Neuropathogenic flaviviruses : isolation and characterization of Zika and tick-borne encephalitis viruses from human brains

Flavivirus is a genus that consists of small RNA viruses transmitted by arthropod vectors, mosquitos and ticks. Flaviviruses cause human diseases of global concern, such as dengue fever, Yellow fever, West Nile disease and Japanese encephalitis, with hundreds of millions of infections every year. Zika virus (ZIKV), a mosquito-borne flavivirus, recently caused a major epidemic with severe and unexpected consequences. Circulating in sylvatic cycles for decades and traditionally causing only mild self-limiting disease, ZIKV quickly spread through the Americas between 2015 and 2016, leading to an unforeseen epidemic that affected hundreds of thousands of people. Following this outbreak of acute ZIKV infections, the number of microcephaly cases reported in neonates accumulated; a causal relationship was suspected and the World Health Organization declared a Public Health Emergency of International Concern in February 2016. We were able to provide a key piece of evidence in proving the causal relationship between ZIKV infection and microcephaly. We described a case of prolonged maternal viremia from an expectant mother infected with ZIKV at the 11th week of gestation, which led to brain damage in the fetus. Subsequently, we isolated the virus FB-FWUH-2016 from the brain tissue of the fetus in human neuroblastoma cells. There is no specific anti-viral treatment for ZIKV infections nor is there a fully effective vaccine. Thus, we screened several anti-cancer compounds known to possess anti-viral activities to determine their anti-Zika virus effectivity. We found that gemcitabine, saliphenylhalamide and obatoclax, the latter of which is a novel compound, inhibited ZIKV replication and virus production in retinal pigment epithelial cells. Furthermore, we found that the compounds differentially affect the metabolism of infected cells. These data provide novel information for anti-virus drug development, as these compounds affect the functions of infected cells instead of the virus itself. We further characterized the new epidemic virus isolate and conducted a study on cell tropism. In this study, we compared four ZIKV isolates, three of which were from the epidemic Asian lineage and the other was a prototypic virus of the African lineage. We found differences in cell susceptibility that favored the new strain and a closely related French Polynesian strain, particularly in cell lines originating from the placenta, umbilical veins, kidney and brain. Tick-borne encephalitis virus (TBEV), another flavivirus that affects the central nervous system, is endemic in many parts of Europe and in the Northern parts of Asia. Three subtypes of the virus, namely, the European, Siberian and Far Eastern subtypes, can cause encephalitis with different degrees of severity. The European subtype is most prominent in most of Europe, but the Siberian subtype is also found in the Baltic countries and Finland. Fatalities are rare, which made the Kotka Archipelago in Finland a new focus after the report of two fatal TBE cases in 2015 from the same island in the archipelago along with two other infected individuals. We isolated the virus from the brain of one of the deceased patients in human neuroblastoma cells and obtained sequence data on both fatal cases. Surprisingly, the viruses were the Siberian and European subtypes. During this and previous