A Single Vaccination of IBDV Subviral Particles Generated by Kluyveromyces marxianus Efficiently Protects Chickens against Novel Variant and Classical IBDV Strains

Infectious bursal disease (IBD), caused by the infectious bursal disease virus (IBDV), is a highly contagious and immunosuppressive disease in chickens worldwide. The novel variant IBDV (nvIBDV) has been emerging in Chinese chicken farms since 2017, but there are no available vaccines that can provide effective protection. Herein, the capsid protein VP2 from nvIBDV strain FJ-18 was expressed in Kluyveromyces marxianus with the aim to produce nvIBDV subviral particles (SVPs). Two recombinant strains constructed for expression of nvIBDV VP2 (nvVP2) and His-tagged VP2 (nvHVP2) formed two types of nvIBDV subviral particles (SVPs), namely nvVP2-SVPs and nvHVP2-SVPs. TEM scans showed that both SVPs were about 25 nm in diameter, but there was a large portion of nvVP2-SVPs showing non-spherical particles. Molecular dynamics simulations indicate that an N-terminal His tag strengthened the interaction of the nvHVP2 monomer and contributed to the assembly of SVPs. Vaccination of chicks with the nvHVP2-SVPs provided 100% protection against novel variant IBDV infection when challenged with the FJ-18 strain, as well as the classical strain BC6/85. By contrast, vaccination with the nvVP2-SVPs only provided 60% protection against their parent FJ-18 strain, suggesting that the stable conformation of subviral particles posed a great impact on their protective efficacy. Our results showed that the nvHVP2-SVPs produced by the recombinant K. marxianus strain is an ideal vaccine candidate for IBDV eradication.

Verification of an algorithm for actin tail detection of subviral particles by comparison with expert validated data

The locomotion of subviral particles of Marburg virus has been shown to be primarily actin based. For this work, a virologist selected 14 subviral particles that show actin tails in fluorescence image sequences. Using the tracked coordinates, examination areas around these subviral particles are defined. The brightness of within the examination area behind the subviral particle is analysed. In addition, the speed of the particle in each frame is calculated to investigate potential correlations between actin activity and particle speed. The results show that actin tracks can be automatically detected and analysed. First hints of a correlation between subviral particle movement and actin activity could be gathered with the presented actin tail quantifier.

Estimation of accuracy loss by training a deep-learning-based cell organelle recognition software using full dataset and a reduced dataset containing only subviral particle distribution information

In collaboration with the Institute of Virology, Philipps University, Marburg, a deep-learning-based method that recognizes and classifies cell organelles based on the distribution of subviral particles in fluorescence microscopy images of virus-infected cells has been further developed. In this work a method to recognize cell organelles by means of partial image information is extended. The focus is on investigating loss of accuracy by only providing information about subviral particles and not all cell organelles to an adopted Mask-R convolutional neural network. Our results show that the subviral particle distribution holds information about the cell morphology, thus making it possible to use it for cell organelle-labelling.

Development of a New Reverse Genetics System for Ebola Virus

ABSTRACT Ebola virus (EBOV) is a highly pathogenic negative-stranded RNA virus that has caused several deadly endemics in the past decades. EBOV reverse genetics systems are available for studying live viruses under biosafety level 4 (BSL-4) or subviral particles under BSL-2 conditions. However, these systems all require cotransfection of multiple plasmids expressing viral genome and viral proteins essential for EBOV replication, which is technically challenging and unable to naturally mimic virus propagation using the subviral particle. Here, we established a new EBOV reverse genetics system only requiring transfection of a single viral RNA genome into an engineered cell line that stably expresses viral nucleoprotein (NP), viral protein 35 (VP35), VP30, and large (L) proteins and has been fine-tuned for its superior permissiveness for EBOV replication. Using this system, subviral particles expressing viral VP40, glycoprotein (GP), and VP24 could be produced and continuously propagated and eventually infect the entire cell population. We demonstrated the authentic response of the subviral system to antivirals and uncovered that the VP35 amount is critical for optimal virus replication. Furthermore, we showed that fully infectious virions can be efficiently rescued by delivering the full-length EBOV genome into the same supporting cell, and the efficiency is not affected by genome polarity or virus variant specificity. In summary, our work provides a new tool for studying EBOV under different biosafety levels. IMPORTANCE Ebola virus is among the most dangerous viral pathogens, with a case fatality rate of up to 90%. Since 2013, the two largest and most complex Ebola outbreaks in Africa have revealed the lack of investigation on this notorious virus. A reverse genetics system is an important tool for studying viruses by producing mutant viruses or generating safer and convenient model systems. Here, we developed an EBOV life cycle modeling system in which subviral particles can spontaneously propagate in cell culture. In addition, this system can be employed to rescue infectious virions of homologous or heterologous EBOV isolates using either sense or antisense viral RNA genomes. In summary, we developed a new tool for EBOV research.

