Viruses

Hepatitis B virus (HBV) is an enveloped pararetrovirus with a DNA genome, which is found in an up to 36 nm-measuring capsid. Replication of the genome occurs via an RNA intermediate, which is synthesized in the nucleus. The virus must have thus ways of transporting its DNA genome into this compartment. This review summarizes the data on hepatitis B virus genome transport and correlates the finding to those from other viruses

Can Robots Earn Our Trust the Same Way Humans Do?

Robots increasingly act as our social counterparts in domains such as healthcare and retail. For these human-robot interactions (HRI) to be effective, a question arises on whether we trust robots the same way we trust humans. We investigated whether the determinants competence and warmth, known to influence interpersonal trust development, influence trust development in HRI, and what role anthropomorphism plays in this interrelation. In two online studies with 2 × 2 between-subjects design, we investigated the role of robot competence (Study 1) and robot warmth (Study 2) in trust development in HRI. Each study explored the role of robot anthropomorphism in the respective interrelation. Videos showing an HRI were used for manipulations of robot competence (through varying gameplay competence) and robot anthropomorphism (through verbal and non-verbal design cues and the robot's presentation within the study introduction) in Study 1 (n = 155) as well as robot warmth (through varying compatibility of intentions with the human player) and robot anthropomorphism (same as Study 1) in Study 2 (n = 157). Results show a positive effect of robot competence (Study 1) and robot warmth (Study 2) on trust development in robots regarding anticipated trust and attributed trustworthiness. Subjective perceptions of competence (Study 1) and warmth (Study 2) mediated the interrelations in question. Considering applied manipulations, robot anthropomorphism neither moderated interrelations of robot competence and trust (Study 1) nor robot warmth and trust (Study 2). Considering subjective perceptions, perceived anthropomorphism moderated the effect of perceived competence (Study 1) and perceived warmth (Study 2) on trust on an attributional level. Overall results support the importance of robot competence and warmth for trust development in HRI and imply transferability regarding determinants of trust development in interpersonal interaction to HRI. Results indicate a possible role of perceived anthropomorphism in these interrelations and support a combined consideration of these variables in future studies. Insights deepen the understanding of key variables and their interaction in trust dynamics in HRI and suggest possibly relevant design factors to enable appropriate trust levels and a resulting desirable HRI. Methodological and conceptual limitations underline benefits of a rather robot-specific approach for future research

The FDA-Approved Drug Cobicistat Synergizes with Remdesivir To Inhibit SARS-CoV-2 Replication In Vitro and Decreases Viral Titers and Disease Progression in Syrian Hamsters

Combinations of direct-acting antivirals are needed to minimize drug resistance mutations and stably suppress replication of RNA viruses. Currently, there are limited therapeutic options against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and testing of a number of drug regimens has led to conflicting results. Here, we show that cobicistat, which is an FDA-approved drug booster that blocks the activity of the drug-metabolizing proteins cytochrome P450-3As (CYP3As) and P-glycoprotein (P-gp), inhibits SARS-CoV-2 replication. Two independent cell-to-cell membrane fusion assays showed that the antiviral effect of cobicistat is exerted through inhibition of spike protein-mediated membrane fusion. In line with this, incubation with low-micromolar concentrations of cobicistat decreased viral replication in three different cell lines including cells of lung and gut origin. When cobicistat was used in combination with remdesivir, a synergistic effect on the inhibition of viral replication was observed in cell lines and in a primary human colon organoid. This was consistent with the effects of cobicistat on two of its known targets, CYP3A4 and P-gp, the silencing of which boosted the in vitro antiviral activity of remdesivir in a cobicistat-like manner. When administered in vivo to Syrian hamsters at a high dose, cobicistat decreased viral load and mitigated clinical progression. These data highlight cobicistat as a therapeutic candidate for treating SARS-CoV-2 infection and as a potential building block of combination therapies for COVID-19

Etude des interactions de la capside du VHB impliquées dans le transport nucléaire

