| 1. |
- Evander, Mikael, et al.
(författare)
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Acoustic trapping of cells in a microfluidic format
- 2005
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Ingår i: Proceedings of µTAS 2005 Conference. ; 1, s. 515-517
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Konferensbidrag (refereegranskat)abstract
- This paper presents, for the first time, non-contact acoustic trapping of cells in a microfluidic format. The employed acoustic force maintains the cells in the center of a fluidic channel while allowing for perfusion of e.g. nutrients or drugs as well as optical monitoring of the cells. Neural stem cells have been acoustically trapped and tested for viability after 15 minutes of ultrasonic radiation. It is also shown that it is possible to grow yeast cells suspended in an acoustic standing wave while perfusing with cell media.
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| 2. |
- Evander, Magnus, et al.
(författare)
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Milder winters in northern Scandinavia may contribute to larger outbreaks of haemorrhagic fever virus
- 2009
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Ingår i: Global Health Action. - : Informa UK Limited. - 1654-9716 .- 1654-9880. ; 2
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Tidskriftsartikel (refereegranskat)abstract
- The spread of zoonotic infectious diseases may increase due to climate factors such as temperature, humidity and precipitation. This is also true for hantaviruses, which are globally spread haemorrhagic fever viruses carried by rodents. Hantaviruses are frequently transmitted to humans all over the world and regarded as emerging viral diseases. Climate variations affect the rodent reservoir populations and rodent population peaks coincide with increased number of human cases of hantavirus infections. In northern Sweden, a form of haemorrhagic fever called nephropathia epidemica (NE), caused by the Puumala hantavirus (PUUV) is endemic and during 2006-2007 an unexpected, sudden and large outbreak of NE occurred in this region. The incidence was 313 cases/100,000 inhabitants in the most endemic areas, and from January through March 2007 the outbreak had a dramatic and sudden start with 474 cases in the endemic region alone. The PUUV rodent reservoir is bank voles and immediately before and during the peak of disease outbreak the affected regions experienced extreme climate conditions with a record-breaking warm winter, registering temperatures 6-9 degrees C above normal. No protective snow cover was present before the outbreak and more bank voles than normal came in contact with humans inside or in close to human dwellings. These extreme climate conditions most probably affected the rodent reservoir and are important factors for the severity of the outbreak.
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| 3. |
- Evander, Mikael, et al.
(författare)
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Noninvasive acoustic cell trapping in a microfluidic perfusion system for online bioassays
- 2007
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 79:7, s. 2984-2991
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Tidskriftsartikel (refereegranskat)abstract
- Techniques for manipulating, separating, and trapping particles and cells are highly desired in today's bioanalytical and biomedical field. The microfluidic chip-based acoustic noncontact trapping method earlier developed within the group now provides a flexible platform for performing cell- and particle-based assays in continuous flow microsystems. An acoustic standing wave is generated in etched glass channels (600x61 microm2) by miniature ultrasonic transducers (550x550x200 microm3). Particles or cells passing the transducer will be retained and levitated in the center of the channel without any contact with the channel walls. The maximum trapping force was calculated to be 430+/-135 pN by measuring the drag force exerted on a single particle levitated in the standing wave. The temperature increase in the channel was characterized by fluorescence measurements using rhodamine B, and levels of moderate temperature increase were noted. Neural stem cells were acoustically trapped and shown to be viable after 15 min. Further evidence of the mild cell handling conditions was demonstrated as yeast cells were successfully cultured for 6 h in the acoustic trap while being perfused by the cell medium at a flowrate of 1 microL/min. The acoustic microchip method facilitates trapping of single cells as well as larger cell clusters. The noncontact mode of cell handling is especially important when studies on nonadherent cells are performed, e.g., stem cells, yeast cells, or blood cells, as mechanical stress and surface interaction are minimized. The demonstrated acoustic trapping of cells and particles enables cell- or particle-based bioassays to be performed in a continuous flow format.
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| 4. |
- Evander, Magnus, et al.