studies, we also found both viruses in ticks from the archipelago, which shows that the subtypes coexist in the same focus.Flavivirukset (suku Flavivirus, heimo Flaviviridae) ovat zoonoottisia viruksia, joista suurin osa tarttuu niveljalkaisten, hyttysten ja puutiaisten, välityksellä. Maailmanlaajuisesti flavivirusten aiheuttamia lievempiä ihottuma- ja kuumetauteja, sekä vakavampia keskushermostoinfektioita todetaan vuosittain satoja miljoonia. Denguekuume, Japanin aivotulehdus eli enkefaliitti, Länsi-Niilin kuume ja keltakuume ovat esimerkkejä flavivirusinfektioista. Hyttysvälitteiset flavivirukset säilyvät luonnossa kädellisten ja hyttysten välillä. Ne voivat myös vaihtaa urbaaniin kiertokulkuun ihmisten ja hyttysten välille, luoden näin mahdollisuuden epidemioille. Viime vuosina Zikavirus (ZIKV) on aiheuttanut laajan epidemian Etelä- ja Väli-Amerikassa. Tätä epidemiaa karakterisoivat erityisesti sikiöillä ja vastasyntyneillä esiintyneet infektiot aiheuttaen eriasteisia keskushermoston kehityshäiriöitä, kuten mikrokefaliaa. Puutiaisten välittämistä flaviviruksista puutiaisaivokuumevirus (TBEV) on kotoperäinen useassa Euroopan maassa. Suomessa sitä esiintyy rannikolla ja itäisessä Suomessa, pohjoisessa aina Simoon asti. Viime vuosina tautitapausten määrä on ollut kasvussa, ja myös kuolemaan johtaneita tapauksia on raportoitu. Tässä väitöskirjatutkimuksessa keskitytään näiden kahden keskushermostoinfektioita aiheuttavien flavivirusten, ZIKV ja TBEV, karakterisointiin. Väitöskirjatyössä kuvailemme raskaana olevan naisen zikavirusinfektion ja sen aiheuttaman vakavan seurauksen sikiölle. Zikavirusinfektion todettiin aiheuttavan potilaalla poikkeuksellisen pitkittyneen viruserityksen. Eristimme zikaviruksen sikiön aivokudoksesta, ja osoitimme näin osaltaan zikaviruksen ja sikiön aivovaurion yhteyden. Uuden sukupolven sekvensointitekniikkaa hyödyntäen selvitimme viruksen perimän. Zikavirusinfektion hoitoon ei ole vielä tehokasta viruslääkettä. Testasimme valikoitujen, jo tunnettujen antiviraalisina yhdisteinä vaikuttavien lääkkeiden tehoa zikavirusinfektioon soluviljelymallissa eristetyn viruksen avulla. Löysimme kolme solun omiin toimintoihin vaikuttavaa lääkeainetta, obatoclax, SaliPhe ja gemsitabiini, jotka estivät tehokkaasti zikaviruksen aiheuttaman solukuoleman. Nämä yhdisteet vaikuttivat viruksen ja isäntäsolun vuorovaikutuksiin viruksen elinkierron eri vaiheissa. Tutkimuksen kohteena olevaa vakavan tautimuodon aiheuttamaa zikaviruskantaa verrattiin soluviljelymallissa muihin lievempiä epidemioita aiheuttaneisiin zikaviruskantoihin. Tutkimuksessa osoitettiin aivokudoksesta eristetyn viruksen lisääntyvän tehokkaasti kohdusta, napasuonista, munuaisista ja aivoista peräisin olevissa solulinjoissa. Kotkan saaristossa on viimeisen vuosikymmenen aikana todettu uusi puutiaisaivokuumefokus. Erityisen merkittävän siitä tekee siellä esiintyneet vakavat TBEV-infektiot. Vuonna 2015 Kuutsalon saaressa esiintyi useita puutiaisaivokuumetapauksia, joista kaksi johtivat kuolemaan. Eristimme viruksen toisen tapauksen aivokudoksesta soluviljelmässä, ja virus osoittautui TBE-viruksen siperialaiseksi alatyypiksi. Aiemmassa tutkimuksessa Kotkan saaristosta on löydetty saman alatyypin TBE-virusta puutiaisita. Toinen tapauksista osoittautui täten hieman yllättäen viruksen eurooppalaisen alatyypin aiheuttamaksi. Löysimme saaristosta myös tätä alatyyppiä puutiaisesta, mikä osoittaa, että kaksi viruksen alatyyppiä esiintyvät poikkeuksellisesti samassa fokuksessa

