| 1. |
- Akaberi, Dario, 1989-
(författare)
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Identification of protease inhibitors against Flaviviruses and Coronaviruses
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Vector-borne flaviviruses and coronaviruses of zoonotic origins are important human pathogens and represent a serious threat to public health worldwide. Flaviviruses can be found on all continents, apart from Antarctica, where they are transmitted by arthropod vectors causing millions of infections every year. While most of the infections are mild or asymptomatic, flaviviruses like dengue and yellow fever viruses can cause potentially lethal hemorrhagic fever and shock syndrome. Neurotropic flaviviruses like West Nile, Japanese encephalitis, and Tick-borne encephalitis (TBEV) can cause meningoencephalitis with long-term symptoms. Coronaviruses, and in particular betacoronaviruses of zoonotic origin like SARS (2003) and MERS (2012), have been periodically emerging since the early 2000s causing outbreaks of severe respiratory syndrome. The latest example is SARS-CoV-2 that after causing a cluster of infection in the Chinese city of Wuhan, spread all over the world causing at present over 6.9 million deaths. Although vaccines are essential in preventing infections or severe disease and hospitalization in the case of SARS-CoV-2, antivirals represent an extremely valuable tool for treatment and prevention of current and future flavivirus and coronavirus infections. In the work presented in this thesis we have used a combination of in silico and in vitro techniques to identify and test the activity of potential inhibitors of viral proteases. In our first study (paper 1) we unexpectedly identified an HIV protease inhibitor with in vitro activity against ZIKV NS2B-NS3 protease. The inhibitor was identified by virtual screening of a library of known protease inhibitors, evaluated by molecular dynamics simulation and finally tested against recombinant ZIKV protease using a FRET-based enzymatic assay. The same combination of molecular docking and molecular dynamics simulations were also used to correctly predict the activity of a known pan-Flavivirus protease inhibitor against TBEV protease (paper 2). As a result, we were the first to report peptide-based compounds with in vitro activity against TBEV. After the outbreak of the COVID-19 we switched our attention to SARS-CoV-2. We first tested the inhibitory effect of the broad-spectrum antiviral nitric oxide (NO) and found that the NO-releasing compound SNAP had a dose dependent inhibitory effect on SARS-CoV-2 replication in cell-based assays (paper 3). We speculated that SNAP could inhibit SARS-COV-2 protease by trans-nitration of the catalytic Cys145 of SARS-CoV-2 main protease and found that SNAP had a dose dependent inhibitory effect on recombinant SARS-CoV-2 Mpro protease activity in an in vitro enzymatic assay. In our last study (paper 4) we identified a new class of potent SARS-CoV-2 protease inhibitors through the affinity screening of DNA-encoded-chemical libraries containing 4.2 billion compounds. The identified compounds inhibited recombinant SARS-CoV-2 protease with IC50 as low as 25 nM and had a dose dependent antiviral effect in the low micromolar range in infected Calu-3 and Caco-2 cell lines.
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| 2. |
- Eriksson, Per
(författare)
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Avian Influenza Virus : Deciphering receptor interactions and their role in interspecies transmission
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Influenza A virus (IAV) annually infects approximately 5–15 % of the human population, causing ~500,000 deaths globally. Novel IAVs have emerged and spread pandemically in the human population, but have over time established endemic circulation with reduced pathogenicity causing seasonal influenza. The natural reservoir of IAVs is wild waterfowl. The past pandemics have been associated with host switch and have partly or entirely originated from birds, or adapted via passage through pigs (postulated IAV mixing vessel). Understanding IAV interspecies transmission mechanisms is essential for pandemic preparedness. Enzootic circulation of avian IAV (AIV) is concentrated to a few waterfowl species, while other bird species seldom are infected. A species barrier preventing IAV interspecies transmission has been suggested. To investigate IAV host range and mixing vessels, histochemistry studies were conducted with tissues from avian species, pigs, and humans. Virus adaptation to new hosts was studied by challenging tufted ducks and chickens with mallard-derived AIVs, together with AIV receptor tropism and glycoproteomic analysis of receptor distribution. Finally, receptor and tissue tropism in ducks was studied systematically for AIV (H1–16). More abundant AIV attachment to human than pig tissues was observed, questioning the pig mixing vessel theory. Attachment patterns of AIVs to bird tissues was generally broad with abundant attachment to trachea. However, among ducks, pronounced attachment was observed to colon of Anas spp., suggesting that intestinal infection might be restricted to Anas spp., whereas other species may be susceptible to respiratory infection. Tufted ducks and chickens could not be infected by intraesophageal inoculation further supporting this hypothesis. Glycan array analysis revealed 3’SLN, 3’STF, and their fucosylated and sulfated analogues as main AIV receptors. Moreover, AIV Neu5Acα2,6 recognition was widespread. Avian respiratory and intestinal tracts glycoproteomic analysis revealed that avian and mammalian receptor structures are much more similar than earlier thought. Furthermore, observed AIV subtype titer variation in challenged tufted ducks and chickens did not correlate with virus receptor tropism. In summary, this thesis suggests that IAV receptor recognition, in particular α2,3 vs. α2,6 sialylated receptor structures, is less important for the IAV interspecies barrier than previously thought.
