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1.	Gillman, Anna, et al. (författare) Oseltamivir-Resistant Influenza A (H1N1) Virus Strain with an H274Y Mutation in Neuraminidase Persists without Drug Pressure in Infected Mallards 2015 Ingår i: Applied and Environmental Microbiology. - : American Society for Microbiology. - 0099-2240 .- 1098-5336. ; 81:7, s. 2378-2383 Tidskriftsartikel (refereegranskat)abstract Influenza A virus (IAV) has its natural reservoir in wild waterfowl and emerging human IAVs often contain gene segments from avian viruses. The active drug metabolite of oseltamivir (oseltamivir carboxylate (OC)), stockpiled as Tamiflu® for influenza pandemic preparedness, is not removed by conventional sewage treatment and has been detected in river water. There, it may there exert evolutionary pressure on avian IAV in waterfowl, resulting in development of resistant viral variants. A resistant avian IAV can circulate among wild birds only if resistance does not restrict viral fitness and if the resistant virus can persist without continuous drug pressure. In this in vivo Mallard (Anas platyrhynchos) study we tested if an OC-resistant avian IAV strain (A(H1N1)/NA-H274Y) could retain resistance while drug pressure was gradually removed. Successively infected Mallards were exposed to decreasing levels of OC, and fecal samples were analyzed for neuraminidase sequence and phenotypic resistance. No reversion to wild-type virus was observed during the experiment, which included 17 days of viral transmission in 10 ducks exposed to OC concentrations below resistance induction levels. We conclude that resistance in avian IAV, induced by OC exposure of the natural host, can persist in absence of the drug. Thus, there is a risk that human pathogenic IAVs that evolve from IAVs circulating among wild birds may contain resistance mutations. An oseltamivir resistant pandemic IAV would be a substantial public health threat. Therefore, our observations underscore the need for prudent oseltamivir use, upgraded sewage treatment and resistance surveillance of IAV in wild birds.
2.	Bacchus, Philip, et al. (författare) Civil-military collaboration to facilitate rapid deployment of a mobile laboratory in early response to covid-19 : A high-readiness exercise 2021 Ingår i: HEALTH SECURITY. - : Mary Ann Liebert. - 2326-5094 .- 2326-5108. ; 19:5, s. 488-497 Tidskriftsartikel (refereegranskat)abstract Rapid and adaptable diagnostic capabilities are of great importance in the face of emerging infectious diseases. In an outbreak, timely establishment of diagnostic routines is crucial to identifying cases and preventing the spread of the disease, especially when faced with high-consequence pathogens. In this article, we describe a multiagency exercise including the rapid deployment and diagnostic adaptation of the Swedish Armed Forces mobile laboratory (biological field analysis laboratory) in the context of COVID-19. This deployment was initiated as a high-readiness exercise at the end of January 2020, when the global development of the outbreak was still uncertain. Through collaboration with the Public Health Agency of Sweden and a civilian hospital, a real-time reverse transcriptase polymerase chain reaction method specific to SARS-CoV-2 was made available and adapted to the mobile laboratory, and the team established and evaluated a functional and efficient diagnostic asset along with a logistical support chain. We also organized and evaluated mobile testing teams, and the method was later used in large-scale, national, cross-sectional COVID-19 surveys in several regions of Sweden. In this article, we focus on the challenges of overbridging the civil-military interface in this context and identifying lessons learned and added values to the response during the early pandemic. We propose that the experiences from this exercise and governmental agency collaboration are valuable in preparation for future outbreaks.
3.	Bindra, Amarinder, et al. (författare) Search for DNA of exogenous mouse mammary tumor virus-related virus in human breast cancer samples 2007 Ingår i: Journal of General Virology. - : Microbiology Society. - 0022-1317 .- 1465-2099. ; 88, s. 1806-1809 Tidskriftsartikel (refereegranskat)abstract Earlier reports of a human exogenous retrovirus (HMTV) related closely to mouse mammary tumor virus (MMTV) led us to search for these viral sequences in breast cancer tissues and normal tissues. A real-time PCR was developed based on MMTV and published HMTV envelope sequences. The real-time PCR method can detect one to ten copies of MMTV target DNA. Tissue samples were collected prospectively from 18 breast cancer patients and 11 non-malignant control cases, as well as peripheral blood leukocytes from the same women. Despite the high sensitivity of the real-time PCR method used, none of the samples were positive for HMTV DNA or RNA. The absence of HMTV DNA in both breast cancer samples and controls indicates either that the concentration of putative HMTV DNA in the breast cancers was too low for detection or that it did not exist there.
