Biology subjects

Vapalahti, O.

Publications and source records attributed to Vapalahti, O..

41 records · Page 3Linked to original sources

bioRxiv2019

Maturation of Neuronal Activity in Caudalized Human Brain Organoids

Human brain organoids are an emerging tool to study functional neuronal networks in health and disease. A critical challenge is the engineering of brain organoids with defined regional identity and developmental stage. Here we describe a protocol for generating hindbrain-like organoids from human pluripotent stem cells. We first generated a stable pool of caudalized stem cells that expressed hindbrain identity transcription factors and differentiated into tissue containing neurons and astrocytes. After maturation, caudalized brain organoids presented synaptically connected networks consisting of glutamate-, GABA-, and serotoninergic postmitotic neurons. These mature neurons displayed electric properties and dendritic trees resembling medulla oblongata neurons. They fired spontaneous and evoked repetitive action potentials, released serotonin and displayed excitatory and inhibitory synaptic currents, functionally resembling the activity patterns observed in normal human fetal brain. Reminiscent of infected human fetal brain, infection with Zika virus hampered organoid development, while the treatment with anticonvulsant drugs - carbamazepine and valproic acid - reduced organoid growth. Neuronal maturation also occurred in the grafted organoids in vivo. In conclusion, our approach enables efficient derivation of caudalized neuronal stem cells that differentiate into mature and functional neurons in organoids with hindbrain identity following human developmental trajectory. The organoids provide excellent model to study congenital abnormalities in brain development and for drug testing.

neuroscience↗

bioRxiv2019

Snake deltavirus utilizes envelope proteins of different viruses to generate infectious particles

Hepatitis D virus (HDV) is the only known human satellite virus, and it requires hepatitis B virus (HBV) to form infectious particles. Until 2018 HDV was the sole representative of the genus Deltavirus. The subsequent identification of HDV-like agents in non-human hosts indicated a much longer evolutionary history of deltaviruses than previously hypothesized. Interestingly, none of the HDV-like agents were found in co-infection with an HBV-like agent suggesting that these viruses use different helper virus(es). Here we show, using snake deltavirus (SDeV), that HBV represents only a single example of helper viruses for deltaviruses. We cloned the SDeV genome into a mammalian expression plasmid, and by transfection could initiate SDeV replication in cultured snake and mammalian cells. By superinfecting SDeV infected cells with arenaviruses, or by transfecting different viral surface proteins, we could induce infectious SDeV particle production. Our findings indicate that deltaviruses can likely use a multitude of helper viruses or even viral glycoproteins to form infectious particles. Persistent and recurrent infections would be beneficial for the spread of deltaviruses. It seems plausible that new human and veterinary disease associations with deltavirus infections will be identified in the future.

microbiology↗

bioRxiv2019

Critical Nodes of Virus-Host Interaction Revealed Through an Integrated Network Analysis

Viruses are one of the major causes of various acute and chronic infectious diseases and thus a major contributor to the global burden of disease. Several studies have shown how viruses have evolved to hijack basic cellular pathways and evade innate immune response by modulating key host factors and signalling pathways. A collective view of these multiple studies could advance our understanding of viral evasion mechanisms and provide new therapeutic perspectives for the treatment of viral diseases. Here, we performed an integrative meta-analysis to elucidate the 17 different host-virus interactomes. Network and bioinformatics analyses showed how viruses with small genomes efficiently achieve the maximal effect by targeting multifunctional and highly connected host proteins with a high occurrence of disordered regions. We also identified the core cellular process subnetworks that are targeted by all the viruses. Integration with functional RNA interference (RNAi) datasets showed that a large proportion of the targets are required for viral replication. Furthermore, we performed an interactome-informed drug re-purposing screen and identified novel activities for broad-spectrum antiviral agents against hepatitis C virus and human metapneumovirus. Altogether, these orthogonal datasets could serve as a platform for hypothesis generation and follow-up studies to broaden our understanding of the viral evasion landscape.

immunology↗

bioRxiv2018

Identification of a novel deltavirus in Boa constrictor

Human hepatitis D virus (hHDV) forms the genus Deltavirus which has not been assigned to any virus family. hHDV is a satellite virus and needs hepatitis B virus (HBV) to make infectious particles. Deltaviruses are thought to have evolved in humans, since they have thus far not been identified elsewhere. Herein we report, prompted by a recent observation of HDV-like agent in birds, the identification of a deltavirus in a snake (Boa constrictor) designated as snake-HDV (sHDV). The circular 1711 nt RNA genome of the newly identified virus resembles hHDV in its coding strategy and size. We discovered sHDV when performing a meta-transcriptomic study on brain samples of two boas with central nervous system signs. We did not identify accompanying HBV-like sequences in the samples. By sequence comparison, the putative hepatitis D antigen (HDAg) of sHDV, encoded by one of the two open reading frames (ORFs), is roughly 50% identical to the previously known HDAgs. We used antiserum raised against recombinant snake HDAg to demonstrate a broad viral target cell spectrum, ranging from neurons over epithelial cells to leukocytes. Using RT-PCR, we detected sHDV RNA also in the liver and blood of the two snakes and could show sHDV infection not only in two of their juvenile offspring, but also in a water python (Liasis mackloti savuensis) in the same snake colony, indicating potentially vertical and horizontal transmission. The finding of abundant sHDAg in several tissues suggests that sHDV actively replicated in the studied animals. Our findings suggest that sHDV spread may not be restricted to hepadnavirus co-infection. This would imply that deltaviruses may employ other enveloped viruses for producing infectious particles.

microbiology↗

bioRxiv2018

Defining of MAbs-neutralizing sites on the surface glycoproteins Gn and Gc of a hantavirus using vesicular stomatitis virus pseudotypes and site-directed mutagenesis

Earlier four Monoclonal antibodies (MAbs) against surface glycoproteins Gn and Gc of Puumala virus (PUUV, genus Orthohantavirus, family Hantaviridae, order Bunyavirales) were generated and for three MAbs with neutralizing capacity the localization of binding epitopes was predicted using pepscan and phage-display techniques. In this work, we produced vesicular stomatitis virus (VSV) particles pseudotyped with the Gn and Gc glycoproteins of PUUV and applied site-directed mutagenesis to dissect the structure of neutralizing epitopes. Replacement of cysteine amino acid (aa) residues with alanines resulted in pseudotype particles with diminished (16 to 18%) neut-titers; double Cys[->]Ala mutants, as well as mutants with bulky aromatic and charged residues replaced with alanines, have shown even stronger reduction in neut-titers (from 25% to the escape phenotype). In silico modelling of the neut-epitopes supported the hypothesis that these critical residues are located on the surface of viral glycoprotein molecules and thus can be recognized by the antibodies indeed. Similar pattern was observed in experiments with mutant pseudotypes and sera collected from patients suggesting that these neut-epitopes are utilized in a course of human PUUV infection.\n\nIMPORTANCENeutralization of viruses by antibodies is one of the key events in infection. We identified a set of mutations in the surface proteins of PUUV that reduced the virus-neutralizing activity of MAbs. Moreover, we found three mutants with escaped phenotype. These data will help understanding the mechanisms of hantavirus neutralization and assist construction of vaccine candidates.

microbiology↗