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  • 1.
    Andersson, Martin O.
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Tamba, Paula
    Inst Diag & Anim Hlth, Romania.
    Stefanache, Mircea
    PAUMI VET Private Vet Clin, Romania.
    Radbea, Gabriel
    Sal Vet Private Vet Clin, Romania.
    Frangoulidis, Dimitrios
    Bundeswehr Inst Microbiol, Germany.
    Tomaso, Herbert
    Friedrich Loeffler Inst, Germany.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Dobler, Gerhard
    Bundeswehr Inst Microbiol, Germany;German Ctr Infect Res DZIF Partner Munich, Germany.
    Chitimia-Dobler, Lidia
    Inst Diag & Anim Hlth, Romania;Bundeswehr Inst Microbiol, Germany;German Ctr Infect Res DZIF Partner Munich, Germany.
    Molecular survey of neglected bacterial pathogens reveals an abundant diversity of species and genotypes in ticks collected from animal hosts across Romania2018In: Parasites & Vectors, ISSN 1756-3305, E-ISSN 1756-3305, Vol. 11, article id 144Article in journal (Refereed)
    Abstract [en]

    Background: Ticks are transmitting a wide range of bacterial pathogens that cause substantial morbidity and mortality in domestic animals. The full pathogen burden transmitted by tick vectors is incompletely studied in many geographical areas, and extensive studies are required to fully understand the diversity and distribution of pathogens transmitted by ticks. Results: We sampled 824 ticks of 11 species collected in 19 counties in Romania. Ticks were collected mainly from dogs, but also from other domestic and wild animals, and were subjected to molecular screening for pathogens. Rickettsia spp. was the most commonly detected pathogen, occurring in 10.6% (87/824) of ticks. Several species were detected: Rickettsia helvetica, R. raoultii, R. massiliae, R. monacensis, R. slovaca and R. aeschlimannii. A single occurrence of the zoonotic bacterium Bartonella vinsonii berkhoffii was detected in a tick collected from a dog. Anaplasma phagocytophilum occurred in four samples, and sequences similar to Anaplasma marginale/ovis were abundant in ticks from ruminants. In addition, molecular screening showed that ticks from dogs were carrying an Ehrlichia species identical to the HF strain as well as the enigmatic zoonotic pathogen "Candidatus Neoehrlichia mikurensis". An organism similar to E. chaffeensis or E. muris was detected in an Ixodes ricinus collected from a fox. Conclusions: We describe an abundant diversity of bacterial tick-borne pathogens in ticks collected from animal hosts in Romania, both on the level of species and genotypes/strains within these species. Several findings were novel for Romania, including Bartonella vinsonii subsp. berkhoffii that causes bacteremia and endocarditis in dogs. "Candidatus Neoehrlichia mikurensis" was detected in a tick collected from a dog. Previously, a single case of infection in a dog was diagnosed in Germany. The results warrant further studies on the consequences of tick-borne pathogens in domestic animals in Romania.

  • 2.
    Andersson, Martin O.
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Tamba, Paula
    Inst Diag & Anim Hlth, Romania.
    Stefanache, Mircea
    PAUMI VET Private Vet Clin, Romania.
    Radbea, Gabriel
    Sal Vet Private Vet Clin, Romania.
    Rubel, Franz
    Univ Vet Med Vienna, Austria.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Dobler, Gerhard
    German Ctr Infect Res DZIF Partner Munich, Germany.
    Chitimia-Dobler, Lidia
    Inst Diag & Anim Hlth, Romania ; German Ctr Infect Res DZIF Partner Munich, Germany.
    Babesia, Theileria, and Hepatozoon species in ticks infesting animal hosts in Romania2017In: Parasitology Research, ISSN 0932-0113, E-ISSN 1432-1955, Vol. 116, no 8, p. 2291-2297Article in journal (Refereed)
    Abstract [en]

    Babesia spp., Theileria spp., and Hepatozoon spp. are tick-transmitted apicomplexan parasites that cause several important diseases in animals. To increase current knowledge about the diversity of tick-transmitted pathogens in Romania, we investigated the occurrence of Babesia spp., Theileria spp., and Hepatozoon spp. in a wide range of tick species infesting animal hosts. We collected 852 ticks from 10 different animal species from 20 counties in Romania. The assessment was based on detection of parasite DNA by PCR. Five different apicomplexan parasite species were detected; among them three different species of Babesia: B. canis, B. microti, and B. ovis. Hepatozoon canis was the most frequently detected parasite, found predominately in Ixodes ricinus ticks collected from domestic dogs. It was also detected in I. ricinus collected from goat, fox, and cat. Furthermore, H. canis was found in Haemaphysalis punctata and Haemaphysalis concinna ticks. In addition, Theileria buffeli was detected in Rhipicephalus bursa ticks collected from cattle.

  • 3.
    Andersson, Martin O.
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Tamba, Paula
    Institute for Diagnosis and Animal Health, Romania.
    Stefanache, Mircea
    PAUMI-VET Private Veterinary Clinics, Romania.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Dobler, Gerhard
    German Center for Infection Research, Germany.
    Chitimia-Dobler, Lidia
    German Center for Infection Research, Germany.
    Canine tick-borne diseases in pet dogs from Romania2017In: Parasites & Vectors, ISSN 1756-3305, E-ISSN 1756-3305, Vol. 10, article id 2092Article in journal (Refereed)
    Abstract [en]

    Background: Tick-borne diseases are of substantial concern worldwide for animals as well as humans. Dogs have been a human companion for millennia, and their significant impact on human life renders disease in dogs to be of great concern. Tick-borne diseases in dogs represent a substantial diagnostic challenge for veterinarians in that clinical signs are often diffuse and overlapping. In addition, co-infections with two or more pathogens enhance this problem further. Molecular methods are useful to disentangle co-infections and to accurately describe prevalence and geographical distribution of tick-borne diseases. At this point, this information is lacking in many areas worldwide. Romania is one such area, where prevalence and distribution of several important pathogens need to be further investigated. To address this, we screened blood samples from 96 sick dogs with molecular methods for eight different pathogens including Babesia spp., Theileria spp., Hepatozoon spp., Anaplasma spp., Ehrlichia spp., "Candidatus Neoehrlichia mikurensis", Mycoplasma spp., and Borrelia spp. Results: As many as 45% (43/ 96) of the dogs in the study were infected with protozoan parasites. Babesia canis was the most frequent of these (28 infected dogs), whereas Hepatozoon canis was detected in 15% (14/ 96) and Babesia gibsoni was found in a single sample. Bacterial infection with Mycoplasma spp. occurred in 18% (17/ 96) of the sampled dogs. Obtained bacterial sequences revealed the occurrence of two species: Mycoplasma canis and "Candidatus Mycoplasma haematoparvum". In several cases co-infection with protozoan parasites and Mycoplasma sp. were detected. All dogs were negative for Anaplasma spp., Ehrlichia spp., "Ca. Neoehrlichia mikurensis", and for Borrelia spp. Conclusions: The results from the present study reinforce the notion that Babesia canis is an important pathogen in the Romanian dog population. However, more surprisingly, another protozoan species, H. canis, seems to be infecting dogs to a larger extent than previously recognized in Romania. Well-known tick-borne bacterial disease agents such as Anaplasma spp. and Borrelia spp. were not detected. In contrast, less wellstudied bacteria such as hemotropic Mycoplasma spp. were detected frequently. Moreover, co-infection might aggravate disease and complicate diagnosis and should be further studied in dogs.

  • 4.
    Andersson, Martin O.
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Vichova, Bronislava
    Slovak Acad Sci, Slovakia.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Krzyzanowska, Sandra
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Karlsson, Maria E.
    Swedish University of Agricultural Sciences.
    Co-infection with Babesia divergens and Anaplasma phagocytophilum in cattle (Bos taurus), Sweden2017In: Ticks and Tick-borne Diseases, ISSN 1877-959X, E-ISSN 1877-9603, Vol. 8, no 6, p. 933-935Article in journal (Refereed)
    Abstract [en]

    Babesiosis is a severe disease in cattle worldwide. In Europe, the main causative agent of bovine babesiosis is Babesia divergens. In some areas, this species is reported to have declined or even disappeared, and its etiological role overtaken by other piroplasmid species. Moreover, co-infection with other tick-transmitted pathogens can be expected to complicate diagnosis in cattle. Hence, molecular identification of the causative agent of babesiosis should be a priority. Therefore, samples from 71 domestic cattle, 39 with clinical signs of babesiosis and 32 without, from southern Sweden were screened for Babesia spp. and Anaplasma spp. using molecular methods Babesia divergens was detected in 38 of the samples, and Anaplasma phagocytophilum in 17. Co-infections with both pathogens were frequent, occurring in 18% of the animals with a B. divergens infection. The possibility of co-infection should be considered in diagnosis and treatment of bovine babesiosis.