The Hepatitis B Virus (HBV) is an enveloped virus containing a partially double stranded DNA genome (rcDNA). HBV causes acute and chronic infections. HBV is not cytotoxic but chronic inflammation leads to liver fibrosis, cirrhosis and hepatocellular carcinoma. HBV replicates via an RNA intermediate, which is transcribed from a covalently closed circular form of the viral DNA (cccDNA). This pregenomic RNA is specifically encapsidated into the capsid by interaction with the viral polymerase, which also interacts with the core protein (Cp), forming the capsid. The polymerase retrotranscribes the pregenomic RNA into single stranded DNA and subsequently partially double stranded DNA resulting in mature capsids (MatC). Cp is an 185 aa long polypeptide comprising a N-terminal assembly domain, and a flexible C-terminal domain (CTD). The CTD includes two overlapping nuclear localization signals (NLS) of eight aa and an Importin ß Binding Domain (IBB) of 34 aa. The CTD is fixed in the interior of the capsid by interacting with single stranded nucleic acids but translocates to the exterior in MatC and empty capsids (EmpC). Cp is over expressed leading to assembly of EmpC. The virus has to deliver its genome into the nucleus of infected cells for replication. Nuclear transport is mediated by the capsid that interacts with nuclear import receptors. The group has recently shown that MatC need either both, importin (Imp.) and importin ß (Imp.ß), or Imp.ß alone for transport of the capsids into the nuclear basket. In this structure where genome liberation likely occurs, the transport of the capsid is arrested by interaction between the capsid and the nucleoporin Nup153. In the thesis we demonstrate that MatC binds to Imp.α but not Imp.ß, suggesting that only the part of the CTD, which contains the NLSs is exposed on capsids’ surface. In collaboration with the Adam Zlotnick (Indiana University, U.S.A.) we showed that EmpC, in contrast, bind Imp.β directly without Imp.α acting as an adaptor. This interaction, which is stronger than the one of Imp. occurs needs IBB exposure, meaning that the entire CTD becomes externalized. Furthermore, exposure to very high Imp.ß concentration led to EmpC destabilization. The genome release within the nuclear basket implies that Nup153 is involved in genome liberation from MatC. To verify this hypothesis we used MatC with a radioactively labeled genome, which were exposed to the capsid binding-Nup153 fragment. Investigating the accessibility of the genome to nucleases we found that the Nup153 fragment had no impact on capsids stability, suggesting the need of cellular factors driving disassembly. This conclusion is in agreement with our observation that MatC added to isolated nuclei resulted in nuclear capsid entry, which requires disassembly. To further study the disassembly step and the consequent release of the viral genome, we developed a system to directly visualize the viral genome allowing investigations of genome uncoating in real time. The system is based on the cooperative binding of a fluorescent fusion protein between the bacterial protein OR with GFP to a double stranded DNA sequence called Anch. Using this model we showed that infection of OR-GFP-expressing hepatoma cells with HBV containing a modified Anch genome allowed monitoring genome release into the nucleus. In future, this system may help identifying factors involved in genome release and repair and to decipher their molecular interactions.Le virus de l'hépatite B (VHB) est un virus enveloppé composé d'un ADN partiellement double brin (ADNrc) contenu dans une capside icosahédrique. Le VHB est responsable d'infections aiguës et chroniques. VHB est non cytopathique mais l’inflammation chronique entraîne une fibrose hépatique, une cirrhose et un carcinome hépatocellulaire. Le VHB se réplique via un intermédiaire à ARN. La transcription nécessite que l'ADNrc soit convertit en un ADN circulaire clos de manière covalente (ADNccc). Cet ADNccc sert de matrice pour la transcription de l'ARN prégénomique (ARNpg), qui est spécifiquement encapsidé grâce aux interactions entre la polymérase virale, l'ARNpg et la protéine core (Cp) qui forme la capside. La polymérase rétrotranscrit l'ARNpg en ADN monocaténaire puis en ADNrc, conduisant à des matrices de capside matures (MatC). Cp avec 185 aa contient un domaine N-terminal structuré, et un domaine C-terminal (CTD) flexible. Le CTD comprend deux signaux de localisation nucléaire (NLS) et un domaine de liaison avec l’importin β (IBB). Le CTD est orienté vers l'intérieur de la capside de part son interaction avec les acides nucléiques simples brins tandis qu'il est exposé vers l'extérieur dans les capsides vides (EmpC) et les MatC. De plus Cp étant surexprimée, cela conduit à l'assemblage des EmpC. Le VHB doit délivrer son génome dans le noyau des cellules infectées pour sa réplication. Le transport nucléaire est médié par la capside qui interagit avec les récepteurs d'import. L’équipe a démontré préalablement que ce transport a besoin des récepteurs Importin α (Imp.α) et Importin β (Imp.β) en induisant le transport des capsides au panier nucléaire où elle est stoppée par l'interaction avec la nucléoporine 153 (Nup153). Nous avons démontré que l’Imp.α, mais pas l'Imp.β, se lie aux MatC suggérant que seule la partie du CTD qui contient les NLS est exposée à l’extérieur des MatC. En comparaison, nous avons analysé les EmpC en collaboration avec Adam Zlotnick (Université d'Indiana, États-Unis) et démontré que les EmpC sont capables de lier directement l'Imp.β. Cette interaction qui est plus forte que l’interaction avec l'Imp.α s'effectue via la reconnaissance du domaine IBB du CTD, ce qui implique une exposition totale du CTD à l'extérieur de la capside. Nous avons aussi montré que la liaison avec l'Imp.β à des concentrations très élevées fournit des forces de déstabilisation menant au désassemblage des EmpC. La libération du génome dans le panier nucléaire implique que l’interaction entre les MatC et Nup153 participe au désassemblage de la capside. Afin de valider cette hypothèse, nous avons exposé des MatC dont le génome est radiomarqué avec un fragment de Nup153 contenant le domaine clé, montré pour interagir avec la capside, en présence de nucléases. Nous avons mis en évidence qu'en présence de ce fragment, les MatC restent stables. Cela suggère la nécessité de facteurs cellulaires additionnels pour le désassemblage des MatC. Cette conclusion est conforme avec nos résultats sur noyaux isolés, dans lesquels nous avons observé une localisation nucléaire des capsides laissant supposer que les facteurs cellulaires nécessaires au désassemblage des MatC sont nucléaires. Afin d'étudier plus en détail l'étape de désassemblage et la libération du génome viral, nous avons mis au point un système permettant de suivre en temps réel le devenir du génome viral