4.	Bröjer, Caroline, et al. (författare) Pathobiology and virus shedding of low-pathogenic avian influenza virus (A/H1N1) infection in mallards exposed to oseltamivir 2013 Ingår i: Journal of Wildlife Diseases. - : Wildlife Disease Association. - 0090-3558 .- 1943-3700. ; 49:1, s. 103-113 Tidskriftsartikel (refereegranskat)abstract Low-pathogenic avian influenza (LPAI) viruses in wild birds are important as they can constitute the basis for the development of highly pathogenic avian influenza viruses or form part of human-adapted strains with pandemic potential. However, the pathogenesis of LPAI viruses is not well characterized in dabbling ducks, one of the natural reservoirs of LPAI viruses. Between 21 September 2009 and 21 December 2009, we used real-time reverse transcriptase polymerase chain reaction (q-PCR), histopathology, and immunohistochemistry (IHC) to study Mallards (Anas platyrhynchos) infected with an influenza A/H1N1 virus isolated from a wild Mallard in Sweden. The ducks were either inoculated intraesophageally ("artificial infection") or infected by virus shed by other ducks in the experiment ("contact infection"). The ducks were subjected to three low concentrations (80 ng/L, 1 mu g/L, and 80 mu g/L) of the active metabolite of oseltamivir (Tamiflu (R)), oseltamivir carboxylate (OC), which resulted in the development of the viral resistance mutation H274Y at 1 and 80 mu g/L. The LPAI virus infection was localized to the intestinal tract and cloacal bursa except in one Mallard. The exception was a duck euthanized 1 day postinoculation, whose infection was located solely in the lung, possibly due to intratracheal deposition of virus. The intestinal infection was characterized by occasional degenerating cells in the lamina propria and presence of viral antigen as detected by IHC, as well as positive q-PCR performed on samples from feces and intestinal contents. Histopathologic changes, IHC positivity, and viral shedding all indicated that the infection peaked early, around 2 days postinfection. Furthermore, more viral antigen and viral RNA were detected with IHC and q-PCR in the proximal parts early in the infection. There was no obvious difference in the course of the infection in artificial versus contact infection, when the level of OC was increased from 80 ng/L to 1 mu g/L (based on IHC and q-PCR), when the level of OC was increased to 80 mu/L, or when the resistance mutation H274Y developed (based on q-PCR).
5.	Elfaitouri, A, et al. (författare) Murine Gammaretrovirus Group G3 Was Not Found in Swedish Patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome and Fibromyalgia 2011 Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:10 Tidskriftsartikel (refereegranskat)abstract Background: The recent report of gammaretroviruses of probable murine origin in humans, called xenotropic murine retrovirus related virus (XMRV) and human murine leukemia virus related virus (HMRV), necessitated a bioinformatic search for this virus in genomes of the mouse and other vertebrates, and by PCR in humans. Results: Three major groups of murine endogenous gammaretroviruses were identified. The third group encompassed both exogenous and endogenous Murine Leukemia Viruses (MLVs), and most XMRV/HMRV sequences reported from patients suffering from myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Two sensitive real-time PCRs for this group were developed. The predicted and observed amplification range for these and three published XMRV/HMRV PCRs demonstrated conspicuous differences between some of them, partly explainable by a recombinatorial origin of XMRV. Three reverse transcription real-time PCRs (RTQPCRs), directed against conserved and not overlapping stretches of env, gag and integrase (INT) sequences of XMRV/HMRV were used on human samples. White blood cells from 78 patients suffering from ME/CFS, of which 30 patients also fulfilled the diagnostic criteria for fibromyalgia (ME/CFS/FM) and in 7 patients with fibromyalgia (FM) only, all from the Gothenburg area of Sweden. As controls we analyzed 168 sera from Uppsala blood donors. We controlled for presence and amplifiability of nucleic acid and for mouse DNA contamination. To score as positive, a sample had to react with several of the XMRV/HMRV PCRs. None of the samples gave PCR reactions which fulfilled the positivity criteria. Conclusions: XMRV/HMRV like proviruses occur in the third murine gammaretrovirus group, characterized here. PCRs developed by us, and others, approximately cover this group, except for the INT RTQPCR, which is rather strictly XMRV specific. Using such PCRs, XMRV/HMRV could not be detected in PBMC and plasma samples from Swedish patients suffering from ME/CFS/FM, and in sera from Swedish blood donors.
6.	Gillman, Anna, et al. (författare) Influenza A(H7N9) Virus Acquires Resistance-Related Neuraminidase I222T Substitution When Infected Mallards Are Exposed to Low Levels of Oseltamivir in Water 2015 Ingår i: Antimicrobial Agents and Chemotherapy. - : American Society for Biochemistry and Molecular Biology. - 0066-4804 .- 1098-6596. ; 59:9, s. 5196-5202 Tidskriftsartikel (refereegranskat)abstract Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and new human IAVs often contain gene segments originating from avian IAVs. Treatment options for severe human influenza are principally restricted to neuraminidase inhibitors (NAIs), among which oseltamivir is stockpiled in preparedness for influenza pandemics. There is evolutionary pressure in the environment for resistance development to oseltamivir in avian IAVs, as the active metabolite oseltamivir carboxylate (OC) passes largely undegraded through sewage treatment to river water where waterfowl reside. In an in vivo mallard (Anas platyrhynchos) model, we tested if low-pathogenic avian influenza A(H7N9) virus might become resistant if the host was exposed to low levels of OC. Ducks were experimentally infected, and OC was added to their water, after which infection and transmission were maintained by successive introductions of uninfected birds. Daily fecal samples were tested for IAV excretion, genotype, and phenotype. Following mallard exposure to 2.5 mu g/liter OC, the resistance-related neuraminidase (NA) I222T substitution, was detected within 2 days during the first passage and was found in all viruses sequenced from subsequently introduced ducks. The substitution generated 8-fold and 2.4-fold increases in the 50% inhibitory concentration (IC50) for OC (P < 0.001) and zanamivir (P = 0.016), respectively. We conclude that OC exposure of IAV hosts, in the same concentration magnitude as found in the environment, may result in amino acid substitutions, leading to changed antiviral sensitivity in an IAV subtype that can be highly pathogenic to humans. Prudent use of oseltamivir and resistance surveillance of IAVs in wild birds are warranted.
