Immune cytokines tumor necrosis factor alpha and type I interferons provide front-line defense against viral infection and are regulated in part by ubiquitin (Ub) and Ub-like molecules. Ubiquitin and Ub-like molecule ISG15 share a conserved C-terminal motif where a terminal glycine residue becomes attached to cellular target proteins. Nairoviruses and arteriviruses contain an ovarian tumor domain-containing protease (OTU protease) that was found to corrupt pathways by removing Ub or ISG15 from target proteins. This broad substrate specificity is unlike mammalian deubiquitinating enzymes, which cannot recognize both substrates. To understand how viral OTU domain-containing proteases remove Ub and ISG15, the crystal structure of the Crimean-Congo Heamorhaggic Fever nairovirus (CCHFV) was determined with Ub to 2.5 Å resolution. A computational model was built of the CCHFV Otu protease bound to ISG15 as well. The CCHFV Otu protease has several structural differences from known OTU proteases, manifesting in its broad substrate recognition capability.

Immune cytokines tumor necrosis factor alpha and type I interferons provide front-line defense against viral infection and are regulated in part by ubiquitin (Ub) and Ub-like molecules. Ubiquitin and Ub-like molecule ISG15 share a conserved C-terminal motif where a terminal glycine residue becomes attached to cellular target proteins. Nairoviruses and arteriviruses contain an ovarian tumor domain-containing protease (OTU protease) that was found to corrupt pathways by removing Ub or ISG15 from target proteins. This broad substrate specificity is unlike mammalian deubiquitinating enzymes, which cannot recognize both substrates. To understand how viral OTU domain-containing proteases remove Ub and ISG15, the crystal structure of the Crimean-Congo Heamorhaggic Fever nairovirus (CCHFV) was determined with Ub to 2.5 Å resolution. A computational model was built of the CCHFV Otu protease bound to ISG15 as well. The CCHFV Otu protease has several structural differences from known OTU proteases, manifesting in its broad substrate recognition capability.

Le virus de la fièvre hémorragique de Crimée-Congo (VFHCC) est un Nairovirus appartenant à la famille des Bunyaviridae, responsable d’une maladie hémorragique sévère chez l’Homme, associée à des symptômes non spécifiques et à une forte mortalité. La transmission se fait par morsure de tique ou par contact direct avec des fluides corporels contaminés. N’ayant ni vaccin ni traitement spécifique, un apport de connaissances sur les interactions cellulaires VFHCC-hôte ainsi que sur les mécanismes développés en réponse à l’infection est nécessaire.Nous avons tout d’abord étudié le potentiel antiviral de molécules sur la réplication du VFHCC. La chloroquine et la chlorpromazine ont été identifiées et inhibent efficacement la réplication virale avec une protection induite chez la souris contre l’infection, en particulier en combinaison avec la ribavirine.De nombreux virus sont connus pour être ciblés par, ou pour détourner la voie de l’autophagie. Nous avons regardé si l’infection par le VFHCC était associée à une modulation de l’autophagie et si la réplication virale était impactée par l’activité autophagique. L’étude de cellules hépatocytaires et épithéliales a montré une mobilisation massive du LC3, principal marqueur des vésicules autophagiques, par le VHFCC. Celle-ci reflète une induction du flux autophagique d’un nouveau type, n’impliquant pas les voies classiques de recrutement du LC3. La réplication virale n’est pas directement modulée par cette autophagie atypique mais des effets indirects sont à étudier. La plupart de ces observations ont été montrées pour le Nairovirus Dugbe avec cependant une cinétique différente.Le dernier axe étudié porte sur l’analyse de l’impact des IFITMs, facteurs de restriction virale connu pour interférer avec les processus de fusion membranaire, sur la réplication du virus Dugbe. L’étude a révélé une inhibition de la réplication virale par certains IFITMs.Des études supplémentaires portant sur l’interaction virus-cellule hôte et les mécanismes moléculaires associés sont nécessaires pour mieux comprendre la physiopathologie induite par le VFHCC et mettre au point de nouvelles stratégies thérapeutiques. / Crimean-Congo hemorrhagic fever virus (CCHFV) belongs to Nairovirus genus and to Bunyaviridae family. It is responsible for a severe hemorrhagic disease in humans, associated with non-specific symptoms and high lethality. Transmission is made by tick’s bite or by direct contact with contaminated body fluids. Since no vaccines or treatments are available, there is a need to accumulate knowledge on all aspects of CCHFV-host cell interaction as well as on response mechanisms that are taking place during infection.We first investigated pharmacological ways to interfere with CCHFV replication. Chloroquine and chlorpromazine (known modulators of some viral infections) were efficiently inhibiting viral replication and induce a protection in mice against CCHFV infection, particularly in the presence of ribavirin. Since several viruses are targeted by, or take advantage of, the autophagy response of infected cells, we explored whether CCHFV infection was associated with modulation of autophagy and whether virus replication was impacted by the autophagic activity of infected cells. By using hepatocytes and epithelial cells, we found that CCHFV induced a massive mobilization of the major marker of autophagic vesicles LC3. This mobilization reflected an induced autophagy flux and was of a novel type since known pathways of LC3 recruitment were not involved. The replication of CCHFV was indeed not directly modulated by this atypical form of autophagy but indirect effects remain to be studied. Most of these observations were found to be valid for the related, Dugbe virus (DUGV) with however, a distinct kinetic.Finally, we analyzed whether DUGV was sensitive to the IFITMs, restriction factors that can interfere with membrane fusion processes. Studies revealed that DUGV replication could be inhibited by some IFITMs. Additional studies on virus host-cell interactions and their associated molecular mechanisms should help to better understand the physiopathology induced by CCHFV and to devise therapeutic strategies.