Nuclear Import of Hepatitis B Virus Capsids and Genome

Hepatitis B virus (HBV) is an enveloped pararetrovirus with a DNA genome, which is found in an up to 36 nm-measuring capsid. Replication of the genome occurs via an RNA intermediate, which is synthesized in the nucleus. The virus must have thus ways of transporting its DNA genome into this compartment. This review summarizes the data on hepatitis B virus genome transport and correlates the finding to those from other viruses

Interactions of HBV capsid involved in nuclear transport

Le virus de l'hépatite B (VHB) est un virus enveloppé composé d'un ADN partiellement double brin (ADNrc) contenu dans une capside icosahédrique. Le VHB est responsable d'infections aiguës et chroniques. VHB est non cytopathique mais l’inflammation chronique entraîne une fibrose hépatique, une cirrhose et un carcinome hépatocellulaire. Le VHB se réplique via un intermédiaire à ARN. La transcription nécessite que l'ADNrc soit convertit en un ADN circulaire clos de manière covalente (ADNccc). Cet ADNccc sert de matrice pour la transcription de l'ARN prégénomique (ARNpg), qui est spécifiquement encapsidé grâce aux interactions entre la polymérase virale, l'ARNpg et la protéine core (Cp) qui forme la capside. La polymérase rétrotranscrit l'ARNpg en ADN monocaténaire puis en ADNrc, conduisant à des matrices de capside matures (MatC). Cp avec 185 aa contient un domaine N-terminal structuré, et un domaine C-terminal (CTD) flexible. Le CTD comprend deux signaux de localisation nucléaire (NLS) et un domaine de liaison avec l’importin β (IBB). Le CTD est orienté vers l'intérieur de la capside de part son interaction avec les acides nucléiques simples brins tandis qu'il est exposé vers l'extérieur dans les capsides vides (EmpC) et les MatC. De plus Cp étant surexprimée, cela conduit à l'assemblage des EmpC. Le VHB doit délivrer son génome dans le noyau des cellules infectées pour sa réplication. Le transport nucléaire est médié par la capside qui interagit avec les récepteurs d'import. L’équipe a démontré préalablement que ce transport a besoin des récepteurs Importin α (Imp.α) et Importin β (Imp.β) en induisant le transport des capsides au panier nucléaire où elle est stoppée par l'interaction avec la nucléoporine 153 (Nup153). Nous avons démontré que l’Imp.α, mais pas l'Imp.β, se lie aux MatC suggérant que seule la partie du CTD qui contient les NLS est exposée à l’extérieur des MatC. En comparaison, nous avons analysé les EmpC en collaboration avec Adam Zlotnick (Université d'Indiana, États-Unis) et démontré que les EmpC sont capables de lier directement l'Imp.β. Cette interaction qui est plus forte que l’interaction avec l'Imp.α s'effectue via la reconnaissance du domaine IBB du CTD, ce qui implique une exposition totale du CTD à l'extérieur de la capside. Nous avons aussi montré que la liaison avec l'Imp.β à des concentrations très élevées fournit des forces de déstabilisation menant au désassemblage des EmpC. La libération du génome dans le panier nucléaire implique que l’interaction entre les MatC et Nup153 participe au désassemblage de la capside. Afin de valider cette hypothèse, nous avons exposé des MatC dont le génome est radiomarqué avec un fragment de Nup153 contenant le domaine clé, montré pour interagir avec la capside, en présence de nucléases. Nous avons mis en évidence qu'en présence de ce fragment, les MatC restent stables. Cela suggère la nécessité de facteurs cellulaires additionnels pour le désassemblage des MatC. Cette conclusion est conforme avec nos résultats sur noyaux isolés, dans lesquels nous avons observé une localisation nucléaire des capsides laissant supposer que les facteurs cellulaires nécessaires