7.	Gillman, Anna, et al. (författare) Oseltamivir Resistance in Influenza A(H6N2) Caused by an R292K Substitution in Neuraminidase Is Not Maintained in Mallards without Drug Pressure 2015 Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:9 Tidskriftsartikel (refereegranskat)abstract Background Wild waterfowl is the natural reservoir of influenza A virus (IAV); hosted viruses are very variable and provide a source for genetic segments which can reassort with poultry or mammalian adapted IAVs to generate novel species crossing viruses. Additionally, wild waterfowl act as a reservoir for highly pathogenic IAVs. Exposure of wild birds to the antiviral drug oseltamivir may occur in the environment as its active metabolite can be released from sewage treatment plants to river water. Resistance to oseltamivir, or to other neuraminidase inhibitors (NAIs), in IAVs of wild waterfowl has not been extensively studied. Aim and Methods In a previous in vivo Mallard experiment, an influenza A(H6N2) virus developed oseltamivir resistance by the R292K substitution in the neuraminidase (NA), when the birds were exposed to oseltamivir. In this study we tested if the resistance could be maintained in Mallards without drug exposure. Three variants of resistant H6N2/R292K virus were each propagated during 17 days in five successive pairs of naive Mallards, while oseltamivir exposure was decreased and removed. Daily fecal samples were analyzed for viral presence, genotype and phenotype. Results and Conclusion Within three days without drug exposure no resistant viruses could be detected by NA sequencing, which was confirmed by functional NAI sensitivity testing. We conclude that this resistant N2 virus could not compete in fitness with wild type subpopulations without oseltamivir drug pressure, and thus has no potential to circulate among wild birds. The results of this study contrast to previous observations of drug induced resistance in an avian H1N1 virus, which was maintained also without drug exposure in Mallards. Experimental observations on persistence of NAI resistance in avian IAVs resemble NAI resistance seen in human IAVs, in which resistant N2 subtypes do not circulate, while N1 subtypes with permissive mutations can circulate without drug pressure. We speculate that the phylogenetic group N1 NAs may easier compensate for NAI resistance than group N2 NAs, though further studies are needed to confirm such conclusions.
8.	Gillman, Anna, et al. (författare) Oseltamivir-Resistant Influenza A (H1N1) Virus Strain with an H274Y Mutation in Neuraminidase Persists without Drug Pressure in Infected Mallards 2015 Ingår i: Applied and Environmental Microbiology. - : American Society for Microbiology. - 0099-2240 .- 1098-5336. ; 81:7, s. 2378-2383 Tidskriftsartikel (refereegranskat)abstract Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and emerging human IAVs often contain gene segments from avian viruses. The active drug metabolite of oseltamivir (oseltamivir carboxylate [OC]), stockpiled as Tamiflu for influenza pandemic preparedness, is not removed by conventional sewage treatment and has been detected in river water. There, it may exert evolutionary pressure on avian IAV in waterfowl, resulting in the development of resistant viral variants. A resistant avian IAV can circulate among wild birds only if resistance does not restrict viral fitness and if the resistant virus can persist without continuous drug pressure. In this in vivo mallard (Anas platyrhynchos) study, we tested whether an OC-resistant avian IAV (H1N1) strain with an H274Y mutation in the neuraminidase (NA-H274Y) could retain resistance while drug pressure was gradually removed. Successively infected mallards were exposed to decreasing levels of OC, and fecal samples were analyzed for the neuraminidase sequence and phenotypic resistance. No reversion to wild-type virus was observed during the experiment, which included 17 days of viral transmission among 10 ducks exposed to OC concentrations below resistance induction levels. We conclude that resistance in avian IAV that is induced by exposure of the natural host to OC can persist in the absence of the drug. Thus, there is a risk that human-pathogenic IAVs that evolve from IAVs circulating among wild birds may contain resistance mutations. An oseltamivir-resistant pandemic IAV would pose a substantial public health threat. Therefore, our observations underscore the need for prudent oseltamivir use, upgraded sewage treatment, and surveillance for resistant IAVs in wild birds.
9.	Gillman, Anna, et al. (författare) Resistance Mutation R292K Is Induced in Influenza A(H6N2) Virus by Exposure of Infected Mallards to Low Levels of Oseltamivir 2013 Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:8 Tidskriftsartikel (refereegranskat)abstract Resistance to neuraminidase inhibitors (NAIs) is problematic as these drugs constitute the major treatment option for severe influenza. Extensive use of the NAI oseltamivir (Tamiflu(R)) results in up to 865 ng/L of its active metabolite oseltamivir carboxylate (OC) in river water. There one of the natural reservoirs of influenza A, dabbling ducks, can be exposed. We previously demonstrated that an influenza A(H1N1) virus in mallards (Anas platyrhynchos) exposed to 1 mu g/L of OC developed oseltamivir resistance through the mutation H274Y (N2-numbering). In this study, we assessed the resistance development in an A(H6N2) virus, which belongs to the phylogenetic N2 group of neuraminidases with distinct functional and resistance characteristics. Mallards were infected with A(H6N2) while exposed to 120 ng/L, 1.2 mu g/L or 12 mu g/L of OC in their sole water source. After 4 days with 12 mu g/L of OC exposure, the resistance mutation R292K emerged and then persisted. Drug sensitivity was decreased approximate to 13,000-fold for OC and approximate to 7.8-fold for zanamivir. Viral shedding was similar when comparing R292K and wild-type virus indicating sustained replication and transmission. Reduced neuraminidase activity and decrease in recovered virus after propagation in embryonated hen eggs was observed in R292K viruses. The initial, but not the later R292K isolates reverted to wild-type during egg-propagation, suggesting a stabilization of the mutation, possibly through additional mutations in the neuraminidase (D113N or D141N) or hemagglutinin (E216K). Our results indicate a risk for OC resistance development also in a N2 group influenza virus and that exposure to one NAI can result in a decreased sensitivity to other NAIs as well. If established in influenza viruses circulating among wild birds, the resistance could spread to humans via re-assortment or direct transmission. This could potentially cause an oseltamivir-resistant pandemic; a serious health concern as preparedness plans rely heavily on oseltamivir before vaccines can be mass-produced.