Development of a highly specific serodiagnostic ELISA for West Nile virus infection using subviral particles

West Nile virus (WNV), a member of the Japanese encephalitis virus (JEV) serocomplex group, causes lethal encephalitis in humans and horses. Because serodiagnosis of WNV and JEV is hampered by cross-reactivity, the development of a simple, secure, and WNV-specific serodiagnostic system is required. The coexpression of prM protein and E protein leads to the secretion of subviral particles (SPs). Deletion of the C-terminal region of E protein is reported to affect the production of SPs by some flaviviruses. However, the influence of such a deletion on the properties and antigenicity of WNV E protein is unclear. We analyzed the properties of full-length E protein and E proteins lacking the C-terminal region as novel serodiagnostics for WNV infection. Deletion of the C-terminal region of E protein suppressed the formation of SPs but did not affect the production of E protein. The sensitivity of an enzyme-linked immunosorbent assay (ELISA) using the full-length E protein was higher than that using the truncated E proteins. Furthermore, in the ELISA using full-length E protein, there was little cross-reactivity with anti-JEV antibodies, and the sensitivity was similar to that of the neutralization test.

A Putative Amphipathic Alpha Helix in Hepatitis B Virus Small Envelope Protein Plays a Critical Role in the Morphogenesis of Subviral Particles

ABSTRACT Hepatitis B virus (HBV) small (S) envelope protein has the intrinsic ability to direct the formation of small spherical subviral particles (SVPs) in eukaryotic cells. However, the molecular mechanism underlying the morphogenesis of SVPs from the monomeric S protein initially synthesized at the endoplasmic reticulum (ER) membrane remains largely elusive. Structure prediction and extensive mutagenesis analysis suggested that the amino acid residues spanning W156 to R169 of S protein form an amphipathic alpha helix and play essential roles in SVP production and S protein metabolic stability. Further biochemical analyses showed that the putative amphipathic alpha helix was not required for the disulfide-linked S protein oligomerization but was essential for SVP morphogenesis. Pharmacological disruption of vesicle trafficking between the ER and Golgi complex in SVP-producing cells supported the hypothesis that S protein-directed SVP morphogenesis takes place at the ER-Golgi intermediate compartment (ERGIC). Moreover, it was demonstrated that S protein is degraded in hepatocytes via a 20S proteasome-dependent but ubiquitination-independent nonclassic ER-associated degradation pathway. Taken together, the results reported here favor a model in which the amphipathic alpha helix at the antigenic loop of S protein attaches to the lumen leaflet to facilitate SVP budding from the ERGIC, whereas the failure of the budding process may result in S protein degradation by 20S proteasome in a ubiquitination-independent manner. IMPORTANCE SVPs are the predominant viral product produced by HBV-infected hepatocytes. Their levels exceed those of virion particles by 10,000- to 100,000-fold in the blood of HBV-infected individuals. The high levels of SVPs, or HBV surface antigen (HBsAg), in the circulation induce immune tolerance and contribute to the establishment of persistent HBV infection. The loss of HBsAg, often accompanied by the appearance of anti-HBsAg antibodies, is the hallmark of durable immune control of HBV infection. Therapeutic induction of HBsAg loss is thus considered to be essential for the restoration of the host antiviral immune response and functional cure of chronic hepatitis B. Our findings on the mechanism of SVP morphogenesis and S protein metabolism will facilitate the rational discovery and development of antiviral drugs to achieve this therapeutic goal.

Deep learning-based recognition of cell structures in fluorescence microscopy sequences with respect to their morphology on cells infected with Marburg virus

The Institute of Virology at the Philipps-Universität Marburg is currently researching possible drugs to combat the Marburg virus. This involves classifying cell structures based on fluoroscopic microscopic image sequences. Conventionally, membranes of cells must be marked for better analysis, which is time consuming. In this work, an approach is presented to identify cell structures in images that are marked for subviral particles. It could be shown that there is a correlation between the distribution of subviral particles in an infected cell and the position of the cell’s structures. The segmentation is performed with a "Mask-R-CNN" algorithm, presented in this work. The model (a region-based convolutional neural network) is applied to enable a robust and fast recognition of cell structures. Furthermore, the network architecture is described. The proposed method is tested on data evaluated by experts. The results show a high potential and demonstrate that the method is suitable.

Automatic Classification of the Movements of Directed and Undirected Subviral Particles

The development of drugs against pathogens that cause hemorrhagic fever, such as Marburg and Ebola virus, requires researchers to gather much information about the virus. The accelerating of the research process is of great interest; therefore a new algorithm was developed to analyze intracellular processes. The algorithm will classify the motion characteristics of subviral particles in fluorescence microscopic image sequences of Ebola or Marburg virusinfected cells. The classification is based on the calculation of mean squared displacement. The results look promising to distinguish different particle tracks in active and passive transport. The paper ends with a discussion.