  • 5.
    Avril, Alexis
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Grosbois, Vladimir
    CIRAD, France.
    Latorre-Margalef, Neus
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science. Univ Georgia, USA.
    Gaidet, Nicolas
    CIRAD, France.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Olsen, Björn
    Uppsala University.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Capturing individual-level parameters of influenza A virus dynamics in wild ducks using multistate models2016In: Journal of Applied Ecology, ISSN 0021-8901, E-ISSN 1365-2664, Vol. 53, no 4, p. 1289-1297Article in journal (Refereed)
    Abstract [en]

    Disease prevalence in wildlife is governed by epidemiological parameters (infection and recovery rates) and response to infection, both of which vary within and among individual hosts. Studies quantifying these individual-scale parameters and documenting their source of variation in wild hosts are fundamental for predicting disease dynamics. Such studies do not exist for the influenza A virus (IAV), despite its strong impact on the global economy and public health. Using capture-recaptures of 3500 individual mallards Anas platyrhynchos during seven migration seasons at a stopover site in southern Sweden, we provide the first empirical description of the individual-based mechanisms of IAV dynamics in a wild reservoir host. For most years, prevalence and risk of IAV infection peaked at a single time during the autumn migration season, but the timing, shape and intensity of the infection curve showed strong annual heterogeneity. In contrast, the seasonal pattern of recovery rate only varied in intensity across years. Adults and juveniles displayed similar seasonal patterns of infection and recovery each year. However, compared to adults, juveniles experienced twice the risk of becoming infected, whereas recovery rates were similar across age categories. Finally, we did not find evidence that infection influenced the timing of emigration.Synthesis and applications. Our study provides robust empirical estimates of epidemiological parameters for predicting influenza A virus (IAV) dynamics. However, the strong annual variation in infection curves makes forecasting difficult. Prevalence data can provide reliable surveillance indicators as long as they catch the variation in infection risk. However, individual-based monitoring of infection is required to verify this assumption in areas where surveillance occurs. In this context, monitoring of captive sentinel birds kept in close contact with wild birds is useful. The fact that infection does not impact the timing of migration underpins the potential for mallards to spread viruses rapidly over large geographical scales. Hence, we strongly encourage IAV surveillance with a multistate capture-recapture approach along the entire migratory flyway of mallards.

  • 6.
    Bengtsson, Daniel
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Avril, Alexis
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Gunnarsson, Gunnar
    Kristianstad University.
    Elmberg, Johan
    Kristianstad University.
    Söderquist, Pär
    Kristianstad University.
    Norevik, Gabriel
    Ottenby Bird Observatory.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Safi, Kamran
    Max Planck Institute for Ornitholology, Germany ; University of Konstanz, Germany.
    Fiedler, Wolfgang
    Max Planck Institute for Ornitholology, Germany ; University of Konstanz, Germany.
    Wikelski, Martin
    Max Planck Institute for Ornitholology, Germany ; University of Konstanz, Germany.
    Olsen, Björn
    Uppsala University.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Movements, Home-Range Size and Habitat Selection of Mallards during Autumn Migration2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 6, article id e100764Article in journal (Refereed)
    Abstract [en]

    The mallard (Anas platyrhynchos) is a focal species in game management, epidemiology and ornithology, but comparably little research has focused on the ecology of the migration seasons. We studied habitat use, time-budgets, home-range sizes, habitat selection, and movements based on spatial data collected with GPS devices attached to wild mallards trapped at an autumn stopover site in the Northwest European flyway. Sixteen individuals (13 males, 3 females) were followed for 15-38 days in October to December 2010. Forty-nine percent (SD = 8.4%) of the ducks' total time, and 85% of the day-time (SD = 28.3%), was spent at sheltered reefs and bays on the coast. Two ducks used ponds, rather than coast, as day-roosts instead. Mallards spent most of the night (76% of total time, SD = 15.8%) on wetlands, mainly on alvar steppe, or in various flooded areas (e.g. coastal meadows). Crop fields with maize were also selectively utilized. Movements between roosting and foraging areas mainly took place at dawn and dusk, and the home-ranges observed in our study are among the largest ever documented for mallards (mean = 6,859 ha; SD = 5,872 ha). This study provides insights into relatively unknown aspects of mallard ecology. The fact that autumn-staging migratory mallards have a well-developed diel activity pattern tightly linked to the use of specific habitats has implications for wetland management, hunting and conservation, as well as for the epidemiology of diseases shared between wildlife and domestic animals.

  • 7.
    Bengtsson, Daniel
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Safi, Kamran
    Max Planck Institute for Ornithology, Germany;University of Konstanz, Germany.
    Avril, Alexis
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Fiedler, Wolfgang
    Max Planck Institute for Ornithology, Germany;University of Konstanz, Germany.
    Wikelski, Martin
    Max Planck Institute for Ornithology, Germany;University of Konstanz, Germany.
    Gunnarsson, Gunnar
    Kristianstad University.
    Elmberg, Johan
    Kristianstad University.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Olsen, Björn
    Uppsala University.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Does influenza A virus infection affect movement behaviour during stopover in its wild reservoir host?2016In: Royal Society Open Science, E-ISSN 2054-5703, Vol. 3, no 2, p. 1-11, article id 150633Article in journal (Refereed)
    Abstract [en]

    The last decade has seen a surge in research on avian influenza A viruses (IAVs), in part fuelled by the emergence, spread and potential zoonotic importance of highly pathogenic virus subtypes. The mallard (Anas platyrhynchos) is the most numerous and widespread dabbling duck in the world, and one of the most important natural hosts for studying IAV transmission dynamics. In order to predict the likelihood of IAV transmission between individual ducks and to other hosts, as well as between geographical regions, it is important to understand how IAV infection affects the host. In this study, we analysed the movements of 40 mallards equipped with GPS transmitters and three-dimensional accelerometers, of which 20 were naturally infected with low pathogenic avian influenza virus (LPAIV), at a major stopover site in the Northwest European flyway. Movements differed substantially between day and night, as well as between mallards returning to the capture site and those feeding in natural habitats. However, movement patterns did not differ between LPAIV infected and uninfected birds. Hence, LPAIV infection probably does not affect mallard movements during stopover, with high possibility of virus spread along the migration route as a consequence.

  • 8.
    Ekström, Jens-Ola
    et al.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Tolf, Conny
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Edman, Kjell
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Lindberg, A Michael
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Physicochemical properties of the Ljungan virus virion in different environments: inactivated by heat but resistant th acidic pH, detergents, and non-physiological environments such as Virkon® containing soulutions2007In: Microbiology and immunology, ISSN 0385-5600, E-ISSN 1348-0421, Vol. 51, p. 841-850Article in journal (Refereed)
  • 9.
    Ekström, Jens-Ola
    et al.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Tolf, Conny
    Edman, Kjell
    Lindberg, Michael
    A novel mechanism for maturation of the VP1 protein of Ljungan virusManuscript (preprint) (Other (popular science, discussion, etc.))
  • 10.
    Ekström, Jens-Ola
    et al.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Tolf, Conny
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Fahlgren, Camilla
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Johansson, Susanne
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Arbrandt, Gustav
    Apodemus, Stockholm.
    Niklasson, Bo
    Apodemus, Stockholm.
    Edman, Kjell
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Lindberg, A. Michael
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Replication of Ljungan virus in cell culture: The genomic 5'-end, infectious cDNA clones and host cell response to viral infections2007In: Virus Research, ISSN 0168-1702, E-ISSN 1872-7492, Vol. 130, no December 2007, p. 129-139Article in journal (Refereed)
    Abstract [en]

    Ljungan virus (LV) is a picornavirus recently isolated from bank voles (Clethrionomys glareolus). The previously uncharacterised 5'-end sequence of the LV genome was determined. Infectious cDNA clones were constructed of the wild type LV prototype strain 87-012 and of the cytolytically replicating cell cultureadapted variant 87-012G. Virus generated from cDNA clones showed identical growth characteristics as uncloned virus stocks. Cell culture adapted LV, 87-012G, showed a clear cytopathic effect (CPE) at 3-4 days post-infection (p.i.). Virus titers, determined by plaque titration, increased however only within the first 18 h p.i. Replication of LV (+) strand RNA was determined by real-time PCR and corresponded in time with increasing titers. In contrast, the amounts of thereplication intermediate, the (-) strand, continued to increase until the cells showed CPE. This indicates separate controlling mechanisms for replication of LV (+) and (-) genome strands. Replication was also monitored by immunofluorescence (IF) staining. IF staining of both prototype 87-012 and the CPE causing 87-012G showed groups of 5-25 infected cells at 48 h p.i., suggesting a, for picornaviruses, not previously described direct cell-to-cell transmission.