10.	Gillman, Anna, 1972- (författare) Tamiflu in the Water : Resistance Dynamics of Influenza A Virus in Mallards Exposed to Oseltamivir 2016 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract The natural reservoir of influenza A virus (IAV) is wild waterfowl, and all human IAVs have their genetic origins from avian viruses. Neuraminidase inhibitors (NAIs) are currently the best drugs for treatment of human influenza; therefore, the orally available NAI oseltamivir (Tamiflu®) has been stockpiled worldwide as part of pandemic preparedness planning. Re-sistance to NAIs is related to worse clinical outcomes and if a new pandemic influenza virus would be oseltamivir-resistant its public health impact would be substantially worsened.The active metabolite oseltamivir carboxylate (OC) is not removed by sewage treatment and ends up in river water, where OC-concentrations up to 0.86µg/L have been detected.We hypothesize that occasional OC exposure of wild waterfowl carrying IAVs may result in circulation of resistant variants that may potentially evolve to become human-pathogenic.We tested the hypothesis in an in vivo Mallard (Anas platyrhynchos) model in which birds were infected with avian IAVs and exposed to OC. Excreted viruses were analyzed regarding genotypic and phenotypic resistance by neuraminidase (NA) sequencing and a functional NA inhibition assay.Two viruses with NAs of the phylogenetic N2-group, H6N2 and H7N9, acquired the NA substitutions R292K and I222T when host ducks were exposed to 12µg/L and 2.5µg/L of OC, respectively. Drug susceptibilities were at previously described levels for the substitutions. To test persistence of resistance, an OC resistant avian H1N1/H274Y virus (with a group N1 NA-protein) from a previous study, and three resistant H6N2/R292K variants were allowed to replicate in Mallards without drug pressure. Resistance was entirely maintained in the H1N1/H274Y virus, but the H6N2/R292K variants were outcompeted by wild type virus, indicating retained fitness of the resistant H1N1 but not the H6N2 variants.We conclude that OC in the environment may generate resistant IAVs in wild birds. Resistant avian IAVs may become a problem to humans, should the resistance trait become part of a new human pathogenic virus. It implies a need for prudent use of available NAIs, optimized sewage treatment and resistance surveillance of avian IAVs of wild birds.
11.	Hoffman, T, et al. (författare) Molecular detection and characterization of Brucella species in raw informally marketed milk from Uganda 2016 Ingår i: Infection ecology & epidemiology. - : Informa UK Limited. - 2000-8686. ; 6, s. 32442- Tidskriftsartikel (refereegranskat)
12.	Järhult, Josef D., et al. (författare) Environmental levels of the antiviral oseltamivir induce development of resistance mutation H274Y in influenza A/H1N1 virus in mallards 2011 Ingår i: PLOS ONE. - San Francisco, CA : Public Library of Science (PLoS). - 1932-6203. ; 6:9 Tidskriftsartikel (refereegranskat)abstract Oseltamivir (Tamiflu®) is the most widely used drug against influenza infections and is extensively stockpiled worldwide as part of pandemic preparedness plans. However, resistance is a growing problem and in 2008-2009, seasonal human influenza A/H1N1 virus strains in most parts of the world carried the mutation H274Y in the neuraminidase gene which causes resistance to the drug. The active metabolite of oseltamivir, oseltamivir carboxylate (OC), is poorly degraded in sewage treatment plants and surface water and has been detected in aquatic environments where the natural influenza reservoir, dabbling ducks, can be exposed to the substance. To assess if resistance can develop under these circumstances, we infected mallards with influenza A/H1N1 virus and exposed the birds to 80 ng/L, 1 µg/L and 80 µg/L of OC through their sole water source. By sequencing the neuraminidase gene from fecal samples, we found that H274Y occurred at 1 µg/L of OC and rapidly dominated the viral population at 80 µg/L. IC₅₀ for OC was increased from 2-4 nM in wild-type viruses to 400-700 nM in H274Y mutants as measured by a neuraminidase inhibition assay. This is consistent with the decrease in sensitivity to OC that has been noted among human clinical isolates carrying H274Y. Environmental OC levels have been measured to 58-293 ng/L during seasonal outbreaks and are expected to reach µg/L-levels during pandemics. Thus, resistance could be induced in influenza viruses circulating among wild ducks. As influenza viruses can cross species barriers, oseltamivir resistance could spread to human-adapted strains with pandemic potential disabling oseltamivir, a cornerstone in pandemic preparedness planning. We propose surveillance in wild birds as a measure to understand the resistance situation in nature and to monitor it over time. Strategies to lower environmental levels of OC include improved sewage treatment and, more importantly, a prudent use of antivirals.
13.	Järhult, Josef, et al. (författare) Environmental levels of oseltamivir induce development of resistance mutation H274Y in influenza A/H1N1 virus in mallards – implications for the risk of an oseltamivir resistant influenza pandemic Annan publikation (övrigt vetenskapligt/konstnärligt)abstract Resistance in influenza is a growing problem. Oseltamivir carboxylate (OC), the active substance of the most widely used antiviral drug oseltamivir (Tamiflu ®), is poorly degraded in sewage treatment plants and surface water. OC has been detected in aquatic environments where the natural influenza reservoir, dabbling ducks, can be exposed to it. To test if resistance can occur in this situation, we infected mallards with influenza A/H1N1 virus and exposed the birds to 0.08 μg /L, 1.00 μg/L and 80.00 μg/L of OC. The resistance mutation H274Y occurred at 1 μg/L and rapidly dominated the viral population at 80 μg/L. The environmental levels of OC are expected to reach this magnitude. IC50 for OC was increased from 1-4 nM to 400-700 nM in H274Y-positive isolates, confirming a resistant phenotype. As influenza viruses can cross the species barrier, resistance to oseltamivir can spread to human-adapted strains with pandemic potential disabling one of the cornerstones in pandemic preparedness planning.