<p>Abstract</p><p>Crimean-Congo Hemorrhagic Fever virus (CCHF) and Lassa virus are giving sources illness to humans. In addition to zoonotic transmission, CCHF and Lassa virus can spread from person to person. After a short incubation period, CCHF and Lassa virus infections are characterized by a sudden onset of high fever, chills, headache and cough just like flu. Even some people are vomiting and have diarrhoea. After a few days of illness hemorrhagic manifestations occur. Treatment options for CCHF and Lassa viruses are limited, and there is no vaccine available for use in humans. The purpose of the present study was to produce recombinant nucleocapsid protein of Lassavirus and CCHF virus including an aminoterminal His-tag by a Semliki Forest Virus Replicon (pSFV 4.2). The recombinant proteins are planned to be used in future development of diagnostic methods.</p>

Abstract Crimean-Congo Hemorrhagic Fever virus (CCHF) and Lassa virus are giving sources illness to humans. In addition to zoonotic transmission, CCHF and Lassa virus can spread from person to person. After a short incubation period, CCHF and Lassa virus infections are characterized by a sudden onset of high fever, chills, headache and cough just like flu. Even some people are vomiting and have diarrhoea. After a few days of illness hemorrhagic manifestations occur. Treatment options for CCHF and Lassa viruses are limited, and there is no vaccine available for use in humans. The purpose of the present study was to produce recombinant nucleocapsid protein of Lassavirus and CCHF virus including an aminoterminal His-tag by a Semliki Forest Virus Replicon (pSFV 4.2). The recombinant proteins are planned to be used in future development of diagnostic methods.

The great variation displayed by nucleic acid molecules in human cells, and the continuous discovery of their impact on life, consequently require continuous refinements of molecular analysis techniques. Padlock probes and rolling circle amplification offer single nucleotide discrimination in situ, a high signal-to-noise ratio and localized detection within cells and tissues. In this thesis, in situ detection of nucleic acids with padlock probes and rolling circle amplification was applied for detection of DNA in the single cell gel electrophoresis assay to detect nuclear and mitochondrial DNA. This assay is used to measure DNA damage and repair.  The behaviour of mitochondrial DNA in the single cell gel electrophoresis assay has earlier been controversial, but it was shown herein that mitochondrial DNA diffuses away early in the assay. In contrast, Alu repeats remain associated with the nuclear matrix throughout the procedure. A new twelve gel approach was also developed with increased throughput of the single cell gel electrophoresis assay. DNA repair of three genes OGG1, XPD and HPRT and of Alu repeats after H2O2 induced damage was further monitored. All three genes and Alu repeats were repaired faster than total DNA. Finally, padlock probes and rolling circle amplification were applied for detection of the single stranded RNA virus Crimean Congo hemorrhagic fever virus. The virus was detected by first reverse transcribing RNA into cDNA.. The virus RNA together with its complementary RNA and the nucleocapsid protein were detected in cultured cells. The work presented here enables studies of gene specific damage and repair as well as viral infections in situ. Detection by ligation offers high specificity and makes it possible to discriminate even between closely related molecules. Therefore, these techniques will be useful for a wide range of applications within research and diagnostics.