(författare)
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Puumala hantavirus viremia diagnosed by real-time reverse transcriptase PCR using samples from patients with hemorrhagic fever and renal syndrome
- 2007
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Ingår i: Journal of Clinical Microbiology. - : American Society for Microbiology. - 0095-1137 .- 1098-660X. ; 45:8, s. 2491-2497
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Tidskriftsartikel (refereegranskat)abstract
- Puumala virus (PUUV) is the endemic hantavirus in northern Sweden and causes nephropathia epidemica (NE), a milder form of hemorrhagic fever with renal syndrome. There is a need for fast and reliable diagnostics to differentiate the disease from other infections. By aligning virus RNA sequences isolated from 11 different bank voles and one human patient, we designed a real-time reverse transcriptase (RT) PCR method for detection of PUUV RNA. The real-time RT-PCR assay showed linearity from 20 to 2 x 10(6) virus copies with a correlation coefficient above 0.98 to 0.99 for all experiments. The detection threshold for PUUV cDNA was two copies per reaction. A two-step qualitative RT-PCR to detect PUUV RNA showed 100% concordance with the real-time RT-PCR assay. PUUV RNA viremia was detected in 33 of 34 PUUV immunoglobulin M (IgM)-positive patients with typical clinical NE disease from the region of endemicity. One PUUV IgM-negative sample had PUUV RNA, and 4 days later, the patient was IgM positive. Of samples with indeterminate IgM, 43% were PUUV RNA positive. The kinetics of antibody titers and PUUV viremia were studied, and five of six NE patients displayed a decrease in PUUV viremia a few days after disease outbreak coupled with an increase in PUUV IgM and IgG. In one patient with continuously high PUUV RNA levels but low IgM and no IgG response, the infection was lethal. These findings demonstrated that real-time RT-PCR is a useful method for diagnosis of PUUV viremia and for detecting PUUV RNA at early time points, before the appearance of IgM antibodies.
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| 5. |
- Hardestam, Jonas, et al.
(författare)
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Antiviral effect of human saliva against hantavirus.
- 2008
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Ingår i: Journal of medical virology. - : Wiley. - 1096-9071 .- 0146-6615. ; 80:12, s. 2122-6
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Tidskriftsartikel (refereegranskat)abstract
- Hemorrhagic fever with renal syndrome (HFRS) and Hantavirus pulmonary syndrome are zoonotic diseases caused by rodent borne hantaviruses. Transmission to humans occurs usually by inhalation of aerozolized virus-contaminated rodent excreta. Although human-to-human transmission of Andes hantavirus has been observed, the mode of transmission is currently not known. Saliva from Puumala hantavirus (PUUV)-infected patients was shown recently to contain viral RNA. To test if human saliva interferes with hantavirus replication, the effect of saliva and salivary proteins on hantavirus replication was studied. It was observed that saliva from healthy individuals reduced Hantaan hantavirus (HTNV) infectivity, although not completely. Furthermore, HTNV was resistant against the antiviral capacity of histatin 5, lysozyme, lactoferrin, and SLPI, but was inhibited by mucin. Inoculation of bank voles (Myodes glareolus) with HFRS-patient saliva, positive for PUUV-RNA, did not induce sero-conversion. In conclusion, no evidence of infectious virus in patient saliva was found. However, the in vitro experiments showed that HTNV, the prototype hantavirus, is insensitive to several antiviral salivary proteins, and is partly resistant to the antiviral effect of saliva. It therefore remains to be shown if human saliva might contain infectious virions early during infection, that is, before seroconversion.
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| 6. |
- Mistry, Nitesh, et al.
(författare)
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Cutaneous and mucosal human papillomaviruses differ in net surface charge, potential impact on tropism.
- 2008
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Ingår i: Virology Journal. - 1743-422X. ; 5, s. 118-
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Tidskriftsartikel (refereegranskat)abstract
- Papillomaviruses can roughly be divided into two tropism groups, those infecting the skin, including the genus beta PVs, and those infecting the mucosa, predominantly genus alpha PVs. The L1 capsid protein determines the phylogenetic separation between beta types and alpha types and the L1 protein is most probably responsible for the first interaction with the cell surface. Virus entry is a known determinant for tissue tropism and to study if interactions of the viral capsid with the cell surface could affect HPV tropism, the net surface charge of the HPV L1 capsid proteins was analyzed and HPV-16 (alpha) and HPV-5 (beta) with a mucosal and cutaneous tropism respectively were used to study heparin inhibition of uptake. The negatively charged L1 proteins were all found among HPVs with cutaneous tropism from the beta- and gamma-PV genus, while all alpha HPVs were positively charged at pH 7.4. The linear sequence of the HPV-5 L1 capsid protein had a predicted isoelectric point (pI) of 6.59 and a charge of -2.74 at pH 7.4, while HPV-16 had a pI of 7.95 with a charge of +2.98, suggesting no interaction between HPV-5 and the highly negative charged heparin. Furthermore, 3D-modelling indicated that HPV-5 L1 exposed more negatively charged amino acids than HPV-16. Uptake of HPV-5 (beta) and HPV-16 (alpha) was studied in vitro by using a pseudovirus (PsV) assay. Uptake of HPV-5 PsV was not inhibited by heparin in C33A cells and only minor inhibition was detected in HaCaT cells. HPV-16 PsV uptake was significantly more inhibited by heparin in both cells and completely blocked in C33A cells.
