Requirements for Assembly and Release of Newcastle Disease Virus-Like Particles: A Dissertation

The final step of paramyxovirus infection requires the assembly of viral structural components at the plasma membrane of infected cells followed by budding of virions. While the matrix (M) protein of some paramyxoviruses has been suggested to play a central role in the assembly and release of virus particles, the specific viral and host protein requirements are still unclear. Using Newcastle disease virus (NDV) as a prototype paramyxovirus, we explored the role of each of the NDV structural proteins in virion assembly and release. For these studies, we established a virus-like particle (VLP) system for NDV. The key viral proteins required for particle formation and the specific viral protein-protein interactions required for assembly and release of particles were explored in chapter 2. First we found that co-expression of all four proteins resulted in the release of VLPs with densities and efficiencies of release (1.18 to 1.16 g/cm3and 83.8%±1.1, respectively) similar to that of authentic virions. Expression of M protein alone, but not NP, F-K115Q or HN proteins individually, resulted in efficient VLP release. No combination of proteins in the absence of M protein resulted in particle release. Expression of any combination of proteins that included M protein yielded VLPs, although with different densities and efficiencies of release. To address the roles of NP, F and HN proteins in VLP assembly, the interactions of proteins in VLPs formed with different combinations of viral proteins were characterized by co-immunoprecipitation. The co-localization of M protein with cell surface F and HN proteins in cells expressing all combinations of viral proteins was characterized. Taken together, the results show that M protein is necessary and sufficient for NDV budding. Furthermore, they suggest that M protein – HN protein and M protein - NP interactions are responsible for incorporation of HN protein and NP proteins into VLPs and that F protein is incorporated indirectly due to interactions with NP and HN protein. Since the vacuolar protein sorting (VPS) system is involved in the release of several enveloped RNA viruses, chapter 3 describes studies which explored the role of the VPS system on NDV particle release. First, we characterized the effects of three dominant negative mutant proteins of the VPS pathway on particle release. Expression of dominant negative mutants of CHMP3, Vps4 and AIP1 proteins inhibited M protein particle release as well as release of complete VLPs. Mutation of a YANL sequence in the NDV M protein to AANA inhibited particle release while replacement of this sequence with either of the classical late domain motifs, PTAP or YPDL, completely restored particle release. The host protein AIP1, which binds YXXL late domain sequences, is incorporated into M protein particles. These results suggest that an intact VPS pathway is necessary for NDV VLP release and that the YANL sequence is an NDV M protein L domain. The sequence and structure of the Newcastle disease virus (NDV) fusion (F) protein are consistent with its classification as a type 1 glycoprotein. We have previously reported, however, that F protein can be detected in at least two topological forms with respect to membranes in both a cell-free protein synthesizing system containing membranes as well as infected COS-7 cells (J. Virol. 2004 77:1951). One form is the classical type 1 glycoprotein while the other is a polytopic form in which approximately 200 amino acids of the amino terminal end as well as the cytoplasmic domain (CT) are translocated across membranes. Furthermore, we detected CT sequences on surfaces of F protein expressing cells and antibodies specific for these sequences inhibited red blood cell fusion to HN and F protein expressing cells suggesting a role for surface expressed CT sequences in cell-cell fusion. In chapter 4, we extended these findings and found that the alternate form of the F protein can also be detected in infected and transfected avian cells, the natural host cells of NDV. Furthermore, the alternate form of F protein was also found in virions released from both infected COS-7 cells and avian cells by Western analysis. Mass spectrometry confirmed its presence in virions released from avian cells. Two different polyclonal antibodies raised against sequences of the CT domain of the F protein slowed plaque formation in both avian and COS-7 cells. Antibody specific for the CT domain also inhibited single cycle infections as detected by immunofluorescence of viral proteins in infected cells. The potential roles of this alternate form of the NDV F protein in infection are discussed. Virus-like particles (VLPs) generated from different viruses have been shown to have potential as good vaccines. Chapter 5 explored the potential of NDV VLPs as a vaccine for NDV or as a vaccine vector for human pathogens. Significant quantities of NDV VLPs can be produced from tissue culture cells. These VLPs are as pure as virions prepared in eggs. In addition, some rules for incorporation of viral proteins into VLPs were also explored. We found that the cytoplasmic domain of the fusion (F) protein is necessary for its incorporation into VLPs. We found that an HN protein with an HA tag at its carboxyl terminus was incorporated into VLPs. We also found that the HN and F proteins of NDV, strain B1, can be incorporated into VLPs with M and NP of strain AV. The demonstration of specific domains required for protein incorporation into particles is important in using NDV VLPs as a vaccine vector for important human pathogens. In conclusion, this dissertation presents results that show that the M protein plays a central role in NDV assembly and release, a finding that is consistent with findings with other paramyxoviruses. More importantly, this work extends the current knowledge of paramyxovirus assembly and release by providing the first direct evidence of interactions between paramyxovirus proteins. These interactions between viral proteins provide a rational basis for incorporation of viral proteins into particles. This work also provides a clearer understanding of the role of the host vacuolar protein sorting machinery in NDV budding. A clear understanding of virus assembly and budding process contributes to the design of strategies for therapeutic intervention and in the development of safer, more economical and effective vaccines