14.	Lagerqvist, Nina, et al. (författare) Evaluation of 11 SARS-CoV-2 antibody tests by using samples from patients with defined IgG antibody titers 2021 Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 11:1 Tidskriftsartikel (refereegranskat)abstract We evaluated the performance of 11 SARS-CoV-2 antibody tests using a reference set of heat-inactivated samples from 278 unexposed persons and 258 COVID-19 patients, some of whom contributed serial samples. The reference set included samples with a variation in SARS-CoV-2 IgG antibody titers, as determined by an in-house immunofluorescence assay (IFA). The five evaluated rapid diagnostic tests had a specificity of 99.0% and a sensitivity that ranged from 56.3 to 81.6% and decreased with low IFA IgG titers. The specificity was > 99% for five out of six platform-based tests, and when assessed using samples collected ≥ 22 days after symptom onset, two assays had a sensitivity of > 96%. These two assays also detected samples with low IFA titers more frequently than the other assays. In conclusion, the evaluated antibody tests showed a heterogeneity in their performances and only a few tests performed well with samples having low IFA IgG titers, an important aspect for diagnostics and epidemiological investigations.
15.	Metreveli, Giorgi, et al. (författare) Comparison of two H1N2 swine influenza A viruses from disease outbreaks in pigs in Sweden during 2009 and 2010 2011 Ingår i: Virus Genes. - : Springer Science and Business Media LLC. - 0920-8569 .- 1572-994X. ; 42, s. 236-244 Tidskriftsartikel (refereegranskat)abstract The influenza A virus subtypes H1N1, H1N2 and H3N2 are prevalent in pig populations worldwide. In the present study, two relatively uncommon swine influenza virus (SIV) H1N2 subtypes, isolated in Sweden in 2009 and 2010, were compared regarding their molecular composition and biological characteristics. The differences regarding markers purportedly related to pathogenicity, host adaptation or replication efficiency. They included a truncated PB1-F2 protein in the earlier isolate but a full length version in the more recent one; differences in the number of haemagglutinin glycosylation sites, including a characteristic human one; and a nuclear export protein with altered export signal. Of particular interest, the NS1 amino acid sequence of swine H1N2-2009 and 2010 has a 'unique or very unusual' PDZ binding domain (RPKV) at the C-terminal of the protein, a motif that has been implicated as a virulence marker. Concerning biological properties, these viruses reached lower titre and showed reduced cytopathogenicity in MDCK cells compared with an avian-like H1N1 isolate A/swine/Lidkoping/1193/2002 belonging to the same lineage as the 2009 and 2010 isolates. The findings should contribute to better understanding of factors related to the survival/extinction of this uncommon reassortant variant.
16.	Muradrasoli, Shaman, et al. (författare) Broadly targeted multiprobe QPCR for detection of coronaviruses : Coronavirus is common among mallard ducks (Anas platyrhynchos) 2009 Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 159:2, s. 277-287 Tidskriftsartikel (refereegranskat)abstract Coronaviruses (CoV) can cause trivial or fatal disease in humans and in animals. Detection methods for a wide range of CoVs are needed, to understand viral evolution, host range, transmission and maintenance in reservoirs. A new concept, "Multiprobe QPCR", which uses a balanced mixture of competing discrete non- or moderately degenerated nuclease degradable (TaqMan((R))) probes was employed. It provides a broadly targeted and rational single tube real-time reverse transcription PCR ("NQPCR") for the generic detection and discovery of CoV. Degenerate primers, previously published, and the new probes, were from a conserved stretch of open reading frame 1b, encoding the replicase. This multiprobe design reduced the degree of probe degeneration, which otherwise decreases the sensitivity, and allowed a preliminary classification of the amplified sequence directly from the QPCR trace. The split probe strategy allowed detection of down to 10 viral nucleic acid equivalents of CoV from all known CoV groups. Evaluation was with reference CoV strains, synthetic targets, human respiratory samples and avian fecal samples. Infectious-Bronchitis-Virus (IBV)-related variants were found in seven of 35 sample pools, from 100 wild mallards (Anas platyrhynchos). Ducks may spread and harbour CoVs. NQPCR can detect a wide range of CoVs, as illustrated for humans and ducks.
17.	Muradrasoli, Shaman, et al. (författare) Broadly targeted triplex real-time PCR detection of influenza A, B and C viruses based on the nucleoprotein gene and a novel "MegaBeacon" probe strategy 2010 Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 163:2, s. 313-322 Tidskriftsartikel (refereegranskat)abstract A PCR assay that covers animal and human influenza A, B and C viruses, i.e., most of Orthomyxoviridae, is needed. Influenza types are distinguished based on differences in the nucleoprotein (NP) present in the virus. Conserved NP regions were therefore used to design a TaqMan (R)-based triplex reverse transcription real-time PCR method. Variability of influenza A within the probe target region mandated the development of a novel molecular beacon, the "Mega" molecular beacon (MegaBeacon: MegB), for the detection of influenza A with this method. MegaBeacon is a mismatch-tolerant molecular beacon that is also a TaqMan (R) probe. The triplex method (3QPCR-MegB) was evaluated with influenza A isolates covering 18 HxNx combinations, two influenza B isolates, and five Japanese influenza C isolates, as well as influenza A, B and C synthetic DNA targets. One to ten viral RNA and cDNA genome equivalents were detected per PCR reaction for influenza A, B and C. Seventy-one human nasopharyngeal aspirates from respiratory infections yielded 30 influenza A, 11 influenza B and 0 influenza C with 3QPCR-MegB, where immunofluorescence (IF) found 28 influenza A and 10 influenza B. 3QPCR-MegB was more mismatch-tolerant than a variant PCR with an influenza A TaqMan (R) probe (3QPCR) and is a sensitive and rational method to detect influenza viruses of animal and human origin. MegaBeacon probes hold promise for variable target nucleic acids.