  • 11.
    Gillman, Anna
    et al.
    Uppsala University.
    Muradrasoli, Shaman
    Uppsala University;Swedish University of Agricultural Sciences.
    Söderström, Hanna
    Umeå University.
    Holmberg, Fredrik
    Uppsala University.
    Latorre-Margalef, Neus
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Gunnarsson, Gunnar
    Kristianstad University.
    Olsen, Björn
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science. Uppsala University.
    Jarhult, Josef D.
    Uppsala University.
    Oseltamivir-Resistant Influenza A (H1N1) Virus Strain with an H274Y Mutation in Neuraminidase Persists without Drug Pressure in Infected Mallards2015In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 81, no 7, p. 2378-2383Article in journal (Refereed)
    Abstract [en]

    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.

  • 12.
    Gullberg, Maria
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Tolf, Conny
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Jonsson, Nina
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Mulders, Mick N
    Savolainen-Kopra, Carita
    Hovi, Tapani
    Van Ranst, Marc
    Lemey, Philippe
    Hafenstein, Susan
    Lindberg, A. Michael
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Characterization of a putative ancestor of coxsackievirus B5.2010In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 84, p. 9695-9708Article in journal (Refereed)
    Abstract [en]

    Like other RNA viruses, coxsackievirus B5 (CVB5) exists as circulating heterogeneous populations of genetic variants. In this study, we present the reconstruction and characterization of a probable ancestral virion of CVB5. Phylogenetic analyses based on capsid protein encoding regions (the VP1 gene of 41 clinical isolates and the entire P1 region of eight clinical isolates) of CVB5 revealed two major co-circulating lineages. Ancestral capsid sequences were inferred from sequences of these contemporary CVB5 isolates using maximum likelihood methods. By using Bayesian phylodynamic analysis, the inferred VP1 ancestral sequence was dated back to 1854 (1807-1898). In order to study the properties of the putative ancestral capsid, the entire ancestral P1 sequence was synthesized de novo and inserted into the replicative backbone of an infectious CVB5 cDNA clone. Characterization of the recombinant virus in cell culture showed that fully functional infectious virus particles were assembled and that these viruses displayed properties similar to those of modern isolates, in terms of receptor preferences, plaque phenotype, growth characteristics and cell tropism. This is the first report describing resurrection and characterization of a picornavirus with a putative ancestral capsid. Our approach, including phylogenetics-based reconstruction of viral predecessors, could serve as a starting point for experimental studies of viral evolution and might also provide an alternative strategy in the development of vaccines.

  • 13.
    Gullberg, Maria
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Tolf, Conny
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Jonsson, Nina
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Polacek, Charlotta
    Precechtelova, Jana
    Badurova, Miriam
    Sojka, Martin
    Mohlin, Camilla
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Israelsson, Stina
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Johansson, Kjell
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Bopegamage, Shubhada
    Hafenstein, Susan
    Lindberg, A. Michael
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    A single coxsackievirus B2 capsid residue controls cytolysis and apoptosis in rhabdomyosarcoma cells.2010In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 84, no 12, p. 5868-5879Article in journal (Refereed)
    Abstract [en]

    Coxsackievirus B2 (CVB2), one of six human pathogens of the group B coxsackieviruses within the enterovirus genus of Picornaviridae, causes a wide spectrum of human diseases ranging from mild upper respiratory illnesses to myocarditis and meningitis. The CVB2 prototype strain Ohio-1 (CVB2O) was originally isolated from a patient with summer grippe in the 1950s. Later on, CVB2O was adapted to cytolytic replication in rhabdomyosarcoma (RD) cells. Here, we present analyses of the correlation between the adaptive mutations of this RD variant and the cytolytic infection in RD cells. Using reverse genetics, we identified a single amino acid change within the exposed region of the VP1 protein (glutamine to lysine at position 164) as the determinant for the acquired cytolytic trait. Moreover, this cytolytic virus induced apoptosis, including caspase activation and DNA degradation, in RD cells. These findings contribute to our understanding of the host cell adaptation process of CVB2O and provide a valuable tool for further studies of virus-host interactions.

  • 14.
    Helin, Anu S.
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Chapman, Joanne R.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science. University of Kansas, USA.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Aarts, Lauren
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Bususu, Isaya
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Rosengren, Johan
    University of Queensland, Australia.
    Andersson, Håkan S.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Uppsala University, Sweden;Karolinska Institutet, Sweden.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Relation between structure and function of three AvBD3b variants from mallard (Anas platyrhynchos)Manuscript (preprint) (Other academic)
    Abstract [en]

    Defensins are multifunctional antimicrobial peptides expressed in several tissue types and leucocytes as part of the innate immune response against microbes. Based on the three-dimensional structure and disulfide connectivity, vertebrate defensins are subdivided into α-, β-, and θ-defensins. While all three types have been found in mammals, only β-defensins have been identified in birds. Genetic studies have revealed dozens of different avian β-defensin (AvBD) genes in different bird species, as well as allelic variation for different genes. Knowledge of the relation between avian peptide structure features and antimicrobial activity is however limited. In this study, the structure-functional relations of three variants of AvBD3b, a mallard (Anas platyrhynchos) defensin of evolutionary interest, was investigated. Gene alleles encoding two of these peptides, AvBD3b:1 and AvBD3b:2 are common in mallards, whereas AvBD3b:3 occurs rare. These β-defensin peptides were synthesized as linear peptides and subjected to oxidative folding. The three-dimensional structure of AvBD3b:1, including disulfide bond connectivity, was determined using NMR, and those of AvBD3b:2 and AvBD3b:3 respectively, were modelled using AvBD3b:1 as the template. The antimicrobial activities of folded peptides were compared to those of linear peptides. The NMR analysis showed that folded AvBD3b adopts a three-dimensional structure typical for β-defensins, including C-terminal antiparallel β-sheets and disulfide bond organization between six cysteine (C) residues: C6-C34, C13-C28, and C18-C35. Analyses of antimicrobial activity showed that both folded and linear variants of the three peptides inhibited bacterial growth. However, differences in activity were observed, suggesting that folded AvBD3b:3 was the most efficient against both Gram-negative and Gram-positive bacteria. Taken together, these findings provide additional insight into the influence of amino acid sequence variation and three-dimensional structure on the antimicrobial activity of mallard AvBD3b.

  • 15.
    Helin, Anu S.
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Chapman, Joanne R.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science. University of Kansas, USA.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Andersson, Håkan S.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Uppsala University, Sweden;Karolinska Institutet, Sweden.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    From genes to function: variation in antimicrobial activity of avian β-defensin peptides from mallardsManuscript (preprint) (Other academic)
    Abstract [en]

    Avian β-defensins are an important class of antimicrobial peptides in birds. These short cationic peptides are directly involved in the clearance of infections by membrane disruption, but can also act as immunomodulators and chemotactic agents recruiting immune cells. Recent genomic studies have shown the presence of several different avian β-defensin (AvBD) genes across the avian phylogeny, but also significant copy number variation and occurrence of pseudogenes. In mallard (Anas platyrhynchos) and other waterfowl AvBD genes are conserved and seem to be maintained by purifying selection. Due to their relatively simple peptide structure and direct mode of action, AvBDs is a potential tractable system to investigate how small differences in the gene sequence translates into differences in immune function. Here, we used genomic information from three different mallard defensin loci (AvBD4, AvBD10 and AvBD13) and synthesized the linear peptides from the most common allele of each locus, plus two rare alleles from AvBD13 locus and measured their antimicrobial activity against Gram-negative (E. coli and S. Typhimurium) and Gram-positive (S. aureus and M. luteus) bacteria. In these assays, AvBD4 showed the most potent antibacterial activity against both Gram-negative and Gram-positive bacteria, with an IC50 value of 0.48 mM against S. Typhimurium. Among AvBD13 peptides, the less frequently observed AvBD13:2 variant, was most potent, with IC50 value against S. aureus approximately 15 times lower than that of the most common AvBD13:1. Interestingly, AvBD10 had no antibacterial effect on the tested bacteria. Thus, antimicrobial function varied substantially among loci, but also within the AvBD13 locus, suggesting a direct link between genetic variation and immune function variation. Interestingly, results from assays with AvBD4 and AvBD13 seem to indicate that a higher positive net charge in peptides is associated with a more potent antibacterial effect.