Ventilation performance of single zone occupied space in ancient Myanmar multistage roof buildings

© 2019 Published under licence by IOP Publishing Ltd. Roof ventilation gives passive cooling effect in removing hot air from tropical climate buildings. However, little is known about the impacts of building parameters on the passive cooling effect in ancient Myanmar multistage roof buildings. The authors believe that this is the first study to assess the ventilation performance of multistage roof buildings taking into account their typologies, ventilation modes and climatic parameters. This paper presents the ventilation performance of two roof typologies by varying defined parameters from studies in computational fluid dynamic simulations. Simulations were generated by using the 3D steady Reynolds Averaged Navier Stokes equations and k-ϵ turbulence models. The results of CFD studies revealed that the presence of gable vents allowed better indoor air movement although the indoor air turbulence was depended on the intensity of exterior wind speed and roof typology. It was found that the single gable roof buildings increased the indoor airspeed with insignificant indoor air temperature improvement for thermal comfort. The results of this CFD study substantiate the findings of another dynamic simulation study and support the conclusion that the three-stage roof buildings have more potential to improve a better thermal and ventilation performance if they have gable vents

Monoclonal anti-envelope antibody AP33 protects humanized mice against a patient-derived hepatitis C virus challenge

End-stage liver disease caused by hepatitis C virus (HCV) infection is a major indication for liver transplantation. However, immediately after transplantation the liver graft of viremic patients universally becomes infected by circulating virus, resulting in accelerated liver disease progression. Currently available direct-acting antiviral therapies have reduced efficacy in patients with end-stage liver disease and prophylactic strategies to prevent HCV recurrence are still highly needed. In this study we compared the ability of two broadly reactive monoclonal antibodies (mAbs), designated 3/11 and AP33, recognizing a distinct but overlapping epitope in the viral E2 glycoprotein to protect humanized mice from a patient-derived HCV challenge. Their neutralizing activity was assessed using the HCVpp and HCVcc systems expressing multiple patient-derived envelopes and a human-liver chimeric mouse model. HCV RNA was readily detected in all control mice challenged with a patient-derived HCV genotype 1b isolate, while three out of four AP33-treated mice were completely protected. In contrast, only one out of four 3/11-treated mice remained HCV RNA negative throughout the observation period, while the other three had a viral load that was indistinguishable from that in the control group. The increased in vivo efficacy of AP33 was in line with its higher affinity and neutralizing capacity observed in vitro. Conclusion: Although mAbs AP33 and 3/11 target the same region in E2, only mAb AP33 can efficiently protect from challenge with a heterologous HCV population in vivo. Since mAb AP33 efficiently neutralizes viral variants that escaped the humoral immune response and re-infected the liver graft of transplant patients, it may be a valuable candidate to prevent HCV recurrence. In addition our data is valuable for the design of a prophylactic vaccine

Analysis of a rotary passive heat recovery device for natural ventilation windcatcher

Based on the design of traditional architecture, windcatchers are devices which provides passive ventilation by manipulating pressure differentials around buildings induced by the movement of wind and difference in temperature. In temperature climates, it is effective in providing passive cooling during summer months. However, during winter months, the low air temperature supplied to the space can cause further thermal discomfort and increase heat loss which lead to higher energy consumption. This limits the capabilities of windcatchers to provide ventilation all year round. To address this issue, the present study proposes incorporating a rotary thermal heat recovery device into the windcatcher channel and investigate its performance using numerical modelling and experimental tests. The study focused on characterizing the design and performance of the copper radial blades of the proposed heat recovery device. The predicted results of the airflow and temperature showed good agreement with the experimental tests. Two types of radial blade designs were assessed in terms of the airflow velocity and distribution, the pressure drop and the heat recovery performance

Coding-Complete Genome Sequence of an African Swine Fever Virus from an Outbreak in 2021 among Domestic Pigs in Pangasinan, Philippines

We report a coding-complete genome sequence of an African swine fever virus from an outbreak in 2021 among domestic pigs in Pangasinan, Philippines using Oxford Nanopore Technologies minION. The linear genome assembly is a single contig with 192,377 bp

Analysis of a rotary passive heat recovery device for natural ventilation windcatcher