Identification of linear human B-cell epitopes of tick-borne encephalitis virus

Peer reviewe

Antidepressant and Antipsychotic Drugs Reduce Viral Infection by SARS-CoV-2 and Fluoxetine Shows Antiviral Activity Against the Novel Variants in vitro

Repurposing of currently available drugs is a valuable strategy to tackle the consequences of COVID-19. Recently, several studies have investigated the effect of psychoactive drugs on SARS-CoV-2 in cell culture models as well as in clinical practice. Our aim was to expand these studies and test some of these compounds against newly emerged variants. Several antidepressants and antipsychotic drugs with different primary mechanisms of action were tested in ACE2/TMPRSS2-expressing human embryonic kidney cells against the infection by SARS-CoV-2 spike protein-dependent pseudoviruses. Some of these compounds were also tested in human lung epithelial cell line, Calu-1, against the first wave (B.1) lineage of SARS-CoV-2 and the variants of concern, B.1.1.7, B.1.351, and B.1.617.2. Several clinically used antidepressants, including fluoxetine, citalopram, reboxetine, imipramine, as well as antipsychotic compounds chlorpromazine, flupenthixol, and pimozide inhibited the infection by pseudotyped viruses with minimal effects on cell viability. The antiviral action of several of these drugs was verified in Calu-1 cells against the B.1 lineage of SARS-CoV-2. By contrast, the anticonvulsant carbamazepine, and novel antidepressants ketamine, known as anesthetic at high doses, and its derivatives as well as MAO and phosphodiesterase inhibitors phenelzine and rolipram, respectively, showed no activity in the pseudovirus model. Furthermore, fluoxetine remained effective against pseudoviruses with common receptor binding domain mutations, N501Y, K417N, and E484K, as well as B.1.1.7 (alpha), B.1.351 (beta), and B.1.617.2 (delta) variants of SARS-CoV-2. Our study confirms previous data and extends information on the repurposing of these drugs to counteract SARS-CoV-2 infection including different variants of concern, however, extensive clinical studies must be performed to confirm our in vitro findings.Peer reviewe

Fatal Tick-Borne Encephalitis Virus Infections Caused by Siberian and European Subtypes, Finland, 2015

In most locations except for Russia, tick-borne encephalitis is mainly caused by the European virus subtype. In 2015, fatal infections caused by European and Siberian tick-borne encephalitis virus subtypes in the same Ixodes ricinus tick focus in Finland raised concern over further spread of the Siberian subtype among widespread tick species.Peer reviewe

Characterization of low-density granulocytes in COVID-19

Author summary The emergence of SARS-COV-2 and the ensuing COVID-19 disease has revealed an unprecedented need to understand the pathological mechanisms of acute respiratory infections in more detail. Granulocytes are highly abundant cells of the innate immunity, and thus first responders towards acute infections. However, their excessive activation can cause unwanted tissue damage and detrimental effects in humans. This study identifies a population of low-density granulocytes (LDGs) in COVID-19 patient samples, which has been poorly described in the context of acute infections so far. These cells were subclassified and found to be mainly of immature phenotypes. Further characterization revealed COVID-19 LDGs as a phenotypically diverse population with immunosuppressive characteristics, which seemed to be in line with an elevated recruitment and activation of granulocytes. Altogether, these findings suggest LDG may play a role in COVID-19 disease progression. Severe COVID-19 is characterized by extensive pulmonary complications, to which host immune responses are believed to play a role. As the major arm of innate immunity, neutrophils are one of the first cells recruited to the site of infection where their excessive activation can contribute to lung pathology. Low-density granulocytes (LDGs) are circulating neutrophils, whose numbers increase in some autoimmune diseases and cancer, but are poorly characterized in acute viral infections. Using flow cytometry, we detected a significant increase of LDGs in the blood of acute COVID-19 patients, compared to healthy controls. Based on their surface marker expression, COVID-19-related LDGs exhibit four different populations, which display distinctive stages of granulocytic development and most likely reflect emergency myelopoiesis. Moreover, COVID-19 LDGs show a link with an elevated recruitment and activation of neutrophils. Functional assays demonstrated the immunosuppressive capacities of these cells, which might contribute to impaired lymphocyte responses during acute disease. Taken together, our data confirms a significant granulocyte activation during COVID-19 and suggests that granulocytes of lower density play a role in disease progression.Peer reviewe

Complete Protection from SARS-CoV-2 Lung Infection in Mice Through Combined Intranasal Delivery of PIKfyve Kinase and TMPRSS2 Protease Inhibitors

Emerging variants of concern of SARS-CoV-2 can significantly reduce the prophylactic and therapeutic efficacy of vaccines and neutralizing antibodies due to mutations in the viral genome. Targeting cell host factors required for infection provides a complementary strategy to overcome this problem since the host genome is less susceptible to variation during the life span of infection. The enzymatic activities of the endosomal PIKfyve phosphoinositide kinase and the serine protease TMPRSS2 are essential to meditate infection in two complementary viral entry pathways. Simultaneous inhibition in cultured cells of their enzymatic activities with the small molecule inhibitors apilimod dimesylate and nafamostat mesylate synergistically prevent viral entry and infection of native SARS-CoV-2 and vesicular stomatitis virus (VSV)-SARS-CoV-2 chimeras expressing the SARS-CoV-2 surface spike (S) protein and of variants of concern. We now report prophylactic prevention of lung infection in mice intranasally infected with SARS-CoV-2 beta by combined intranasal delivery of very low doses of apilimod dimesylate and nafamostat mesylate, in a formulation that is stable for over 3 months at room temperature. Administration of these drugs up to 6 hours post infection did not inhibit infection of the lungs but substantially reduced death of infected airway epithelial cells. The efficiency and simplicity of formulation of the drug combination suggests its suitability as prophylactic or therapeutic treatment against SARS-CoV-2 infection in households, point of care facilities, and under conditions where refrigeration would not be readily available

