TaqMan PCR in 29/37 (78%); of these PCR-positive samples, 93% (27/29) were found IgM negative. By a combination of IgM ELISA and PCR assays, 84% (48/57) of the acute-phase samples were found to be positive. Our results demonstrated that detection of dengue viral RNA by reverse transcription-PCR and Taq-Man PCR is an excellent tool for the early diagnoses of dengue fever and that the IgM assay is a reliable complement for samples collected from day 5 post onset

Additional file 2: of One-step real-time RT-PCR assays for serotyping dengue virus in clinical samples

Overview of the theoretical specificity towards non-targeted serotypes. The sequences of the primers and probe in the (A) DENV1, (B) DENV2, (C) DENV3, and (D) DENV4 RT-PCR plotted against their respective non-targeted serotypes. Numbers above the primers and probe indicate genomic positions. (PDF 87 kb

Universal Single-Probe RT-PCR Assay for Diagnosis of Dengue Virus Infections

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