| 1. |
- Borgmästars, Emmy, et al.
(författare)
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Improved Detection of Norovirus and Hepatitis A Virus in Surface Water by Applying Pre-PCR Processing
- 2017
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Ingår i: Food and Environmental Virology. - : Springer Science and Business Media LLC. - 1867-0334 .- 1867-0342. ; 9:4, s. 395-405
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Tidskriftsartikel (refereegranskat)abstract
- Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) detection of waterborne RNA viruses generally requires concentration of large water volumes due to low virus levels. A common approach is to use dead-end ultrafiltration followed by precipitation with polyethylene glycol. However, this procedure often leads to the co-concentration of PCR inhibitors that impairs the limit of detection and causes false-negative results. Here, we applied the concept of pre-PCR processing to optimize RT-qPCR detection of norovirus genogroup I (GI), genogroup II (GII), and hepatitis A virus (HAV) in challenging water matrices. The RT-qPCR assay was improved by screening for an inhibitor-tolerant master mix and modifying the primers with twisted intercalating nucleic acid molecules. Additionally, a modified protocol based on chaotropic lysis buffer and magnetic silica bead nucleic acid extraction was developed for complex water matrices. A validation of the modified extraction protocol on surface and drinking waters was performed. At least a 26-fold improvement was seen in the most complex surface water studied. The modified protocol resulted in average recoveries of 33, 13, 8, and 4% for mengovirus, norovirus GI, GII, and HAV, respectively. The modified protocol also improved the limit of detection for norovirus GI and HAV. RT-qPCR inhibition with Cq shifts of 1.6, 2.8, and 3.5 for norovirus GI, GII, and HAV, respectively, obtained for the standard nucleic acid extraction were completely eliminated by the modified protocol. The standard nucleic acid extraction method worked well on drinking water with no RT-qPCR inhibition observed and average recoveries of 80, 124, 89, and 32% for mengovirus, norovirus GI, GII, and HAV, respectively.
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| 2. |
- 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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| 3. |
- 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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| 4. |
- Mousavi-Jazi, Mehrdad, et al.
(författare)
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Healthy individuals immune response to the Bulgarian Crimean-Congo hemorrhagic fever virus vaccine
- 2012
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Ingår i: Vaccine. - : Elsevier. - 0264-410X .- 1873-2518. ; 30:44, s. 6225-6229
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Tidskriftsartikel (refereegranskat)abstract
- Crimean-Congo hemorrhagic fever virus (CCHFV) poses a great threat to public health due to its high mortality and transmission rate and wide geographical distribution. There is currently no specific antiviral therapy for CCHF. This study provides the first in-depth analysis of the cellular and humoral immune response in healthy individuals following injection of inactivated Bulgarian vaccine, the only CCHFV vaccine available at present. Vaccinated individuals developed robust, anti-CCHFV-specific T-cell activity as measured by IFN-gamma ELISpot assay. The frequency of IFN-gamma secreting T-cells was 10-fold higher in individuals after vaccination with four doses than after one single dose. High levels of CCHFV antibodies were observed following the first dose, but repeated doses were required to achieve antibodies with neutralizing activity against CCHFV. However, the neutralizing activity in these groups was low.
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| 5. |
- Mousavi-Jazi, Mehrdad
(författare)
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Human cytomegalovirus : development of resistance to antiviral drugs and mechanisms of NK-cell evasion
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Human cytomegalovirus (HCMV), a member of the herpesviridae, is ubiquitous and causes persistent infections mostly without any clinical symptoms. However, HCMV infection is a major cause of morbidity and mortality among immunocompromised patients, especially transplant recipients and patients with AIDS. Antiviral agents currently licensed for the treatment of HCMV infections include ganciclovir, foscarnet, and cidofovir. Clinical management of HCMV infections in severely immunocompromised patients often requires prolonged antiviral treatment, which is associated with the risk of development of resistanct virus. HCMV isolates resistant to antiviral agents have been selected in the laboratory and have also been recovered from immunocompromised patients treated with antiviral drugs. Studies performed in the last 5 years have contributed significantly to the current understanding of the mechanisms of resistance of HCMV to antiviral drugs. We could identify isolates from AIDS patients, that had alterations within the viral phosphotransferase (pUL97) and/or DNA polymerase (pUL54) and which conferred drug resistance. However, we found no evidence that short term antiviral treatment of HCMV disease in renal transplant recipients induced resistance. By in vitro mutagenesis and induction of HCMV mutants in cell culture, it was determined that mutations in UL54 and UL97 genes induced reduced susceptibility to PFA and/or GCV. The mutated Towne strains had the same growth phenotype as the original HCMW Towne. Clinical HCMV isolates were shown to replicate either rapidly or slowly. Slow or fast replication was neither related to major immediate early gene exon 4, gB genotypes, nor to antiviral susceptibility. The NK cells are of importance for the innate immune response to viral infections and may play a key role in the control of HCMV infections. In humans, low NK cell cytotoxicity is associated with increased sensitivity to herpesviruses infections, including CMV. The mechanism by which NK cells may recognize and eliminate virus-infected cells is still unclear. HCMV has evolved strategies to protect infected cells from lysis caused by NK cells, a feature that may have a role in the pathogenesis of HCMV disease. We found that the clinical isolates confer a strong NK resistance, whereas only marginal or variable effects in recognition were found when laboratory strains were used. The NK cell inhibition did not correlate with HLA class I expression and was independent of the leukocyte Ig-like receptor- I (LIR-1). The upregulation of HLA-E mediated by CMV pUL40 and IFN-gamma is one possible mechanism to avoid NK cell recognition of HCMV infected cells.
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| 6. |
- Mousavi-Jazi, Mehrdad, et al.
(författare)
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Sequence analysis of UL54 and UL97 genes and evaluation of antiviral susceptibility of human cytomegalovirus isolates obtained from kidney allograft recipients before and after treatment
- 2001
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Ingår i: Transplant Infectious Disease. - : Wiley. - 1398-2273 .- 1399-3062. ; 3:4, s. 195-202
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Tidskriftsartikel (refereegranskat)abstract
- The frequency of infections caused by drug-resistant cytomegalovirus (CMV) in solid-organ transplant recipients is not known. Only a few resistant strains have been described in transplant recipients. Antiviral susceptibility to ganciclovir (GCV) and foscarnet (PFA) of CMV isolates from 24 renal transplant patients with CMV viremia and CMV disease before and after therapy were investigated by a solid phase ELISA. The CMV DNA polymerase (UL54) and viral phosphotransferase (UL97) genes were also sequenced. Ten patients did not receive antiviral treatment; five and nine patients were treated with PFA and GCV, respectively. No appearance of drug-resistant viruses was observed in the present study, but one isolate showed a reduced sensitivity to PFA after treatment with GCV. This finding could not be explained by the presence or development of mutations that have been associated with drug resistance in UL54. We found no evidence that short-term treatment of CMV with PFA- or GCV-induced resistance.
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| 7. |
- Papa, Anna, et al.
(författare)
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Crimean-Congo Hemorrhagic Fever Virus, Greece
- 2014
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Ingår i: Emerging Infectious Diseases. - : U.S. National Center for Infectious Diseases. - 1080-6040 .- 1080-6059. ; 20:2, s. 288-290
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Tidskriftsartikel (refereegranskat)abstract
- Seroprevalence of Crimean-Congo hemorrhagic fever virus (CCHFV) is high in some regions of Greece, but only 1 case of disease has been reported. We used 4 methods to test 118 serum samples that were positive for CCHFV IgG by commercial ELISA and confirmed the positive results. A nonpathogenic or low-pathogenicity strain may be circulating.
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