18.	Muradrasoli, Shaman, 1972- (författare) Detection and Quantification of Variable Viral RNA by Real-Time PCR Assays 2008 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract As the area of nucleic acid based technologies develops, so will our understanding of how structural variations in DNA and RNA pathogens are associated with disease. The overall goal of this thesis is the development of broadly targeted measurement techniques for variable viral RNA by Real-Time PCR (here referred to as quantitative reverse transcriptase PCR, QRT-PCR). In papers I & II, broadly targeted and specific QRT-PCRs were used to study expression of endogenous and exogenous betaretrovirus sequences in human tissues. Results from human tissues demonstrated endogenous betaretrovirus expression in a tissue-specific manner, highest in reproductive tissues. Despite the high sensitivity, no exogenous betaretrovirus was found in human breast cancer samples. The limits of primer and probe degeneracy for detection of a diverse set of retroviral sequences was evaluated. These methods are useful for further investigations on the pathophysiological contribution(s) of endogenous betaretrovirus and to investigate whether an exogenous betaretrovirus is involved in human breast cancer. In papers III & IV, we developed and applied broadly targeted one-step QRT-PCRs for influenza viruses and coronaviruses. In addition to the generic primers, two novel probe design strategies were used in order to be able to broadly amplify these diverse sets of viruses: A triplex system for simultaneous detection and quantification of influenza A, B and C (3QRT-PCR and further developed 3QRT-PCR-MegB; where MegB stands for MegaBeacon) based on TaqMan® and MegB probes, and a pan-CoV QRT-PCR, based on three TaqMan® probes i.e., degeneracy was distributed on three probes. Probe fault tolerance was thus increased in two ways, either with short probes with/without locked nucleic acid (LNA) nucleotides concentrated to conserved stretches, or with long probes (MegB), compensating mismatching positions with many matching ones. Clinical samples, negative by antigen detection with immunofluorescence (IFA), were influenza A positive with 3QPCR-MegB. Avian pooled samples, negative with an earlier pan-CoV QPCR, came out positive with the triple-probe system. Assay evaluation with clinical samples and reference strains revealed good clinical diagnostic potential. Thus, the thesis describes several strategies to counteract sequence variation of RNA viruses and describes a set of broadly targeted QRT-PCRs useful for scientific screening or diagnostics of betaretroviruses and respiratory viruses.
19.	Muradrasoli, Shaman, et al. (författare) Development of real-time PCRs for detection and quantitation of human MMTV-Iike (HML) sequences HML expression in human tissues 2006 Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 136:1-2, s. 83-92 Tidskriftsartikel (refereegranskat)abstract The human genome contains around 1000 betaretrovirus-like copies, human mouse mammary tumour virus (MMTV)-like (HML) groups 1 - 10, also referred to as human endogenous retroviruts "HERV-K". Despite many efforts, it is not established whether betaretroviruses, exo- or endogenous, are involved in the etiology of breast cancer, or other cancer diseases, in humans. Quantitative real-time PCR (QPCR) TaqMan((R))-based assays for HML groups 1-7, targeting the conserved reverse transcriptase (RT) and integrase (IN) domains of the pol gene were designed. Plasmids containing the entire pol gene of HML 1-7 were used as standards. The RT and IN based QPCRs could detect 10(0)-10(3) copies per PCR reaction of the plasmids. However, not all plasmids gave a signal in both RT and IN QPCRs, probably due to mismatches. Furthermore, RT and IN based HML6 specific QPCRs were developed. They were specific for amplification of transcripts for the whole HML6 group. The methods allow the monitoring in body fluids and tissues of expression of a wide range of betaretrovirus-like sequences. Betaretrovirus-like RNA was studied in normal human tissues and of HML6 in brains of multiple sclerosis (MS) patients. Brain, adrenal gland and testis had a high betaretrovirus-like expression. Multiple sclerosis plaques contained the same HML6 RNA concentration as control tissue. These assays are expected to enhance studies on involvement of betaretroviruses in physiology and disease.
20.	Muradrasoli, Shaman, et al. (författare) Prevalence and phylogeny of coronaviruses in wild birds from the Bering Strait area (Beringia) 2010 Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 5:10 Tidskriftsartikel (refereegranskat)abstract Coronaviruses (CoVs) can cause mild to severe disease in humans and animals, their host range and environmental spread seem to have been largely underestimated, and they are currently being investigated for their potential medical relevance. Infectious bronchitis virus (IBV) belongs to gamma-coronaviruses and causes a costly respiratory viral disease in chickens. The role of wild birds in the epidemiology of IBV is poorly understood. In the present study, we examined 1,002 cloacal and faecal samples collected from 26 wild bird species in the Beringia area for the presence of CoVs, and then we performed statistical and phylogenetic analyses. We detected diverse CoVs by RT-PCR in wild birds in the Beringia area. Sequence analysis showed that the detected viruses are gamma-coronaviruses related to IBV. These findings suggest that wild birds are able to carry gamma-coronaviruses asymptomatically. We concluded that CoVs are widespread among wild birds in Beringia, and their geographic spread and frequency is higher than previously realised. Thus, Avian CoV can be efficiently disseminated over large distances and could be a genetic reservoir for future emerging pathogenic CoVs. Considering the great animal health and economic impact of IBV as well as the recent emergence of novel coronaviruses such as SARS-coronavirus, it is important to investigate the role of wildlife reservoirs in CoV infection biology and epidemiology.