Geographically, Pakistan is in the western part of south Asia at about 24-37 °N latitudes and62-75 °E longitudes. Livestock and agriculture are two major sectors in Pakistan and play animportant role in the country economy.The tick infestation in livestock is not only devastating for animals and their products but alsobecome the cause of transmission of pathogens into humans. Crimean Congo fever (CCHF) isa viral tick-borne fatal disease. The dissemination of ticks and amplification of Crimean Congofever (CCHF) pathogen throughout the tick-animals-tick cycle, increases risk of transmissionto humans many times. In Pakistan, cases are reported in all areas, particularly those areaswhich lie on the border to CCHF endemic countries. There is a high prevalence of CCHF inboth Baluchistan and Khyber Pakhtunkhwa regions. Baluchistan is bordering with Afghanistanand Iran and Khyber Pakhtunkhwa with Afghanistan. Linear regression analysis revealed apositive significant association of high level of CCHF cases in livestock, with camels, goatsand sheep. The literacy rate is negatively significantly corelated with the numbers of cases.Statistical analysis of border effect revealed a high positive significant correlation of CCHFprevalence in areas near to borders. Both Baluchistan and Khyber Pakhtunkhwa (KPK) haslow literacy rate than other regions of Pakistan. Islamabad (capital city) has a higher literacyrate than all other regions but there is still a high CCHF prevalence. This is not only becauseof high population density but people from other regions, particularly from Baluchistan andKPK come here for animals selling or to seeking medical facilities in the large city hospitals.The study gives a proof that illiteracy and borders are the major respondent factors in theCCHF incidences and prevalence in an area. There is a need to raise awareness about ticksand tick-borne disease in the public and establishment of monitoring system across the bordersto prevent the spread of CCHF virus.