  • 16. Jääskeläinen, Anne J
    et al.
    Kolehmainen, Pekka
    Voutilainen, Liina
    Hauffe, Heidi C
    Kallio-Kokko, Hannimari
    Lappalainen, Maija
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Lindberg, A. Michael
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Henttonen, Heikki
    Vaheri, Antti
    Tauriainen, Sisko
    Vapalahti, Olli
    Evidence of ljungan virus specific antibodies in humans and rodents, Finland.2013In: Journal of Medical Virology, ISSN 0146-6615, E-ISSN 1096-9071, Vol. 85, no 11, p. 2001-2008Article in journal (Refereed)
    Abstract [en]

    Ljungan virus (LV, genus Parechovirus, family Picornaviridae) is considered currently to be a rodent-borne virus. Despite suggested human disease associations, its zoonotic potential remains unclear. To date, LV antibody prevalence in both humans and rodents has not been studied. In this study, two different LV immunofluorescence assays (LV IFAs) were developed with LV genotypes 1 (LV strain 87-012G) and 2 (LV strain 145SLG), and cross-neutralization and -reaction studies were carried out with LV strain 145SLG. Finally, a panel of 37 Finnish sera was screened for anti-LV antibodies using two different LV IFAs (LV 145SLG and LV 87-012G) and a neutralization (NT) assay (LV 145SLG), and 50 samples from Myodes glareolus by LV IFA (LV 145SLG). The LV seroprevalence study showed 38% and 18% positivity in humans and M. glareolus, respectively. LV IFAs and NT assays were compared, and the results were in good agreement. The data are the first evidence of humans and rodents coming into contact with LV in Finland. Additional studies are required in order to acquire a better understanding of the prevalence, epidemiological patterns and possible disease association of LV infections.

  • 17.
    Kanagarajan, Selvaraju
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Tolf, Conny
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Lundgren, Anneli
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Waldenström, Jonas
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Brodelius, Peter E.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Transient Expression of Hemagglutinin Antigen from Low Pathogenic Avian Influenza A (H7N7) in Nicotiana benthamiana2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 3, article id e33010Article in journal (Refereed)
    Abstract [en]

    The influenza A virus is of global concern for the poultry industry, especially the H5 and H7 subtypes as they have the potential to become highly pathogenic for poultry. In this study, the hemagglutinin (HA) of a low pathogenic avian influenza virus of the H7N7 subtype isolated from a Swedish mallard Anas platyrhynchos was sequenced, characterized and transiently expressed in Nicotiana benthamiana. Recently, plant expression systems have gained interest as an alternative for the production of vaccine antigens. To examine the possibility of expressing the HA protein in N. benthamiana, a cDNA fragment encoding the HA gene was synthesized de novo, modified with a Kozak sequence, a PR1a signal peptide, a C-terminal hexahistidine (6xHis) tag, and an endoplasmic retention signal (SEKDEL). The construct was cloned into a Cowpea mosaic virus (CPMV)-based vector (pEAQ-HT) and the resulting pEAQ-HT-HA plasmid, along with a vector (pJL3:p19) containing the viral gene-silencing suppressor p19 from Tomato bushy stunt virus, was agro-infiltrated into N. benthamiana. The highest gene expression of recombinant plant-produced, uncleaved HA (rHA0), as measured by quantitative real-time PCR was detected at 6 days post infiltration (dpi). Guided by the gene expression profile, rHA0 protein was extracted at 6 dpi and subsequently purified utilizing the 6xHis tag and immobilized metal ion adsorption chromatography. The yield was 0.2 g purified protein per kg fresh weight of leaves. Further molecular characterizations showed that the purified rHA0 protein was N-glycosylated and its identity confirmed by liquid chromatography-tandem mass spectrometry. In addition, the purified rHA0 exhibited hemagglutination and hemagglutination inhibition activity indicating that the rHA0 shares structural and functional properties with native HA protein of H7 influenza virus. Our results indicate that rHA0 maintained its native antigenicity and specificity, providing a good source of vaccine antigen to induce immune response in poultry species.

  • 18. Kim, M-C
    et al.
    Kwon, Y-K
    Joh, S-J
    Kim, S-J
    Tolf, Conny
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Kim, J-H
    Sung, H-W
    Lindberg, A Michael
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Kwon, J-H
    Recent Korean isolates of duck hepatitis virus reveal the presence of a novel geno- and serotype comparing to duck hepatitis virus type 1 strains2007In: Archives of Virology, ISSN 0304-8608, E-ISSN 1432-8798, Vol. 152, p. 2059-2072Article in journal (Refereed)
  • 19.
    Latorre-Margalef, Neus
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science. Univ Georgia, USA.
    Avril, Alexis
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Olsen, Björn
    Uppsala University.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    How Does Sampling Methodology Influence Molecular Detection and Isolation Success in Influenza A Virus Field Studies?2016In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 82, no 4, p. 1147-1153Article in journal (Refereed)
    Abstract [en]

    Wild waterfowl are important reservoir hosts for influenza A virus (IAV) and a potential source of spillover infections in other hosts, including poultry and swine. The emergence of highly pathogenic avian influenza (HPAI) viruses, such as H5N1 and H5N8, and subsequent spread along migratory flyways prompted the initiation of several programs in Europe, North America, and Africa to monitor circulation of HPAI and low-pathogenicity precursor viruses (low-pathogenicity avian influenza [LPAI] viruses). Given the costs of maintaining such programs, it is essential to establish best practice for field methodologies to provide robust data for epidemiological interpretation. Here, we use long-term surveillance data from a single site to evaluate the influence of a number of parameters on virus detection and isolation of LPAI viruses. A total of 26,586 samples (oropharyngeal, fecal, and cloacal) collected from wild mallards were screened by real-time PCR, and positive samples were subjected to isolation in embryonated chicken eggs. The LPAI virus detection rate was influenced by the sample type: cloacal/fecal samples showed a consistently higher detection rate and lower cycle threshold (Ct) value than oropharyngeal samples. Molecular detection was more sensitive than isolation, and virus isolation success was proportional to the number of RNA copies in the sample. Interestingly, for a given Ct value, the isolation success was lower in samples from adult birds than in those from juveniles. Comparing the results of specific real-time reverse transcriptase (RRT)-PCRs and of isolation, it was clear that coinfections were common in the investigated birds. The effects of sample type and detection methods warrant some caution in interpretation of the surveillance data.

  • 20.
    Latorre-Margalef, Neus
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Grosbois, Vladimir
    International Research Center in Agriculture for Development, France.
    Wahlgren, John
    Karolinska Institutet.
    Munster, Vincent J.
    Erasmus Medical Center, The Netherlands ; National Institutes of Health, USA.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Fouchier, Ron A. M.
    Erasmus Medical Center, The Netherlands.
    Osterhaus, Albert D. M. E.
    Erasmus Medical Center, The Netherlands.
    Olsen, Björn
    Uppsala University.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Heterosubtypic Immunity to Influenza A Virus Infections in Mallards May Explain Existence of Multiple Virus Subtypes2013In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 9, no 6, article id e1003443Article in journal (Refereed)
    Abstract [en]

    Wild birds, particularly duck species, are the main reservoir of influenza A virus (IAV) in nature. However, knowledge of IAV infection dynamics in the wild bird reservoir, and the development of immune responses, are essentially absent. Importantly, a detailed understanding of how subtype diversity is generated and maintained is lacking. To address this, 18,679 samples from 7728 Mallard ducks captured between 2002 and 2009 at a single stopover site in Sweden were screened for IAV infections, and the resulting 1081 virus isolates were analyzed for patterns of immunity. We found support for development of homosubtypic hemagglutinin (HA) immunity during the peak of IAV infections in the fall. Moreover, re-infections with the same HA subtype and related prevalent HA subtypes were uncommon, suggesting the development of natural homosubtypic and heterosubtypic immunity (p-value = 0.02). Heterosubtypic immunity followed phylogenetic relatedness of HA subtypes, both at the level of HA clades (p-value = 0.04) and the level of HA groups (p-value = 0.05). In contrast, infection patterns did not support specific immunity for neuraminidase (NA) subtypes. For the H1 and H3 Clades, heterosubtypic immunity showed a clear temporal pattern and we estimated within-clade immunity to last at least 30 days. The strength and duration of heterosubtypic immunity has important implications for transmission dynamics of IAV in the natural reservoir, where immune escape and disruptive selection may increase HA antigenic variation and explain IAV subtype diversity.

