Engineering the Nicotiana benthamiana secretory pathway for the production of Lujo virus vaccines and diagnostic tools

Sub-Saharan Africa is severely deficient in vaccine manufacturing facilities that can keep up with the rate of emergence of viral pathogens. As seen with the Covid-19 pandemic, outbreaks of disease can be extremely detrimental to economies and put severe strain on the public health sector. Vaccination offers a solution to reduce the difficulties that accompany viral outbreaks. Lujo virus, an emerging arenavirus responsible for causing a devastating haemorrhagic fever with an 80% mortality rate, currently has no vaccines or diagnostic tools available. In this study we developed a production pipeline in HEK293T cells and N. benthamiana for the protein LUJV GP-C∆TM (Lujo virus glycoprotein precursor without the transmembrane region). The LUJV GP-C∆TM was constructed from gene sequences of the envelope glycoprotein of Lujo virus and then adapted to production in HEK293T cells using the DNA expression vector pTHpCapR. An N. benthamiana plant protein expression system was developed in parallel to compare the production utility of both systems with an emphasis being placed on the plant system. Plant expression systems are arguably cheaper and more easily automatable than traditional mammalian expression technologies. This makes them suitable for vaccine protein production in lower socio-economic countries that have an overwhelming burden of disease and poor health care systems. LUJV GP-C∆TM was successfully expressed in both HEK293T cells and N. benthamiana. It was confirmed that the LUJV GP-C∆TM undergoes the post translational modifications glycosylation and proteolytic cleavage in recombinant HEK293T cells and was produced in a conformation that allowed successful purification via a His tag sequence that was inserted into the LUJV GP-C∆TM protein gene sequence. On the other hand, N. benthamiana does not endogenously express the Site-1 protease needed for cleavage of the Lujo glycoprotein, and thus this needed to be co-expressed. Attempts were made to detect the Site-1 protease including extraction into buffers with different pHs and ammonium sulfate precipitation to concentrate the protein. However, I was not able elicit proteolytic cleavage of the LUJV GP-C∆TM nor detect the Site-1 protease in N. benthamiana. This is probably attributable to the innate responses against the hostile nature of proteases to non-native expression hosts. Proteins were expressed in N. benthamiana through the use of previously established protocols utilizing recombinant Agrobacterium tumefaciens to deliver synthesized genes to plant cells and induce their expression. It was determined that co-expression of the molecular folding chaperone calreticulin is necessary for LUJV GP-C∆TM to accumulate at detectable levels. Co-expression of an oligossacharyl transferase LmSTT3D, isolated from Leishmania major, may increase the glycan occupancy of the LUJV GP-C∆TM protein, indicated by a molecular mass shift. However, further experimental lines of evidence are needed -such as glycosylation mapping to determine if this is the case. Other parameters such as the optimal day of protein harvest and optical density of recombinant Agrobacterium tumefaciens strains were recorded. Collectively these findings serve as a prototype pipeline for the production of commercially relevant immunogens, diagnostic tools and virus-like particles. This study narrows the focus for bottlenecks in plant protein production, not just for Lujo virus proteins, but for arenaviruses generally and potentially also other haemorrhagic fever-causing viruses. Future efforts should be directed towards addressing the barriers to plant production of complex viral antigens, and to further investigate the utility of mammalian cell-produced LUJV GPC∆TM in animal studies

The Fecal Viral Flora of Wild Rodents

The frequent interactions of rodents with humans make them a common source of zoonotic infections. To obtain an initial unbiased measure of the viral diversity in the enteric tract of wild rodents we sequenced partially purified, randomly amplified viral RNA and DNA in the feces of 105 wild rodents (mouse, vole, and rat) collected in California and Virginia. We identified in decreasing frequency sequences related to the mammalian viruses families Circoviridae, Picobirnaviridae, Picornaviridae, Astroviridae, Parvoviridae, Papillomaviridae, Adenoviridae, and Coronaviridae. Seventeen small circular DNA genomes containing one or two replicase genes distantly related to the Circoviridae representing several potentially new viral families were characterized. In the Picornaviridae family two new candidate genera as well as a close genetic relative of the human pathogen Aichi virus were characterized. Fragments of the first mouse sapelovirus and picobirnaviruses were identified and the first murine astrovirus genome was characterized. A mouse papillomavirus genome and fragments of a novel adenovirus and adenovirus-associated virus were also sequenced. The next largest fraction of the rodent fecal virome was related to insect viruses of the Densoviridae, Iridoviridae, Polydnaviridae, Dicistroviriade, Bromoviridae, and Virgaviridae families followed by plant virus-related sequences in the Nanoviridae, Geminiviridae, Phycodnaviridae, Secoviridae, Partitiviridae, Tymoviridae, Alphaflexiviridae, and Tombusviridae families reflecting the largely insect and plant rodent diet. Phylogenetic analyses of full and partial viral genomes therefore revealed many previously unreported viral species, genera, and families. The close genetic similarities noted between some rodent and human viruses might reflect past zoonoses. This study increases our understanding of the viral diversity in wild rodents and highlights the large number of still uncharacterized viruses in mammals