au désassemblage des MatC sont nucléaires. Afin d'étudier plus en détail l'étape de désassemblage et la libération du génome viral, nous avons mis au point un système permettant de suivre en temps réel le devenir du génome viral.The Hepatitis B Virus (HBV) is an enveloped virus containing a partially double stranded DNA genome (rcDNA). HBV causes acute and chronic infections. HBV is not cytotoxic but chronic inflammation leads to liver fibrosis, cirrhosis and hepatocellular carcinoma. HBV replicates via an RNA intermediate, which is transcribed from a covalently closed circular form of the viral DNA (cccDNA). This pregenomic RNA is specifically encapsidated into the capsid by interaction with the viral polymerase, which also interacts with the core protein (Cp), forming the capsid. The polymerase retrotranscribes the pregenomic RNA into single stranded DNA and subsequently partially double stranded DNA resulting in mature capsids (MatC). Cp is an 185 aa long polypeptide comprising a N-terminal assembly domain, and a flexible C-terminal domain (CTD). The CTD includes two overlapping nuclear localization signals (NLS) of eight aa and an Importin ß Binding Domain (IBB) of 34 aa. The CTD is fixed in the interior of the capsid by interacting with single stranded nucleic acids but translocates to the exterior in MatC and empty capsids (EmpC). Cp is over expressed leading to assembly of EmpC. The virus has to deliver its genome into the nucleus of infected cells for replication. Nuclear transport is mediated by the capsid that interacts with nuclear import receptors. The group has recently shown that MatC need either both, importin (Imp.) and importin ß (Imp.ß), or Imp.ß alone for transport of the capsids into the nuclear basket. In this structure where genome liberation likely occurs, the transport of the capsid is arrested by interaction between the capsid and the nucleoporin Nup153. In the thesis we demonstrate that MatC binds to Imp.α but not Imp.ß, suggesting that only the part of the CTD, which contains the NLSs is exposed on capsids’ surface. In collaboration with the Adam Zlotnick (Indiana University, U.S.A.) we showed that EmpC, in contrast, bind Imp.β directly without Imp.α acting as an adaptor. This interaction, which is stronger than the one of Imp. occurs needs IBB exposure, meaning that the entire CTD becomes externalized. Furthermore, exposure to very high Imp.ß concentration led to EmpC destabilization. The genome release within the nuclear basket implies that Nup153 is involved in genome liberation from MatC. To verify this hypothesis we used MatC with a radioactively labeled genome, which were exposed to the capsid binding-Nup153 fragment. Investigating the accessibility of the genome to nucleases we found that the Nup153 fragment had no impact on capsids stability, suggesting the need of cellular factors driving disassembly. This conclusion is in agreement with our observation that MatC added to isolated nuclei resulted in nuclear capsid entry, which requires disassembly. To further study the disassembly step and the consequent release of the viral genome, we developed a system to directly visualize the viral genome allowing investigations of genome uncoating in real time. The system is based on the cooperative binding of a fluorescent fusion protein between the bacterial protein OR with GFP to a double stranded DNA sequence called Anch. Using this model we showed that infection of OR-GFP-expressing hepatoma cells with HBV containing a modified Anch genome allowed monitoring genome release into the nucleus. In future, this system may help identifying factors involved in genome release and repair and to decipher their molecular interactions