  • 21.
    Latorre-Margalef, Neus
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science. Univ Georgia, Dept Populat Hlth, Coll Vet Med, Southeastern Cooperat Wildlife Dis Study, Athens, GA 30602 USA.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Grosbois, Vladimir
    Int Res Ctr Agr Dev CIRAD UPR AGIRs, Anim & Integrate Risk Management, F-34398 Montpellier, France.
    Avril, Alexis
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Bengtsson, Daniel
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Wille, Michelle
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Osterhaus, Albert D M E
    Erasmus MC, Dept Virol, Rotterdam, Netherlands.
    Fouchier, Ron A M
    Erasmus MC, Dept Virol, Rotterdam, Netherlands.
    Olsen, Björn
    Uppsala Univ.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Long-term variation in influenza A virus prevalence and subtype diversity in migratory mallards in northern Europe.2014In: Proceedings of the Royal Society of London. Biological Sciences, ISSN 0962-8452, E-ISSN 1471-2954, Vol. 281, no 1781, p. Article ID: UNSP 20140098-Article in journal (Refereed)
    Abstract [en]

    Data on long-term circulation of pathogens in wildlife populations are seldom collected, and hence understanding of spatial-temporal variation in prevalence and genotypes is limited. Here, we analysed a long-term surveillance series on influenza A virus (IAV) in mallards collected at an important migratory stopover site from 2002 to 2010, and characterized seasonal dynamics in virus prevalence and subtype diversity. Prevalence dynamics were influenced by year, but retained a common pattern for all years whereby prevalence was low in spring and summer, but increased in early autumn with a first peak in August, and a second more pronounced peak during October-November. A total of 74 haemagglutinin (HA)/neuraminidase (NA) combinations were isolated, including all NA and most HA (H1-H12) subtypes. The most common subtype combinations were H4N6, H1N1, H2N3, H5N2, H6N2 and H11N9, and showed a clear linkage between specific HA and NA subtypes. Furthermore, there was a temporal structuring of subtypes within seasons based on HA phylogenetic relatedness. Dissimilar HA subtypes tended to have different temporal occurrence within seasons, where the subtypes that dominated in early autumn were rare in late autumn, and vice versa. This suggests that build-up of herd immunity affected IAV dynamics in this system.

  • 22.
    Lawson, Becki
    et al.
    Zool Soc London, UK.
    Robinson, Robert A.
    British Trust Ornithol, UK.
    Fernandez, Julia Rodriguez-Ramos
    IDEXX Labs Ltd, UK.
    John, Shinto K.
    Zool Soc London, UK.
    Benitez, Laura
    Univ Complutense Madrid, Spain.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Risely, Kate
    British Trust Ornithol, UK.
    Toms, Mike P.
    British Trust Ornithol, UK.
    Cunningham, Andrew A.
    Zool Soc London, UK.
    Williams, Richard A. J.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science. Univ Complutense Madrid, Spain.
    Spatio-temporal dynamics and aetiology of proliferative leg skin lesions in wild British finches2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 14670Article in journal (Refereed)
    Abstract [en]

    Proliferative leg skin lesions have been described in wild finches in Europe although there have been no large-scale studies of their aetiology or epizootiology to date. Firstly, disease surveillance, utilising public reporting of observations of live wild finches was conducted in Great Britain (GB) and showed proliferative leg skin lesions in chaffinches (Fringilla coelebs) to be widespread. Seasonal variation was observed, with a peak during the winter months. Secondly, pathological investigations were performed on a sample of 39 chaffinches, four bullfinches (Pyrrhula pyrrhula), one greenfinch (Chloris chloris) and one goldfinch (Carduelis carduelis) with proliferative leg skin lesions and detected Cnemidocoptes sp. mites in 91% (41/45) of affected finches and from all species examined. Fringilla coelebs papillomavirus (FcPV1) PCR was positive in 74% (23/31) of birds tested: a 394 base pair sequence was derived from 20 of these birds, from all examined species, with 100% identity to reference genomes. Both mites and FcPV1 DNA were detected in 71% (20/28) of birds tested for both pathogens. Histopathological examination of lesions did not discriminate the relative importance of mite or FcPV1 infection as their cause. Development of techniques to localise FcPV1 within lesions is required to elucidate the pathological significance of FcPV1 DNA detection.

  • 23.
    Persson, Malin
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Gullberg, Maria
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Lindberg, A. Michael
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Kocer, Armagan
    University of Groningen, The Netherlands.
    Transportation of Nanoscale Cargoes by Myosin Propelled Actin Filaments2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 2, article id e55931Article in journal (Refereed)
    Abstract [en]

    Myosin II propelled actin filaments move ten times faster than kinesin driven microtubules and are thus attractive candidates as cargo-transporting shuttles in motor driven lab-on-a-chip devices. In addition, actomyosin-based transportation of nanoparticles is useful in various fundamental studies. However, it is poorly understood how actomyosin function is affected by different number of nanoscale cargoes, by cargo size, and by the mode of cargo-attachment to the actin filament. This is studied here using biotin/fluorophores, streptavidin, streptavidin-coated quantum dots, and liposomes as model cargoes attached to monomers along the actin filaments ("side-attached") or to the trailing filament end via the plus end capping protein CapZ. Long-distance transportation (> 100 mu m) could be seen for all cargoes independently of attachment mode but the fraction of motile filaments decreased with increasing number of side-attached cargoes, a reduction that occurred within a range of 10-50 streptavidin molecules, 1-10 quantum dots or with just 1 liposome. However, as observed by monitoring these motile filaments with the attached cargo, the velocity was little affected. This also applied for end-attached cargoes where the attachment was mediated by CapZ. The results with side-attached cargoes argue against certain models for chemomechanical energy transduction in actomyosin and give important insights of relevance for effective exploitation of actomyosin-based cargo-transportation in molecular diagnostics and other nanotechnological applications. The attachment of quantum dots via CapZ, without appreciable modulation of actomyosin function, is useful in fundamental studies as exemplified here by tracking with nanometer accuracy.

  • 24.
    Sandelin, Lisa Labbe
    et al.
    Uppsala University ; Kalmar County Hospital.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Larsson, Sara
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Wilhelmsson, Peter
    Linköping University.
    Salaneck, Erik
    Uppsala University.
    Jaenson, Thomas G. T.
    Uppsala University.
    Lindgren, Per-Eric
    Linköping University.
    Olsen, Björn
    Uppsala University.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Candidatus Neoehrlichia mikurensis in Ticks from Migrating Birds in Sweden2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 7, article id e0133250Article in journal (Refereed)
    Abstract [en]

    Candidatus Neoehrlichia mikurensis (CNM; family Anaplasmataceae) was recently recognized as a potential tick-borne human pathogen. The presence of CNM in mammals, in host-seeking Ixodes ticks and in ticks attached to mammals and birds has been reported recently. We investigated the presence of CNM in ornithophagous ticks from migrating birds. A total of 1,150 ticks (582 nymphs, 548 larvae, 18 undetermined ticks and two adult females) collected from 5,365 birds captured in south-eastern Sweden was screened for CNM by molecular methods. The birds represented 65 different species, of which 35 species were infested with one or more ticks. Based on a combination of morphological and molecular species identification, the majority of the ticks were identified as Ixodes ricinus. Samples were initially screened by real-time PCR targeting the CNM 16S rRNA gene, and confirmed by a second real-time PCR targeting the groEL gene. For positive samples, a 1260 base pair fragment of the 16S rRNA gene was sequenced. Based upon bacterial gene sequence identification, 2.1% (24/1150) of the analysed samples were CNM-positive. Twenty-two out of 24 CNM-positive ticks were molecularly identified as I. ricinus nymphs, and the remaining two were identified as I. ricinus based on morphology. The overall CNM prevalence in I. ricinus nymphs was 4.2%. None of the 548 tested larvae was positive. CNM-positive ticks were collected from 10 different bird species. The highest CNM-prevalences were recorded in nymphs collected from common redpoll (Carduelis flammea, 3/7), thrush nightingale (Luscinia luscinia, 2/29) and dunnock (Prunella modularis, 1/17). The 16S rRNA sequences obtained in this study were all identical to each other and to three previously reported European strains, two of which were obtained from humans. It is concluded that ornithophagous ticks may be infected with CNM and that birds most likely can disperse CNM-infected ticks over large geographical areas.