Novel flaviviruses from mosquitoes : Mosquito-specific evolutionary lineages within the phylogenetic group of mosquito-borne flaviviruses

Peer reviewe

Kinetics of Neutralizing Antibodies of COVID-19 Patients Tested Using Clinical D614G, B.1.1.7, and B 1.351 Isolates in Microneutralization Assays

Increasing evidence suggests that some newly emerged SARS-CoV-2 variants of concern (VoCs) resist neutralization by antibodies elicited by the early-pandemic wild-type virus. We applied neutralization tests to paired recoveree sera (n = 38) using clinical isolates representing the first wave (D614G), VoC1, and VoC2 lineages (B.1.1.7 and B 1.351). Neutralizing antibodies inhibited contemporary and VoC1 lineages, whereas inhibition of VoC2 was reduced 8-fold, with 50% of sera failing to show neutralization. These results provide evidence for the increased potential of VoC2 to reinfect previously SARS-CoV-infected individuals. The kinetics of NAbs in different patients showed similar decline against all variants, with generally low initial anti-B.1.351 responses becoming undetectable, but with anti-B.1.1.7 NAbs remaining detectable (>20) for months after acute infection

Kinetics of Neutralizing Antibodies of COVID-19 Patients Tested Using Clinical D614G, B.1.1.7, and B 1.351 Isolates in Microneutralization Assays

Increasing evidence suggests that some newly emerged SARS-CoV-2 variants of concern (VoCs) resist neutralization by antibodies elicited by the early-pandemic wild-type virus. We applied neutralization tests to paired recoveree sera (n = 38) using clinical isolates representing the first wave (D614G), VoC1, and VoC2 lineages (B.1.1.7 and B 1.351). Neutralizing antibodies inhibited contemporary and VoC1 lineages, whereas inhibition of VoC2 was reduced 8-fold, with 50% of sera failing to show neutralization. These results provide evidence for the increased potential of VoC2 to reinfect previously SARS-CoV-infected individuals. The kinetics of NAbs in different patients showed similar decline against all variants, with generally low initial anti-B.1.351 responses becoming undetectable, but with anti-B.1.1.7 NAbs remaining detectable (>20) for months after acute infection

Molecular detection and phylogenetic analysis of Borrelia miyamotoi strains from ticks collected in the capital region of Finland

Borrelia miyamotoi is an emerging pathogen that shares high similarity with relapsing fever Borrelia, but has an atypical clinical presentation. Within the framework of tick-borne disease surveillance in Finland, human serum samples suspected for tick-borne encephalitis (n=974) and questing ticks (n=739) were collected from the capital region in Finland to determine the prevalence of B. miyamotoi. All tested human samples were negative and 5 (0.68 %) Ixodes ricinus ticks were positive for B. miyamotoi. Partial sequencing of the flagellin (flaB) gene of 3 positive samples and 27 B. miyamotoi-positive tick samples obtained from previous studies across Finland were amplified, sequenced, and included in the phylogenetic analysis.The phylogenetic tree revealed that most B. miyamotoi strains isolated from ticks in Finland share high similarity with other European strains, including strains related to human infection. Possible disease transmission may occur during exposure to tick bites. A single strain collected from an I. persulcatus tick in Pajujärvi grouped with an outlier of B. miyamotoi strains isolated from Russia and Far East Asian countries. Further studies should investigate the pathogen’s role in human infection in Finland.Another important finding is the occurrence of I. persulcatus ticks (8%) collected by crowdsourcing from the coastal southern part of Finland. This suggests a regular introduction and a possible wide expansion of this tick species in the country. This could be associated with transmission of new pathogens.</p