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| 3. |
- Hesson, Jenny C., 1980-
(författare)
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Clearing up Culex Confusion : A Basis for Virus Vector Discrimination in Europe
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mosquito species of the Culex genus are the enzootic vectors for several bird-associated viruses that cause disease in humans. In Europe, these viruses include Sindbis (SINV), West Nile and Usutu viruses. The morphologically similar females of Cx. torrentium and Cx. pipiens are potential vectors of these viruses, but difficulties in correctly identifying the mosquito species have caused confusion regarding their respective distribution, abundance, ecology, and consequently their importance as vectors. Species-specific knowledge from correctly identified field material is however of crucial importance since previous research shows that the relatively unknown Cx. torrentium is a far more efficient SINV vector than the widely recognized Cx. pipiens. The latter is involved in the transmission of several other viruses, but its potential importance for SINV transmission is debated.In this thesis I describe the development of a molecular method for species identification, based on reliably identified males of Cx. torrentium and Cx. pipiens. This identification method was then used in consecutive studies on the distribution and relative abundance of the two species in Sweden and 12 other European countries, SINV field infection rates in mosquitoes identified to species level, and evaluation of potential trap bias associated with common sampling techniques.The results showed that Cx. torrentium is a far more common species in Europe than previously assumed. In Sweden and Finland, it is the dominant species, accounting for 89% of the sampled Culex population. In central Europe, it is equally common to Cx. pipiens, while Cx. pipiens dominates south of the Alps Mountain range. Larvae of both species are often found together in both artificial containers (e.g. car tires) and natural sites. Also, a trapping bias against Cx. torrentium was revealed for CDC-traps. For the first time, SINV was isolated from species-identified Cx. torrentium and Cx. pipiens mosquitoes caught in the field, with Cx. torrentium being superior in infection rates (36/1,000 vs. 8.2/1,000). Future studies on SINV, as well as other mosquito-borne bird viruses in Europe, can hopefully gain from the baseline information provided here, and from principles of vector discrimination discussed in the thesis.
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| 4. |
- Hoffman, Tove
(författare)
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Dispersal of ticks and their microorganisms by African-Western Palaearctic migratory birds
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In Europe, tick-borne diseases are the most widespread and common vector-borne diseases and their geographical distribution is increasing. The dispersal of ticks depends on the movements of their vertebrate hosts. Avian hosts are more likely to be involved in long-distance range expansion of ticks due to their migration pattern. Billions of birds in the African-Palaearctic migration system migrate biannually between breeding grounds in the Palaearctic and wintering grounds in Africa and thereby create natural links between Africa, Europe, and Asia. In this thesis the dispersal of ticks and their microorganisms by northbound migratory birds utilizing flyways in the African-Western Palaearctic region has been investigated and the association between bird ecology and tick taxon addressed. The results suggest that long-distance migratory birds with wintering regions in Africa are involved in northward dispersal of the tick species Hyalomma rufipes, a known vector or Crimean-Congo hemorrhagic fever virus, and that birds with an open or wetland habitat have more H. rufipes in comparison to birds with a winter habitat comprising forest and shrubs. The results also suggest a role for birds in the ecology of Alkhurma hemorrhagic fever virus, a hemorrhagic flavivirus, and a potential mechanism for dispersal of the virus to new regions, including Europe and Asia Minor. The results did not provide evidence for immature ticks of the Hyalomma marginatum complex and birds having a major role in the ecology and northward dispersal of tick-borne Anaplasma phagocytophilum, a zoonotic bacterium causing febrile illness in humans and domestic animals. However, the results give support to the idea of a divergent enzootic cycle of A. phagocytophilum involving birds as hosts. Finally, the results of this thesis suggest that H. rufipes do not serve as vectors or contribute to the transmission of the tularemia-causing bacterium Francisella tularensis and that migratory birds do not contribute to northward dispersal of F. tularensis-infected ticks. However, the results suggest that migratory birds contribute to northward dispersal of H. rufipes carrying both Francisella and spotted fever group Rickettsia species, including Francisella-like endosymbionts and Rickettsia aeschlimannii. In conclusion, this thesis helps to clarify the knowledge about the dispersal of ticks and the microorganisms they carry by northbound migrating birds in the African-Western Palaearctic region. Furthermore, it highlights the need of establishing surveillance programs for monitoring the risk of introduction and establishment of important exotic tick species, such as H. rufipes, and tick-borne pathogens in the Western Palaearctic.