  • 25.
    Stedt, Johan
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Bonnedahl, Jonas
    Kalmar County Hospital;Uppsala University.
    Hernandez, Jorge
    Uppsala University.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    McMahon, Barry J.
    University College Dublin, UK.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Olsen, Björn
    Uppsala University.
    Drobni, Mirva
    Uppsala University;Östersund Hospital.
    Carriage of CTX-M type extended spectrum beta-lactamases (ESBLs) in gulls across Europe2015In: Acta Veterinaria Scandinavica, ISSN 0044-605X, Vol. 57, article id 74Article in journal (Refereed)
    Abstract [en]

    Background: Extended spectrum beta-lactamases (ESBLs), a group of enzymes conferring resistance to third generation cephalosporins have rapidly increased in Enterobacteriacae and pose a major challenge to human health care. Resistant isolates are common in domestic animals and clinical settings, but prevalence and genotype distribution varies on a geographical scale. Although ESBL genes are frequently detected in bacteria isolated from wildlife samples, ESBL dissemination of resistant bacteria to the environment is largely unknown. To address this, we used three closely related gull species as a model system and collected more than 3000 faecal samples during breeding times in nine European countries. Samples were screened for ESBL-producing bacteria, which were characterized to the level of ESBL genotype groups (SHV, TEM), or specific genotypes (CTX-M). Results: ESBL-producing bacteria were frequently detected in gulls (906 of 3158 samples, 28.7 %), with significant variation in prevalence rates between countries. Highest levels were found in Spain (74.8 %), The Netherlands (37.8 %) and England (27.1 %). Denmark and Poland represented the other extreme with no, or very few positive samples. Genotyping of CTX-M isolates identified 13 different variants, with bla(CTX-M-1) and bla(CTX-M-14) as the most frequently detected. In samples from England, Spain and Portugal, blaCTX-M-14 dominated, while in the rest of the sampled countries blaCTX-M-1 (except Sweden where bla(CTX-M-15) was dominant) was the most frequently detected genotype, a pattern similar to what is known from studies of human materials. Conclusions: CTX-M type ESBLs are common in the faecal microbiota from gulls across Europe. The gull ESBL genotype distribution was in large similar to published datasets from human and food-production animals in Europe. The data suggests that the environmental dissemination of ESBL is high from anthropogenic sources, and widespread occurrence of resistant bacteria in common migratory bird species utilizing urban and agricultural areas suggests that antibiotic resistance genes may also be spread through birds.

  • 26.
    Stoltz, Malin
    et al.
    Karolinska Institutet ; Swedish Institute for Communicable Disease Control.
    Sundström, Karin B.
    Karolinska Institutet ; Swedish Institute for Communicable Disease Control.
    Hidmark, Åsa
    University of Heidelberg, Germany.
    Tolf, Conny
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Vene, Sirkka
    Swedish Institute for Communicable Disease Control.
    Ahlm, Clas
    Umeå University.
    Lindberg, A. Michael
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Lundkvist, Åke
    Karolinska Institutet ; Swedish Institute for Communicable Disease Control.
    Klingström, Jonas
    Karolinska Institutet ; Swedish Institute for Communicable Disease Control.
    A model system for in vitro studies of bank vole borne viruses2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 12, article id e28992Article in journal (Refereed)
    Abstract [en]

    The bank vole (Myodes glareolus) is a common small mammal in Europe and a natural host for several important emerging zoonotic viruses, e.g. Puumala hantavirus (PUUV) that causes hemorrhagic fever with renal syndrome (HFRS). Hantaviruses are known to interfere with several signaling pathways in infected human cells, and HFRS is considered an immune-mediated disease. There is no in vitro-model available for infectious experiments in bank vole cells, nor tools for analyses of bank vole immune activation and responses. Consequently, it is not known if there are any differences in the regulation of virus induced responses in humans compared to natural hosts during infection. We here present an in vitro-model for studies of bank vole borne viruses and their interactions with natural host cell innate immune responses. Bank vole embryonic fibroblasts (VEFs) were isolated and shown to be susceptible for PUUV-infection, including a wild-type PUUV strain (only passaged in bank voles). The significance of VEFs as a model system for bank vole associated viruses was further established by infection studies showing that these cells are also susceptible to tick borne encephalitis, cowpox and Ljungan virus. The genes encoding bank vole IFN-β and Mx2 were partially sequenced and protocols for semi-quantitative RT-PCR were developed. Interestingly, PUUV did not induce an increased IFN-β or Mx2 mRNA expression. Corresponding infections with CPXV and LV induced IFN-β but not Mx2, while TBEV induced both IFN-β and Mx2.In conclusion, VEFs together with protocols developed for detection of bank vole innate immune activation provide valuable tools for future studies of how PUUV and other zoonotic viruses affect cells derived from bank voles compared to human cells. Notably, wild-type PUUV which has been difficult to cultivate in vitro readily infected VEFs, suggesting that embryonic fibroblasts from natural hosts might be valuable for isolation of wild-type hantaviruses.

  • 27.
    Tolf, Conny
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Bengtsson, Daniel
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Rodrigues, David
    Instituto Politécnico de Coimbra, Portugal.
    Latorre-Margalef, Neus
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Wille, Michelle
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Figueiredo, Maria Ester
    Instituto Politécnico de Coimbra, Portugal.
    Jankowska-Hjortaas, Monika
    Norwegian Veterinary Institute, Norway.
    Germundsson, Anna
    Norwegian Veterinary Institute, Norway.
    Duby, Pierre-Yves
    Instituto Politécnico de Coimbra, Portugal.
    Lebarbenchon, Camille
    Universite de La Reunion, France.
    Gauthier-Clerc, Michel
    Centre de Recherche de la Tour du Valat, France.
    Olsen, Björn
    Uppsala University.
    Waldenström, Jonas
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Birds and viruses at a crossroad: surveillance of influenza a virus in portuguese waterfowl2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 11, article id e49002Article in journal (Refereed)
    Abstract [en]

    During recent years, extensive amounts of data have become available regarding influenza A virus (IAV) in wild birds in northern Europe, while information from southern Europe is more limited. Here, we present an IAV surveillance study conducted in western Portugal 2008-2009, analyzing 1653 samples from six different species of waterfowl, with the majority of samples taken from Mallards (Anas platyrhynchos). Overall 4.4% of sampled birds were infected. The sampling results revealed a significant temporal variation in the IAV prevalence, including a pronounced peak among predominantly young birds in June, indicating that IAV circulate within breeding populations in the wetlands of western Portugal. The H10N7 and H9N2 subtypes were predominant among isolated viruses. Phylogenetic analyses of the hemagglutinin and neuraminidase sequences of H10N7, H9N2 and H11N3 virus showed that sequences from Portugal were closely related to viral sequences from Central Europe as well as to IAVs isolated in the southern parts of Africa, reflecting Portugal's position on the European-African bird migratory flyway. This study highlights the importance of Portugal as a migratory crossroad for IAV, connecting breeding stationary waterfowl with birds migrating between continents which enable transmission and spread of IAV.

  • 28.
    Tolf, Conny
    et al.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Ekström, Jens-Ola
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Gullberg, Maria
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Arbrandt, Gustav
    Apodemus AB.
    Niklasson, Bo
    Apodemus AB ; Uppsala University.
    Frisk, Gun
    Uppsala University.
    Liljeqvist, Jan-Ake
    Göteborg University.
    Edman, Kjell
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Lindberg, A Michael
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Characterization of polyclonal antibodies against the capsid proteins of Ljungan virus2008In: Journal of Virological Methods, ISSN 0166-0934, E-ISSN 1879-0984, Vol. 150, no 1-2, p. 34-40Article in journal (Refereed)
    Abstract [en]

    Ljungan virus (LV) is a suspected human pathogen isolated from voles in Sweden and North America. To enable virus detection and studies of localization and activity of virion proteins, polyclonal antibodies were produced against bacterially expressed capsid proteins of the LV strain, 87-012G. Specific detection of proteins corresponding to viral antigens in lysates of LV infected cells was demonstrated by immunoblotting using each one of the generated polyclonal antibodies. In addition, native viral antigens present in cell culture infected with LV strains 87-012G or 145SLG were detected in ELISA and by immunofluorescence using the antibodies against the VP0 and VP1 proteins. The anti-VP3 antibody did not react with native proteins of the LV virion, suggesting that the VP3 is less potent in evoking humoral response and may have a less exposed orientation in the virus capsid. No activity of the antibodies was observed against the closely related human parechovirus type 1. The polyclonal antibody against the VP1 protein was further used for detection of LV infected myocytes in a mouse model of LV-induced myocarditis. Thus, polyclonal antibodies against recombinant viral capsid proteins enabled detection of natural LV virions by several different immunological methods.