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| 7. |
- Mistry, Nitesh, 1978-
(författare)
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Human papillomavirus tropism : determinants of viral tissue specificity
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cervical cancer is the second most common cancer among women worldwide and human papillomavirus (HPV) is a prerequisit for the development of this cancer. HPV belongs to the Papillomaviridae family and infects the basal layer of epithelial cells where it generally progresses into warts or condylomas. HPV can only reproduce in differentiating epithelia and it is therefore difficult to study the natural infection of HPV. More than 100 HPV types exist and they are divided into different genera based on their L1 open reading frame sequence. Most of the HPV types in the alpha-papillomavirus genus infect the mucosal epithelium while HPVs from the beta-papillomavirus genus usually infect cutaneous epithelial cells. Presently, it is not known what decides the anatomical tropism and our aim was to study determinants of this tropism. By using HPV virus like particles (VLP) and pseudovirus we found that VLPs from the two alpha-papillomaviruses HPV-6 and HPV-16 interacted with cell-surface heparan sulfate (HS) for initial attachment. When we labelled HPV VLPs with a fluorescent dye to study internalization HPV-6 was more strongly inhibited than HPV-16. Furthermore, a pseudovirus infection assay demonstrated that the beta-papillomavirus HPV-5 was less dependent on HS for infection than HPV-16. By analyzing the isoelectric point (p1) of the HPV L1 capsid protein we found that alpha HPV types were more positively charged than beta HPV types. Also, HPV-6 had a higher positive charge than HPV-16. Thus, the inhibition of the negatively charged heparin against HPV infection was clearly related to the charge of the HPV L1 capsid. This suggested that the initial interaction could be one of the determinants of tropism although not the sole factor. Lactoferrin is a protein found in milk, saliva, semen, tear fluid and endocervical secretions that has antiviral activities. Both human and bovine lactoferrin inhibited HPV infection but we found no significant differences in inhibition of alpha- and beta-papillomavirus infection. We could however demonstrate that different lactoferricins, small peptide derivates from the N-terminal part of lactoferrin, were able to inhibit HPV infection. This antiviral activity depended on lactoferricin peptide, HPV type and cell origin. The regulation of HPV gene expression in the host cell could also determine HPV tropism. The HPV long control region (LCR) contains cis-responsive elements that regulate HPV transcription and the epithelial tropism of HPV is determined by epithelial specific constitutive enhancers in the LCR. It has been hypothesized that the combination of transcription factors in the host cell determines the cell-type-specific expression. In cells with a skin origin the HPV-5 LCR was twice as efficient in transcriptional activation compared to HPV-16 LCR, while in cervical cells the HPV-16 LCR was almost twice as effective in activating transcription compared to HPV-5 LCR. To conclude, alpha- and beta-papillomaviruses differed regarding their ability to infect cells and regulate viral gene expression. These abilities corresponded with their natural host cells and suggested that HPV anatomical tropism could be determined at several steps in the HPV life cycle.
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| 8. |
- Mistry, Nitesh, et al.
(författare)
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The anti-papillomavirus activity of human and bovine lactoferricin.
- 2007
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Ingår i: Antiviral Research. - : Elsevier BV. - 0166-3542 .- 1872-9096. ; 75:3, s. 258-265
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Tidskriftsartikel (refereegranskat)abstract
- Human papillomavirus (HPV) cause common warts, laryngeal papilloma and genital condylomata and is necessary for the development of cervical cancer. We have previously found that lactoferrin has antiviral activity against HPV-16 and others have demonstrated that lactoferricin, an N-terminal fragment of lactoferrin, has inhibitory activities against several viruses. Two cell lines and two virus types, HPV-5 and HPV-16, were used to study if lactoferrin and lactoferricin could inhibit HPV pseudovirus (PsV) infection. We demonstrated that bovine lactoferrin (bLf) and human lactoferrin (hLf) were both potent inhibitors of HPV-5 and -16 PsV infections. Among the four lactoferricin derivatives we analyzed, a 15 amino acid peptide from bovine lactoferricin (bLfcin) 17-31 was the most potent inhibitor of both HPV-5 and HPV-16 PsV infection. Among the other derivatives, the human lactoferricin (hLfcin) 1-49 showed some antiviral activity against HPV PsV infection while bLfcin 17-42 inhibited only HPV-5 PsV infection in one of the cell lines. When we studied initial attachment of HPV-16, only bLfcin 17-42 and hLfcin 1-49 had an antiviral effect. This is the first time that lactoferricin was demonstrated to have an inhibitory effect on HPV infection and the antiviral activity differed depending on size, charge and structures of the lactoferricin.
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| 9. |
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| 10. |
- Näslund, Jonas, 1979-, et al.