21.	Orozovic, Goran, 1965-, et al. (författare) Degenerate primers for PCR amplification and sequencing of the avian influenza A neuraminidase gene 2010 Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 170:1-2, s. 94-98 Tidskriftsartikel (refereegranskat)abstract This study describes the design of degenerate primers and their use for synthesis of full-length avian influenza A neuramindase (NA). Each reaction was performed using either two forward primers and one reverse primer, or one forward primer and one reverse primer. Both primer combinations had comparable amplification efficiencies for all NA subtypes (1-9). A total of 11 virus strains, including both field isolates and reference strains, were amplified successfully using these degenerate primer sets. Of the sequences amplified, 108 strains (93.9%) resulted in near full-length NA cDNAs after two readings with one forward primer and one reverse primer. Of the remaining sequences, five strains (4.3%) yielded reads with enough information for subtype categorization by BLAST although they were of insufficient quality for assembly. One strain (0.9%) yielded different subtypes from both sequence reads whereas the other one (0.9%) was not possible to assemble and subtype. This successful demonstration of these degenerate primers for the amplification and sequencing of all avian NA subtypes suggests that these primers could be employed in the avian influenza surveillance program as well as studies of antiviral resistance, virus ecology or viral phylogeny.
22.	Rwabiita Mugizi, Denis, et al. (författare) Isolation and Molecular Characterization of Brucella Isolates in Cattle Milk in Uganda 2015 Ingår i: BioMed Research International. - : Hindawi Limited. - 2314-6133 .- 2314-6141. Tidskriftsartikel (refereegranskat)abstract Brucellosis is endemic in livestock and humans in Uganda and its transmission involves amultitude of risk factors like consumption of milk from infected cattle. To shed new light on the epidemiology of brucellosis in Uganda the present study used phenotypic and molecular approaches to delineate the Brucella species, biovars, and genotypes shed in cattle milk. Brucella abortus without a biovar designation was isolated from eleven out of 207 milk samples from cattle in Uganda. These isolates had a genomic monomorphism at 16 variable number tandem repeat (VNTR) loci and showed in turn high levels of genetic variation when compared with other African strains or other B. abortus biovars from other parts of the world. This study further highlights the usefulness of MLVA as an epidemiological tool for investigation of Brucella infections.
23.	Vågsholm, Ivar, et al. (författare) Achievement V - Methods for breaking the transmission of pathogens along the food chain: Detection of viruses in food 2011 Ingår i: Trends in Food Science and Technology. - : Elsevier BV. - 0924-2244. ; 22, s. S49-S57 Tidskriftsartikel (refereegranskat)abstract Traditionally the focus for control of food-borne disease has been bacteria. During the last decade viruses have emerged as important sources of food borne human disease. Since the traditional bacteriological indicators, are not reliable for viral contamination, new methods are needed. PCR has enhanced the detection of virus in food. A challenge for developing detection reliable methods for viruses in food is that food matrices vary in composition, high sequence variability and inhibitors may be present. Therefore it is necessary to develop assays that have high diagnostic sensitivity, are broad and robust, and combine sample concentration and removal of inhibitors
24.	Westman, Gabriel, 1977-, et al. (författare) Torque teno virus viral load is related to age, CMV infection and HLA type but not to Alzheimer's disease 2020 Ingår i: PLOS ONE. - : PUBLIC LIBRARY SCIENCE. - 1932-6203. ; 15:1 Tidskriftsartikel (refereegranskat)abstract Torque teno virus (TTV) is an unenveloped, circular, single stranded DNA virus with a genome size of approximately 3.8 kb. Previous studies have demonstrated varying grades of association between TTV DNA levels and immune deficiencies related to age, chronic infections and cancer. Alzheimer's disease (AD) has been related to persistent viral infections such as HSV-1 and CMV, but it is not known whether TTV viral load could serve as a functional biomarker of cellular immunity in this setting. Therefore, the objective of this study was to investigate whether TTV infection and viral load is related to AD status, CMV immunity, systemic inflammation or HLA types connected to anti-viral immunity. A total of 50 AD subjects and 51 non-demented controls were included in the study. AD subjects were diagnosed according to NINCDS-ADRDA and DSM-IV criteria and neuroradiologic findings were consistent with the diagnosis. TTV viral load was analyzed in plasma samples using a quantitative real-time PCR. Using a cut-off for TTV status at 200 copies/ml, 88% (89/101) of the study subjects were classified as TTV positive. TTV viral load significantly increased with age (beta 0.049 per year, p< 0.001) but significantly decreased in relation to CMV IgG levels (beta -0.022 per 1000 units, p = 0.005) and HLA-B27 positivity (beta -0.53, p = 0.023). In conclusion, TTV immune control is not significantly affected by AD status, but appears related to age, CMV humoral immune response and HLA type.