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| 5. |
- Nguyen-Tien, Thang, 1988-
(författare)
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An epidemiological and virological study on mosquito-borne flavivirus transmission : The role of urban livestock keeping and the risk for mosquito-borne zoonotic diseases in humans in Hanoi city, Vietnam
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mosquito-borne diseases (MBDs), which are transmitted by the bite of mosquitoes to humans, are causing high morbidity and mortality worldwide and are emerging in many tropical cities. Urban livestock keeping is still growing in cities around the world, which can itself contribute to increasing the mosquito populations. In addition, the animals may also serve as reservoirs for zoonotic MBDs, which increase the risks for humans. In this thesis, knowledge and practices related to MBDs were assessed in 513 households, with and without livestock. The perceptions of the community and health care sector were explored, with a focus on the association of MBDs and livestock keeping in Hanoi city of Vietnam. Generally, the participants had relatively low awareness on MBDs prevention and control. Those who had better knowledge did also use more adequate preventive practices against MBDs (p<0.001). An entomological survey was performed to explore how livestock keeping in and around a large city was associated with the presence of mosquitoes and their risk for spreading flaviviruses. The findings indicated that the distribution of vector populations in Hanoi city was heterogeneous, and that livestock played a role in influencing this. Households with better preventive practices reduced the presence of mosquitoes. Pig keeping particularly lead to more mosquitoes – especially of the Culex genus. 12,861 adult mosquitoes were collected and pooled into 385 pools, whereof one pool of Culex tritaeniorhynchus was found positive for Japanese encephalitis virus (JEV) by a pan-flavivirus qPCR. Phylogenetic analysis indicated that the detected virus belonged to genotype I. A hospital-based study with 98 dengue patients and 99 non-dengue patients was conducted to investigate risk factors of dengue infection and dengue virus (DENV) variants circulating in Hanoi. Age group and living district were the identified risk factors that correlated with dengue status. Serum and blood samples were analysed by a panflavivirus qPCR. The results showed that DENV serotypes 1, 2, and 4 were co-circulating during this outbreak, of which DENV serotype 1 was the predominant genotype.This thesis provides insight into the risk of flavivirus-related diseases such as dengue fever and Japanese encephalitis for those living in Hanoi metropolitan city, Vietnam, and the links between mosquitoes, animals and humans. Expanded and effective vector control measures and vaccination programs need to be taken to prevent the transmission of mosquito-borne flavivirus due to the significant dispersion of Culex and Aedes mosquitoes.
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| 6. |
- Pham-Thanh, Long, 1975-
(författare)
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The influence of urban livestock-keeping on the epidemiology of mosquito-borne zoonotic flaviviruses in Hanoi city of Vietnam
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In rapid urbanizing cities, the high density of livestock populations in close vicinity to humans, and high mosquito vector abundance have provided suitable conditions for transmission of mosquito-borne zoonotic flaviviruses, that cause several million cases of human disease annually. This thesis explores the urban livestock-keeping in Hanoi and its potential influence on the epidemiology of flavivirus infections. Four major components were investigated: livestock development of the city, knowledge and practices of local people on mosquito-borne diseases (MBDs), mosquito vector abundance, and risk factors for flavivirus exposure in animals.The analyses of the livestock development policy, animal data and key informant interviews showed that the livestock production system of Hanoi had increased in line with the governmental policy during 2014 to 2018. However, some major challenges were identified such as the high proportion of small-scale livestock farms, animal diseases, and a slow progress of translocation of livestock farms out of the urban areas.A cross-sectional study was conducted in 513 households in six districts of Hanoi (two urban, two peripheral, and two peri-urban). Knowledge and practices were evaluated; mosquitoes and larvae inside and outside the houses were collected and identified; blood samples of pigs and dogs were tested by a West Nile virus competitive enzyme-linked immunosorbent assay (cELISA), a kit that allowed for detection of antibodies against several flaviviruses of different animal species. JEV-specific antibodies were confirmed by plaque reduction neutralization test (PRNT). Logistic regression models using seropositivity of households as the outcome were built to identify significant risk factors.A low level of knowledge and preventive practices against zoonotic MBDs in community was indicated in the study. Among 12,861 adult mosquitoes and 2,427 larvae collected, Culex mosquitoes were the most abundant (93.01%), followed by Anopheles (3.82%), Mansonia (1.21%), Armigeres (1.18%) and Aedes mosquitoes (0.78%). In contrast, Aedes genus larvae were prominent at 87.02%, followed by Culex spp. (12.2%). There was a positive association between pig-keeping and Culex mosquitoes (p<0.001). In total, 475 dogs and 636 pigs were tested by a cELISA. The overall flavivirus seroprevalences in dogs and pigs were 70.7% and 88.5%, respectively. The PRNT results for a subset of 50 dog sera (34 positive, 6 doubtful and 10 negative samples by cELISA), and 50 pig sera (26 positive, 6 doubtful and 18 negative samples by cELISA) showed a lower proportion of JEV-specific antibodies (28 positives for dog samples, and 13 positives for pig samples). In conclusion, this thesis shows associations between livestock-keeping and risks of mosquito-borne zoonotic flavivirus infections to humans in Hanoi.