Based on the design of traditional architecture, windcatchers are devices which provides passive ventilation by manipulating pressure differentials around buildings induced by the movement of wind and difference in temperature. In temperature climates, it is effective in providing passive cooling during summer months. However, during winter months, the low air temperature supplied to the space can cause further thermal discomfort and increase heat loss which lead to higher energy consumption. This limits the capabilities of windcatchers to provide ventilation all year round. To address this issue, the present study proposes incorporating a rotary thermal heat recovery device into the windcatcher channel and investigate its performance using numerical modelling and experimental tests. The study focused on characterizing the design and performance of the copper radial blades of the proposed heat recovery device. The predicted results of the airflow and temperature showed good agreement with the experimental tests. Two types of radial blade designs were assessed in terms of the airflow velocity and distribution, the pressure drop and the heat recovery performance

Glycan shifting on hepatitis C virus (HCV) E2 glycoprotein is a mechanism for escape from broadly neutralizing antibodies

Hepatitis C virus (HCV) infection is a major cause of liver disease and hepatocellular carcinoma. Glycan shielding has been proposed to be a mechanism by which HCV masks broadly neutralizing epitopes on its viral glycoproteins. However, the role of altered glycosylation in HCV resistance to broadly neutralizing antibodies is not fully understood. Here, we have generated potent HCV neutralizing antibodies hu5B3.v3 and MRCT10.v362 that, similar to the previously described AP33 and HCV1, bind to a highly conserved linear epitope on E2. We utilize a combination of in vitro resistance selections using the cell culture infectious HCV and structural analyses to identify mechanisms of HCV resistance to hu5B3.v3 and MRCT10.v362. Ultra deep sequencing from in vitro HCV resistance selection studies identified resistance mutations at asparagine N417 (N417S, N417T and N417G) as early as 5 days post treatment. Comparison of the glycosylation status of soluble versions of the E2 glycoprotein containing the respective resistance mutations revealed a glycosylation shift from N417 to N415 in the N417S and N417T E2 proteins. The N417G E2 variant was glycosylated neither at residue 415 nor at residue 417 and remained sensitive to MRCT10.v362. Structural analyses of the E2 epitope bound to hu5B3.v3 Fab and MRCT10.v362 Fab using X-ray crystallography confirmed that residue N415 is buried within the antibody–peptide interface. Thus, in addition to previously described mutations at N415 that abrogate the β-hairpin structure of this E2 linear epitope, we identify a second escape mechanism, termed glycan shifting, that decreases the efficacy of broadly neutralizing HCV antibodies

Efficient Cellular Release of Rift Valley Fever Virus Requires Genomic RNA

The Rift Valley fever virus is responsible for periodic, explosive epizootics throughout sub-Saharan Africa. The development of therapeutics targeting this virus is difficult due to a limited understanding of the viral replicative cycle. Utilizing a virus-like particle system, we have established roles for each of the viral structural components in assembly, release, and virus infectivity. The envelope glycoprotein, Gn, was discovered to be necessary and sufficient for packaging of the genome, nucleocapsid protein and the RNA-dependent RNA polymerase into virus particles. Additionally, packaging of the genome was found to be necessary for the efficient release of particles, revealing a novel mechanism for the efficient generation of infectious virus. Our results identify possible conserved targets for development of anti-phlebovirus therapies

Vaccine Potential of Nipah Virus-Like Particles

Nipah virus (NiV) was first recognized in 1998 in a zoonotic disease outbreak associated with highly lethal febrile encephalitis in humans and a predominantly respiratory disease in pigs. Periodic deadly outbreaks, documentation of person-to-person transmission, and the potential of this virus as an agent of agroterror reinforce the need for effective means of therapy and prevention. In this report, we describe the vaccine potential of NiV virus-like particles (NiV VLPs) composed of three NiV proteins G, F and M. Co-expression of these proteins under optimized conditions resulted in quantifiable amounts of VLPs with many virus-like/vaccine desirable properties including some not previously described for VLPs of any paramyxovirus: The particles were fusogenic, inducing syncytia formation; PCR array analysis showed NiV VLP-induced activation of innate immune defense pathways; the surface structure of NiV VLPs imaged by cryoelectron microscopy was dense, ordered, and repetitive, and consistent with similarly derived structure of paramyxovirus measles virus. The VLPs were composed of all the three viral proteins as designed, and their intracellular processing also appeared similar to NiV virions. The size, morphology and surface composition of the VLPs were consistent with the parental virus, and importantly, they retained their antigenic potential. Finally, these particles, formulated without adjuvant, were able to induce neutralizing antibody response in Balb/c mice. These findings indicate vaccine potential of these particles and will be the basis for undertaking future protective efficacy studies in animal models of NiV disease