Ο ιός του αιμορραγικού πυρετού Κριμαίας-Κογκό (Crimean-Congo Hemorrhagic Fever Virus, CCHFV), καθώς και οι χανταϊοί (hantaviruses) προκαλούν στον άνθρωπο αιμορραγικό πυρετό. Αυτοί παρουσιάζουν ευρεία γεωγραφική κατανομή και αποτελούν απειλή για τη δημόσια υγεία, λόγω του υψηλού ποσοστού θνητότητας που σημειώνουν και της απουσίας αποτελεσματικής θεραπευτικής αγωγής. Παρότι επιδημιολογικές μελέτες δείχνουν την παρουσία αντισωμάτων στον ελληνικό πληθυσμό, περιορισμένες είναι οι αναφορές κλινικών περιστατικών CCHF και HFRS στην Ελλάδα. Σκοπός της παρούσας μελέτης είναι να προσδιορίσει τον επιπολασμό της μόλυνσης με τον CCHFV και τους χανταϊούς στον Ν. Αχαΐας, που αν και παρουσιάζει ευνοϊκές συνθήκες για την κυκλοφορία των δύο ιών, δεν έχει μελετηθεί στο παρελθόν. Σχεδιάσθηκε διατμηματική μελέτη και συγκεντρώθηκαν προοπτικά 207 δείγματα ορού φαινομενικά υγιών ατόμων-κατοίκων της περιοχής, τα οποία εξετάστηκαν με τη μέθοδο ELISA και έμμεσου ανοσοφθορισμού για την ύπαρξη αντισωμάτων έναντι του CCHFV και των χανταϊών. Ο επιπολασμός για τη μόλυνση με CCHFV βρέθηκε 3,4% και 9,7% για τη μόλυνση με χανταϊούς, ενώ κανένα από τα οροθετικά άτομα δεν ανακαλούσε συμπτώματα παρόμοια με αυτά του CCHF ή του HFRS. Για τον CCHFV, βρέθηκε ότι η ηλικία, η αγροτοκτηνοτροφική ενασχόληση, η κατοχή/εκτροφή αιγοπροβάτων, το ιστορικό νύγματος κρότωνα, η μόνιμη διαμονή σε υψόμετρο ≥400μ., η μόνιμη διαμονή σε μη αρδευόμενες αρόσιμες εκτάσεις ή σε αγροτικές εκτάσεις με σημαντικό ποσοστό φυσικής βλάστησης, καθώς και η μόνιμη διαμονή σε αγροτική περιοχή είναι σημαντικοί παράγοντες κινδύνου. Από αυτούς, το νύγμα κρότωνα, η αγροτοκτηνοτροφική ενασχόληση και η μόνιμη διαμονή σε υψόμετρο ≥400μ. βρέθηκαν να προβλέπουν καλύτερα την οροθετικότητα ενός ατόμου. Επίσης, βρέθηκε ότι παράγοντες που σχετίζονται με τη μόλυνση με χανταϊούς είναι: η ηλικία, η θέαση τρωκτικών σε ακτίνα <200μ. γύρω από την οικία και η ιδιοκτησία υπόγειας αποθήκης. Από αυτούς, μόνο η ηλικία βρέθηκε να προβλέπει καλύτερα την οροθετικότητα ενός ατόμου. Επιπλέον, παρατηρήθηκε ότι σχεδόν το 75% των θετικών ατόμων για αντισώματα έναντι των χανταϊών παρουσίαζαν ήπια επηρεασμένη νεφρική λειτουργία. Εντοπίστηκαν, επίσης, ενδημικές εστίες των ιών στον νομό: ο Δ. Ερυμάνθου για τον CCHFV και ο Δ. Δυτικής Αχαΐας για του χανταϊούς. Λαμβάνοντας υπόψη τα παραπάνω αποτελέσματα, θα πρέπει οι κλινικοί γιατροί της περιοχής να συμπεριλαμβάνουν τον CCHF και τον HFRS στη διαφορική διάγνωση εμπύρετων νοσημάτων, ιδίως όταν αυτά συνοδεύονται από θρομβοπενία ή επηρεασμένη νεφρική λειτουργία. / Crimean-Congo hemorrhagic fever virus (CCHFV) and hantaviruses cause to humans fever with hemorrhagic manifestations. These viruses present wide geographic distribution and represent major threats for public health, because of the high fatality rate that they present and the lack of appropriate treatment. Although seroprevalence studies show the presence of antibodies against CCHFV and hantaviruses in the greek population, only some reports of human cases have been reported to date in Greece. The aim of the present study is to estimate seroprevalence for CCHFV and hantaviruses in humans in the prefecture of Achaia, where the local conditions potentially favor the circulation of these viruses and which has not been previously studied. A cross-sectional study was designed and 207 human sera were collected from apparently healthy individuals living in Achaia, which were tested for CCHFV and hantaviruses IgG antibodies by ELISA and by indirect immunofluorescence assay (IFA). Seroprevalence for CCHFV infection was estimated at 3.4%, whereas for hantaviruses at 9.7%; none recalled any illness resembling CCHF or HFRS. For CCHFV, it was found that age, agro-pastoral occupation, tending sheep and/or goats, tick bite, living in areas at an altitude of ≥400m., living at rural areas, living on non-irrigated arable land or on land principally occupied by agriculture, with significant areas of natural vegetation are significantly related to seropositivity. Among them, tick bite, agro-pastoral occupation and living in areas at an altitude of ≥400m. better predict seropositivity of an individual. For hantaviruses, it was found that age, rodent sighting around home and the ownership of an underground shed are significantly related to seropositivity. Among them, it seems that only age can predict seropositivity of an individual. Moreover, it was observed that almost 75% of the seropositive for hantaviruses individuals presented mild renal dysfunction. In this study, endemic foci were also detected: the municipality of Erimanthos for CCHFV and the municipality of Western Achaia for hantaviruses. Clinicians should include CCHF and HFRS in the differential diagnosis of an acute febrile case, especially when thrombocytopenia or impaired renal function is encountered.