Approaching phantom complex after limb amputation in the canine species

The objective of this study was to describe the presence, prevalence, clinical manifestations, and risk factors of phantom complex and its effect on the quality of life for dogs that underwent amputation of a limb. An online questionnaire was developed containing 3 sections with a total of 69 questions. Clinical cases were recruited from a web site for 3-legged dog owners. Data were acquired from February to March 2015. Descriptive statistics and frequency distribution analyses were performed on the collected data. Chi-squared test or Fisher's exact test were used for assessment of the associations between categorical variables. One hundred seven questionnaires were completed by owners of dogs with limb amputation. The most frequent reason for amputation was related to neoplasia (54%). Pain after limb amputation was commonly experienced by dogs, and the time of onset and clinical manifestations of pain after limb amputation were found to resemble those of phantom complex. The duration of pre-amputation pain and time between diagnosis and amputation were identified as risk factors for a higher frequency of post-amputation pain episodes. This pilot study introduces previously unreported signs that may be interpreted as expressions of pain in amputee dogs

Identification of Hammondia heydorni oocysts by a heminested-PCR (hnPCR-AP10) based on the H-heydorni RAPD fragment AP10

Toxoplasma gondii, Hammondia hammondi, Neospora caninum, Neospora hughesi and Hammondia heydorni are members of the Toxoplasmatinae sub-family. They are closely related coccidians with similarly sized oocysts. Molecular diagnostic techniques, especially those based on polymerase chain reaction (PCR), can be successfully applied for the differentiation of Hammondia-like oocysts. In this paper, we describe a rapid and simple method for the identification of H. heydorni oocysts among other members of the Toxoplasmatinae sub-family, using a heminested-PCR (hnPCR-AP10) based on a H. heydorni RAPD fragment available in molecular database. DNA of oocysts of H. heydorni yielded a specific fragment of 289-290 bp in the heminested-PCR assay. No product was yielded when the primers were used for the amplification of DNA extracted from T. gondii, N. caninum, N. hughesi and H. hammondi, thus allowing the differentiation of H. heydorni among other members of the Toxoplasmatinae sub-family. The hnPCR-AP10 was capable of detecting H. heydorni genetic sequences from suspensions with at least 10 oocysts. In conclusion, the hnPCR-AP10 proved to be a reliable method to be used in the identification of H. heydorni oocysts from feces of dogs. (C) 2010 Elsevier B.V. All rights reserved.CNP