  • 29.
    Tolf, Conny
    et al.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Gullberg, Maria
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Ekström, Jens-Ola
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Jonsson, Nina
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Lindberg, A. Michael
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Identification of Ljungan virus VP0 and VP1 amino acid residues associated with cytolytic replication in cultured cells2009In: Archives of Virology, ISSN 0304-8608, E-ISSN 1432-8798, Vol. 154, no 8, p. 1271-1284Article in journal (Refereed)
    Abstract [en]

    Ljungan virus is a picornavirus isolated from Swedish and North American rodents. Replication of Ljungan virus in cultured cells normally induces a weak and delayed cytopathic effect compared to that of many other picornaviruses. However, efficiently replicating Ljungan virus variants may evolve during serial passages in cell culture. In this study, we evaluate the significance of three substitutions in capsid protein VP0 and VP1 of a cell-culture-adapted variant of the Swedish Ljungan virus 145SL strain. In contrast to the parental strain, this 145SLG variant grows to high titers in green monkey kidney cells and induces a distinct cytopathic effect. Reverse genetic analyses demonstrated that each one of the individual capsid substitutions contributes to lytic replication in cell culture, but also that expression of all three substitutions results in a 100- to 500-fold increase in viral titers compared to viruses encoding single capsid substitutions. In addition, as indicated by detection of activated caspase-3 and DNA fragmentation, there seems to be an association between increased replication efficiency of lytic Ljungan virus variants and induction of an apoptotic response in infected green monkey kidney cells.

  • 30.
    Tolf, Conny
    et al.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Gullberg, Maria
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Johansson, E Susanne
    Tesh, Robert B
    Andersson, Björn
    Lindberg, A. Michael
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Molecular characterization of a novel Ljungan virus (Parechovirus; Picornaviridae) reveals a fourth genotype and indications of ancestral recombination2009In: Journal of General Virology, ISSN 0022-1317, E-ISSN 1465-2099, Vol. 90, p. 843-853Article in journal (Refereed)
    Abstract [en]

    Ljungan virus (LV) was discovered 20 years ago in Swedish bank voles (Myodes glareolus, previously referred to as Clethrionomys glareolus) during the search for an infectious agent causing lethal myocarditis in young athletes. To date, the genomes of four LV isolates, including the prototype 87-012 strain, have been characterized. Three of these LV strains were isolated from bank voles trapped in Sweden. Sequence analysis of an American virus (M1146), isolated from a montane vole (Microtus montanus) in western USA, indicates that this strain represents a genotype that is different from the Swedish strains. Here, we present genomic analyses of a fifth LV strain (64-7855) isolated from a southern red-backed vole (Myodes gapperi) trapped during arbovirus studies in New York state in the north-eastern USA in the 1960s. Sequence analysis of the 64-7855 genome showed an LV-like genome organization and sequence similarity to other LV strains. Genetic and phylogenetic analyses of the evolutionary relationship between the 64-7855 strain and other viruses within the family Picornaviridae, including previously published LV strains, demonstrated that the 64-7855 strain constitutes a new genotype within the LV species. Analyses also showed that different regions of the 64-7855 genome have different phylogenetic relationships with other LV strains, indicating that previous recombination events have been involved in the evolution of this virus.

  • 31.
    Tolf, Conny
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Latorre-Margalef, Neus
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Wille, Michelle
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Bengtsson, Daniel
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Gunnarsson, Gunnar
    Kristianstad University.
    Grosbois, Vladimir
    Centre de coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), France.
    Hasselquist, Dennis
    Lund University.
    Olsen, Björn
    Uppsala University.
    Elmberg, Johan
    Kristianstad University.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Individual Variation in Influenza A Virus Infection Histories and Long-Term Immune Responses in Mallards2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 4, article id e61201Article in journal (Refereed)
    Abstract [en]

    Wild dabbling ducks (genus Anas) are the main reservoir for influenza A virus (IAV) in the Northern Hemisphere. Current understanding of disease dynamics and epidemiology in this virus-host system has primarily been based on population-level surveillance studies and infection experiments conducted in laboratory settings. Using a combined experimental-natural approach with wild-strain captive mallards (Anas platyrhynchos), we monitored individual IAV infection histories and immunological responses of 10 birds over the course of 15 months. This is the first detailed study to track natural IAV infection histories over several seasons amongst the same individuals growing from juvenile to adults. The general trends in the infection histories of the monitored birds reflected seasonal variation in prevalence at the population level. However, within the study group there were significant differences between individuals in infection frequency as well as in short and long term anti-IAV antibody response. Further observations included individual variation in the number of infecting virus subtypes, and a strong tendency for long-lasting hemagglutinin-related homosubtypic immunity. Specifically, all infections in the second autumn, except one, were of different subtypes compared to the first autumn. The variation among birds concerning these epidemiologically important traits illustrates the necessity for IAV studies to move from the level of populations to examine individuals in order to further our understanding of IAV disease and epidemiology.

  • 32.
    Tolf, Conny
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Wille, Michelle
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Haidar, Ann-Katrin
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Avril, Alexis
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Zohari, Siamak
    National Veterinary Institute.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Prevalence of avian paramyxovirus type 1 in Mallards during autumn migration in the western Baltic Sea region2013In: Virology Journal, ISSN 1743-422X, E-ISSN 1743-422X, Vol. 10, article id 285Article in journal (Refereed)
    Abstract [en]

    Background: Newcastle disease virus (NDV) is the causative agent of the Newcastle disease, a severe disease in birds associated with substantial economic losses to the poultry industry worldwide. Sweden is situated along the Western European waterfowl flyway and applies a non-vaccination policy combined with directives of immediate euthanisation of NDV infected flocks. During the last decades there have been several outbreaks with NDV in poultry in Sweden. However, less is known about the virus prevalence in the wild bird population including waterfowl, a well-established reservoir of avian paramyxovirus type 1 (APMV-1), the paramyxovirus serotype that include pathogenic NDV. Methods: The survey constituted of 2332 samples from Mallards (Anas platyrhynchos), trapped in the southern part of Sweden during autumn migration in 2010. These samples were screened for APMV-1 by real-time reverse transcription PCR, and viral strains from positive samples were isolated and characterized by sequence analysis of the fusion gene and by phylogenetic analysis. Conclusions: Twenty of these samples were positive for APMV-1, hence a virus prevalence of 0.9% (95% Confidence Interval [95% CI]=0.54%, 1.35%). The highest APMV-1 prevalence was detected in juvenile Mallards sampled in November (n=887, prevalence 1.24% ([95% CI])=0.67%, 2.24%). Sequence analysis and evaluation of phylogenetic relatedness indicated that isolated APMV-1 strains were lentogenic, and phylogenetically most closely related to genotype Ib strains within the clade of class II viruses. The sampling system employed enabled us to follow APMV-1 infections and the shedding of one particular viral strain in one individual bird over several days. Furthermore, combining previous screening results with the APMV-1 detections in this study showed that more than 50% of Mallards that tested positive for APMV-1 RNA were co-infected with influenza A virus.