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| 7. |
- Wallensten, Anders, 1974-
(författare)
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Influenza A virus in wild birds
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Influenza virus is a RNA virus that exists as different types and subtypes. Influenza A virus strains are known to cause disease in several bird and mammalian species. Wild birds are believed to constitute the natural reservoir for influenza A virus.In humans, influenza A virus causes yearly seasonal influenza epidemics of respiratory disease resulting in high morbidity and severe economic consequences. Due to the virus’ ability to change its antigenic properties by mutation, yearly vaccination is required for protection from the disease.There are many different subtypes of influenza virus which are characterized according to two surface structures - the hemagglutinin and neuraminidase proteins - , for example; H5N1. These subtypes have the ability to recombine, and thereby creating new variant combinations. If a subtype that the living population of humans has not encountered before starts to spread among humans, it can result in a pandemic. Pandemic outbreaks have occurred at irregular intervals throughout history and have had a devastating impact on mankind. For example the Spanish influenza pandemic of 1918 is thought to have killed more than 50 million people.Influenza A virus is also an important cause of disease in poultry where virus strains of some subtypes may change into forms that are highly pathogenic. These virus strains may transmit directly to man and multiple other species. This has been the case in the ongoing outbreak that started in South East Asia in 2003. All known subtypes of influenza A virus have been isolated from wild birds living in aquatic environments, mainly dabbling ducks. These species are considered to be the reservoir for influenza A virus. The virus causes sub clinical gastrointestinal infection in ducks. High amounts of virus are excreted in the feces and spread via the fecal-oral route through water where it can persist for a prolonged time.There are still many unknowns about the ecology of influenza virus in the wild bird reservoir. This thesis includes five articles where data are presented that add new knowledge on this subject. We add proof that wild ducks are indeed the host for most influenza A virus subtypes by presenting data from a meta-analysis on all published screening data from wild birds and by presenting data from a four year screening of migratory ducks that were caught and sampled at Ottenby Bird Observatory. Our investigations have shown that the prevalence of influenza virus in the wild duck population of western Eurasia shows temporal differences in comparison to the results found in studies in North America. The prevalence in western Eurasian ducks is high during the period August to December and also rises in the spring. These findings are of importance for the understanding of how influenza virus is perpetuated in nature. During the course of the study only low pathogenic subtypes were isolated. Of concern is the high frequency of isolation of virus strains of the H5 and H7 subtypes that are prone to change into highly pathogenic variants in poultry. Many of the strains isolated in our study are similar to the ones that have caused influenza outbreaks in poultry in Europe during the last seven years. This indicates that wild bird surveillance for influenza A virus can be of major value as a sentinel system to prevent outbreaks in domestic poultry.Studies on Black-headed Gulls (Larus ridibundus) revealed a previously unknown subtype, H16. This finding widened the spectra of known influenza A virus subtypes in nature.Influenza A virus was also isolated in samples from Guillemots (Uria aalge) in the Baltic Sea. This was the first time influenza A virus was isolated from this species in Europe. The isolated virus strains contained a mix of genes, some of which must have been derived from influenza A virus strains present in the North American bird population. This finding proves that limited exchanges between the virus strains present on the American and the Eurasian continents exist, which is of concern for evaluating the risk of spread of highly pathogenic virus strains by wild birds to the Americas.
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