25.	Widén, Frederik, et al. (författare) Development of improved analytical methods for use in animal health and in foodborne disease surveillance for source attribution 2013 Ingår i: Revue Scientifique et Technique- Office International des Epizooties. - 0253-1933 .- 1608-0637. ; 32, s. 549-558 Tidskriftsartikel (refereegranskat)abstract Considering the 'One Health' principles, the links between animal and human health are very strong. Both domestic and wild animals are sources of infectious agents that cause diseases in humans. Poor animal health may also indirectly affect human health, through reduced access to food. A large number of infectious diseases of animals,the transboundary animal diseases, spread rapidly across borders. Robust and accurate diagnostic assays are needed to detect the infectious agents rapidly and to limit their spread. A large arsenal of novel assays has been developed during the last three decades, with a tremendous impact on the detection of infectious agents. The new diagnostic methods are mostly laboratory-based and expensive, requiring sophisticated equipment and special skills. However, rapid and cheap field-based assays have also been developed. Herein, the authors give several examples of the development of novel assays, with special focus on the 'One Health' principles.
26.	Wille, Michelle, et al. (författare) High Prevalence and Putative Lineage Maintenance of Avian Coronaviruses in Scandinavian Waterfowl 2016 Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 11:3 Tidskriftsartikel (refereegranskat)abstract Coronaviruses (CoVs) are found in a wide variety of wild and domestic animals, and constitute a risk for zoonotic and emerging infectious disease. In poultry, the genetic diversity, evolution, distribution and taxonomy of some coronaviruses have been well described, but little is known about the features of CoVs in wild birds. In this study we screened 764 samples from 22 avian species of the orders Anseriformes and Charadriiformes in Sweden collected in 2006/2007 for CoV, with an overall CoV prevalence of 18.7%, which is higher than many other wild bird surveys. The highest prevalence was found in the diving duck-smainly Greater Scaup (Aythya marila; 51.5%)-and the dabbling duck Mallard (Anas platyrhynchos; 19.2%). Sequences from two of the Greater Scaup CoV fell into an infrequently detected lineage, shared only with a Tufted Duck (Aythya fuligula) CoV. Coronavirus sequences from Mallards in this study were highly similar to CoV sequences from the sample species and location in 2011, suggesting long-term maintenance in this population. A single Black-headed Gull represented the only positive sample from the order Charadriiformes. Globally, Anas species represent the largest fraction of avian CoV sequences, and there seems to be no host species, geographical or temporal structure. To better understand the eitiology, epidemiology and ecology of these viruses more systematic surveillance of wild birds and subsequent sequencing of detected CoV is imperative.
27.	Wille, Michelle, et al. (författare) RNAlater (R) is a viable storage option for avian influenza sampling in logistically challenging conditions 2018 Ingår i: Journal of Virological Methods. - : ELSEVIER SCIENCE BV. - 0166-0934 .- 1879-0984. ; 252, s. 32-36 Tidskriftsartikel (refereegranskat)abstract Surveillance of wild birds is critical in monitoring for highly pathogenic avian influenza A viruses (ATVs). However, a successful surveillance regime requires proper treatment of samples in the field - rapid placement of samples in -80 degrees C and subsequent maintenance of cold-chain. Given the logistical difficulties of this, many avian taxa and/or geographic locations are not sampled, or, when sampled may result in false negatives due to poor sample treatment in the field. Here, we assessed the utility of RNAlater (R) as a stabilization agent for AIV sampling. We found no difference in real time PCR performance between virus transport media at optimal conditions and RNAlater (R) at -80 degrees C, -20 degrees C, 4 degrees C or room temperature up to two weeks, at either low or high virus load. Not only was RNAlater (R) useful in comparison of spiked samples or those from duck experiments, it was employed successfully in a field study of backyard birds in China. We detected AIV in cloacal and oropharyngeal samples from chickens and a sample with a low Cq was successfully subtyped as H9, although sample storage conditions were suboptimal. Thus, despite limitations in downstream characterization such virus isolation and typing, RNAlater (R) is a viable option for AIV sampling under logistically challenging circumstances.
28.	Wille, Michelle, et al. (författare) Urbanization and the dynamics of RNA viruses in Mallards (Anas platyrhynchos) 2017 Ingår i: Infection, Genetics and Evolution. - : Elsevier BV. - 1567-1348 .- 1567-7257. ; 51, s. 89-97 Tidskriftsartikel (refereegranskat)abstract Urbanization is intensifying worldwide, and affects the epidemiology of infectious diseases. However, the effect of urbanization on natural host-pathogen systems remains poorly understood. Urban ducks occupy an interesting niche in that they directly interact with both humans and wild migratory birds, and either directly or indirectly with food production birds. Here we have collected samples from Mallards (Anas platyrhynchos) residing in a pond in central Uppsala, Sweden, from January 2013 to January 2014. This artificial pond is kept ice-free during the winter months, and is a popular location where the ducks are fed, resulting in a resident population of ducks year-round. Nine hundred and seventy seven (977) fecal samples were screened for RNA viruses including: influenza A virus (IAV), avian paramyxovirus 1, avian coronavirus (CoV), and avian astrovirus (AstroV). This intra-annual dataset illustrates that these RNA viruses exhibit similar annual patterns to IAV, suggesting similar ecological factors are at play. Furthermore, in comparison to wild ducks, autumnal prevalence of IAV and CoV are lower in this urban population. We also demonstrate that AstroV might be a larger burden to urban ducks than IAV, and should be better assessed to demonstrate the degree to which wild birds contribute to the epidemiology of these viruses. The presence of economically relevant viruses in urban Mallards highlights the importance of elucidating the ecology of wildlife pathogens in urban environments, which will become increasingly important for managing disease risks to wildlife, food production animals, and humans.

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