  • 33.
    Wille, Michelle
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Avril, Alexis
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science. CIRAD, F-34398 Montpellier, France.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Schager, Anna
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Larsson, Sara
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Borg, Olivia
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Olsen, Björn
    Uppsala Univ.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Temporal dynamics, diversity, and interplay in three components of the virodiversity of a Mallard population: Influenza A virus, avian paramyxovirus and avian coronavirus2015In: Infection, Genetics and Evolution, ISSN 1567-1348, E-ISSN 1567-7257, Vol. 29, p. 129-137Article in journal (Refereed)
    Abstract [en]

    Multiple infections, or simultaneous infection of a host with multiple parasites, are the rule rather than the exception. Interactions between co-occurring pathogens in a population may be mutualistic, competitive or facilitative. For some pathogen combinations, these interrelated effects will have epidemiological consequences; however this is as yet poorly incorporated into practical disease ecology. For example, screening of Mallards for influenza A viruses (IAV) have repeatedly revealed high prevalence and large subtype diversity in the Northern Hemisphere. Other studies have identified avian paramyxovirus type 1 (APMV-1) and coronaviruses (CoVs) in Mallards, but without making inferences on the larger viral assemblage. In this study we followed 144 wild Mallards across an autumn season in a natural stopover site and constructed infection histories of IAV, APMV-1 and CoV. There was a high prevalence of IAV, comprising of 27 subtype combinations, while APMV-1 had a comparatively low prevalence (with a peak of 2%) and limited strain variation, similar to previous findings. Avian CoVs were common, with prevalence up to 12%, and sequence analysis identified different putative genetic lineages. An investigation of the dynamics of co-infections revealed a synergistic effect between CoV and IAV, whereby Coy prevalence was higher given that the birds were co-infected with IAV. There were no interactive effects between IAV and APMV-1. Disease dynamics are the result of an interplay between parasites, host immune responses, and resources; and is imperative that we begin to include all factors to better understand infectious disease risk. (C) 2014 Elsevier B.V. All rights reserved.

  • 34.
    Wille, Michelle
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science. Peter Doherty Inst Infect & Immun, Australia.
    Latorre-Margalef, Neus
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science. Lund University.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Halpin, Rebecca
    J Craig Venter Inst, USA.
    Wentworth, David
    J Craig Venter Inst, USA.
    Fouchier, Ron A. M.
    Erasmus MC, Netherlands.
    Raghwani, Jayna
    Univ Oxford, UK.
    Pybus, Oliver G.
    Univ Oxford, UK.
    Olsen, Bjorn
    Uppsala University.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Where do all the subtypes go?: Temporal dynamics of H8-H12 influenza A viruses in waterfowl2018In: Virus Evolution, E-ISSN 2057-1577, Vol. 4, no 2, article id vey025Article in journal (Refereed)
    Abstract [en]

    Influenza A virus (IAV) is ubiquitous in waterfowl. In the northern hemisphere IAV prevalence is highest during the autumn and coincides with a peak in viral subtype diversity. Although haemagglutinin subtypes H1-H12 are associated with waterfowl hosts, subtypes H8-H12 are detected very infrequently. To better understand the role of waterfowl in the maintenance of these rare subtypes, we sequenced H8-H12 viruses isolated from Mallards (Anas platyrhynchos) from 2002 to 2009. These rare viruses exhibited varying ecological and phylodynamic features. The Eurasian clades of H8 and H12 phylogenies were dominated by waterfowl sequences; mostly viruses sequenced in this study. H11, once believed to be a subtype that infected charadriiformes (shorebirds), exhibited patterns more typical of common virus subtypes. Finally, subtypes H9 and H10, which have maintained lineages in poultry, showed markedly different patterns: H10 was associated with all possible NA subtypes and this drove HA lineage diversity within years. Rare viruses belonging to subtypes H8-H12 were highly reassorted, indicating that these rare subtypes are part of the broader IAV pool. Our results suggest that waterfowl play a role in the maintenance of these rare subtypes, but we recommend additional sampling of non-traditional hosts to better understand the reservoirs of these rare viruses.

  • 35.
    Wille, Michelle
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science. Uppsala University.
    Latorre-Margalef, Neus
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science. University of Georgia, USA.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Stallknecht, D. E.
    University of Georgia, USA.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    No evidence for homosubtypic immunity of influenza H3 in Mallards following vaccination in a natural experimental system2017In: Molecular Ecology, ISSN 0962-1083, E-ISSN 1365-294X, Vol. 26, no 5, p. 1420-1431Article in journal (Refereed)
    Abstract [en]

    The Mallard (Anas platyrhynchos) is an important reservoir species for influenza A viruses (IAV), and in this host, prevalence and virus diversity are high. Studies have demonstrated the presence of homosubtypic immunity, where individuals are unlikely to be reinfected with the same subtype within an autumn season. Further, evidence for heterosubtypic immunity exists, whereby immune responses specific for one subtype offer partial or complete protection against related HA subtypes. We utilized a natural experimental system to determine whether homo- or heterospecific immunity could be induced following experimental vaccination. Thirty Mallards were vaccinated with an inactivated H3, H6 or a sham vaccine and after seroconversion were exposed to naturally infected wild conspecifics. All ducks were infected within 2days and had both primary and secondary infections. Overall, there was no observable difference between groups; all individuals were infected with H3 and H10 IAV. At the cessation of the experiment, most individuals had anti-NP antibodies and neutralizing antibodies against H10. Not all individuals had H3 neutralizing antibodies. The isolated H3 IAVs revealed genetic dissimilarity to the H3 vaccine strain, specifically substitutions in the vicinity of the receptor-binding site. There was no evidence of vaccine-induced homosubtypic immunity to H3, a likely result of both a poor H3 immune response in the ducks and H3 immune escape. Likewise, there was no observed heterosubtypic protection related to H6 vaccination. This study highlights the need for experimental approaches to assess how exposure to pathogens and resulting immune processes translates to individual and population disease dynamics.

  • 36.
    Wille, Michelle
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Latorre-Margalef, Neus
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Stallknecht, D.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Putative escape and poor humoral response to inactivatedvaccine in Mallards results in no homo- or heterosubtypic immunity againstH3 influenza A in a natural-experimental system.Manuscript (preprint) (Other academic)
  • 37.
    Wille, Michelle
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Avril, Alexis
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science. CIRAD, Campusinternational de Baillarguet, Montpellier 34398, France.
    Latorre-Margalef, Neus
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Wallerström, Sofie
    Karolinska institutet.
    Olsen, Björn
    Uppsala universitet.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Frequency and patterns of reassortment in natural influenza A virus infection in a reservoir host2013In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 443, no 15, p. 150-160Article in journal (Refereed)
    Abstract [en]

    Influenza A viruses (IAV) can dramatically alter both genotype and phenotype at a rapid rate as a product of co-infection and reassortment Avian IAV exhibit high levels of phylogenetic incongruence, suggesting high levels of reassortment in the virus reservoir. Using a natural-experimental system, we reconstructed relationships amongst 92 viruses across 15 subtypes from 10 Mallards in an autumn season. Phylogenetic analyses estimated that 56% of the isolated viruses were reassorted. Network analysis demonstrated different patterns of reassortment and limited exchange of segments between primary and secondary infections. No clear patterns of linkage between segments were found, and patterns within a season were likely the consequence of continued introduction ofnew constellations, high viral load and diversity in the wild bird reservoir, and co-infections. This is the first IAV study to implement multiple tools available for elucidating factors governing reassortment patterns in naturally infected Mallards.

  • 38.
    Wille, Michelle
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Latorre-Margalef, Neus
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Fouchier, R. A. M.
    Halpin, R. A.
    Wentworth, D. E.
    Ragwani, J.
    Pybus, O.
    Olsen, Björn
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Limited diffusion of genome constellations and pervasive reassortment arefeatures of long-term circulation of H4N6 influenza A in European waterfowlManuscript (preprint) (Other academic)
  • 39.
    Williams, Richard A. J.
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Molecular identification of papillomavirus in ducks2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 9096Article in journal (Refereed)
    Abstract [en]

    Papillomaviruses infect many vertebrates, including birds. Persistent infections by some strains can cause malignant proliferation of cells (i.e. cancer), though more typically infections cause benign tumours, or may be completely subclinical. Sometimes extensive, persistent tumours are recorded-notably in chaffinches and humans. In 2016, a novel papillomavirus genotype was characterized from a duck faecal microbiome, in Bhopal, India; the sixth papillomavirus genotype from birds. Prompted by this finding, we screened 160 cloacal swabs and 968 faecal samples collected from 299 ducks sampled at Ottenby Bird Observatory, Sweden in 2015, using a newly designed real-time PCR. Twenty one samples (1.9%) from six individuals (2%) were positive. Eighteen sequences were identical to the published genotype, duck papillomavirus 1. One additional novel genotype was recovered from three samples. Both genotypes were recovered from a wild strain domestic mallard that was infected for more than 60 days with each genotype. All positive individuals were adult (P = 0.004). Significantly more positive samples were detected from swabs than faecal samples (P < 0.0001). Sample type data suggests transmission may be via direct contact, and only infrequently, via the oral-faecal route. Infection in only adult birds supports the hypothesis that this virus is sexually transmitted, though more work is required to verify this.

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