(författare)
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Kinetics of Rift Valley fever virus in experimentally infected mice using quantitative real-time RT-PCR
- 2008
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 151:2, s. 277-282
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Tidskriftsartikel (refereegranskat)abstract
- Rift Valley Fever (RVF) is an important viral zoonosis in Africa affecting animals and humans. Since no protective vaccines or effective treatments are available for human use, accurate and reliable diagnostic methods are essential for surveillance of the disease in order to implement adequate public health actions. To study the kinetics of the RVF Virus (RVFV) infection, a SYBR Green-based quantitative real-time RT-PCR assay was developed. By using primers targeting the S-segment of RVFV, the detection limit of this assay was estimated to 30 RNA templates. Blood and organs of experimentally infected mice were sampled at different time points and RVFV RNA was quantified. High amounts of RVFV RNA were found in blood, brain, and liver samples shortly after infection with a 1-4 days post infection window for viral RNA detection. Mice developed symptoms after the appearance of serum antibodies, indicating that the host response plays an important role in the outcome of the disease. The RVFV quantitative RT-PCR proved to be a valuable diagnostic tool during the first days of infection, before detectable antibody levels and visual symptoms of RVF were observed.
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| 11. |
- Näslund, Jonas, 1979-, et al.
(författare)
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Vaccination with virus-like particles protects mice from lethal infection of Rift Valley fever virus
- 2009
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Ingår i: Virology. - : Elsevier BV. - 0042-6822 .- 1096-0341. ; 385:2, s. 409-415
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Tidskriftsartikel (refereegranskat)abstract
- Rift Valley Fever virus (RVFV) regularly accounts for severe and often lethal outbreaks among livestock and humans in Africa. Safe and effective veterinarian and human vaccines are highly needed. We present evidence that administration of RVF virus-like particles (VLPs) induces protective immunity in mice. In an accompanying paper, (Habjan, M., Penski, N., Wagner, V., Spiegel, M., Overby, A.K., Kochs, G., Huiskonen, J., Weber, F., 2009. Efficient production of Rift Valley fever virus-like particles: the antiviral protein MxA can inhibit primary transcription of Bunyaviruses. Virology 385, 400-408) we report the production of these VLPs in mammalian cells. After three subsequent immunizations with 1x10(6) VLPs/dose, high titers of virus-neutralizing antibodies were detected; 11 out of 12 mice were protected from challenge and only 1 out of 12 mice survived infection in the control groups. VLP vaccination efficiently suppressed replication of the challenge virus, whereas in the control animals high RNA levels and increasing antibody titers against the nucleocapsid protein indicated extensive viral replication. Our study demonstrates that the RVF VLPs are highly immunogenic and confer protection against RVFV infection in mice. In the test groups, the vaccinated mice did not exhibit any side effects, and the lack of anti-nucleocapsid protein antibodies serologically distinguished vaccinated animals from experimentally infected animals.
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| 12. |
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| 13. |
- Pettersson, Lisa, et al.
(författare)
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Outbreak of Puumala virus infection, Sweden.
- 2008
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Ingår i: Emerging infectious diseases. - 1080-6059. ; 14:5, s. 808-10
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Tidskriftsartikel (refereegranskat)abstract
- An unexpected and large outbreak of Puumala virus infection in Sweden resulted in 313 nephropathia epidemica patients/100,000 persons in Västerbotten County during 2007. An increase in the rodent population, milder weather, and less snow cover probably contributed to the outbreak.
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| 14. |
- Somberg, Monika, et al.
(författare)
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Polypyrimidine tract binding protein induces human papillomavirus type 16 late gene expression by interfering with splicing inhibitory elements at the major late 5' splice site, SD3632
- 2008
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Ingår i: Journal of Virology. - 0022-538X .- 1098-5514. ; 82:7, s. 3665-3678
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Tidskriftsartikel (refereegranskat)abstract
- We have initiated a screen for cellular factors that can induce human papillomavirus type 16 (HPV-16) late gene expression in human cancer cells. We report that the overexpression of polypyrimidine tract binding protein (PTB), also known as heterologous nuclear ribonucleoprotein I (hnRNP I), induces HPV-16 late gene expression in cells transfected with subgenomic HPV-16 plasmids or with full-length HPV-16 genomes and in persistently HPV-16-infected cells. In contrast, other hnRNPs such as hnRNP B1/A2, hnRNP F, and hnRNP Q do not induce HPV-16 late gene expression. PTB activates SD3632, the only 5' splice site on the HPV-16 genome that is used exclusively by late mRNAs. PTB interferes with splicing inhibitory sequences located immediately upstream and downstream of SD3632, thereby activating late gene expression. One AU-rich PTB-responsive element was mapped to a 198-nucleotide sequence located downstream of SD3632. The deletion of this element induced HPV-16 late gene expression in the absence of PTB. Our results suggest that the overexpression of PTB interferes with cellular factors that interact with the inhibitory sequences. One may speculate that an increase in PTB levels or a reduction in the concentration of a PTB antagonist is required for the activation of HPV-16 late gene expression during the viral life cycle.
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