Antibody-based inhibition of pathogenic new world hemorrhagic fever mammarenaviruses by steric occlusion of the human transferrin receptor 1 apical domain

Pathogenic clade B New World mammarenaviruses (NWM) can cause Argentine, Venezuelan, Brazilian, and Bolivian hemorrhagic fevers. Sequence variability among NWM glycoproteins (GP) poses a challenge to the development of broadly neutralizing therapeutics against the entire clade of viruses. However, blockade of their shared binding site on the apical domain of human transferrin receptor 1 (hTfR1/CD71) presents an opportunity for the development of effective and broadly neutralizing therapeutics. Here, we demonstrate that the murine monoclonal antibody OKT9, which targets the apical domain of hTfR1, can sterically block cellular entry by viral particles presenting clade B NWM glycoproteins (GP1-GP2). OKT9 blockade is also effective against viral particles pseudotyped with glycoproteins of a recently identified pathogenic Sabia-like virus. With nanomolar affinity for hTfR1, the OKT9 antigen binding fragment (OKT9-Fab) sterically blocks clade B NWM-GP1s and reduces infectivity of an attenuated strain of Junin virus. Binding of OKT9 to the hTfR1 ectodomain in its soluble, dimeric state produces stable assemblies that are observable by negative-stain electron microscopy. A model of the OKT9-sTfR1 complex, informed by the known crystallographic structure of sTfR1 and a newly determined structure of the OKT9 antigen binding fragment (Fab), suggests that OKT9 and the Machupo virus GP1 share a binding site on the hTfR1 apical domain. The structural basis for this interaction presents a framework for the design and development of high-affinity, broadly acting agents targeting clade B NWMs. IMPORTANCE Pathogenic clade B NWMs cause grave infectious diseases, the South American hemorrhagic fevers. Their etiological agents are Junin (JUNV), Guanarito (GTOV), Sabiá (SABV), Machupo (MACV), Chapare (CHAV), and a new Sabiá-like (SABV-L) virus recently identified in Brazil. These are priority A pathogens due to their high infectivity and mortality, their potential for person-to-person transmission, and the limited availability of effective therapeutics and vaccines to curb their effects. While low homology between surface glycoproteins of NWMs foils efforts to develop broadly neutralizing therapies targeting NWMs, this work provides structural evidence that OKT9, a monoclonal antibody targeting a single NWM glycoprotein binding site on hTfR1, can efficiently prevent their entry into cells.Fil: Ferrero, Sol. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Flores, Maria D.. University of California at Los Angeles; Estados UnidosFil: Short, Connor. University of California at Los Angeles; Estados UnidosFil: Vázquez, Cecilia Alejandra. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Clark, Lars E.. Harvard Medical School; Estados UnidosFil: Ziegenbein, James. University of California at Los Angeles; Estados UnidosFil: Zink, Samantha. University of California at Los Angeles; Estados UnidosFil: Fuentes, Daniel. University of California at Los Angeles; Estados UnidosFil: Payés, Cristian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Batto, María V.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Collazo, Michael. University of California at Los Angeles; Estados UnidosFil: García, Cybele C.. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Abraham, Jonathan. Harvard Medical School; Estados Unidos. Brigham and Women's Hospital; Estados UnidosFil: Cordo, Sandra Myriam. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Rodriguez, Jose A.. University of California at Los Angeles; Estados UnidosFil: Helguera, Gustavo Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Reptarenavirus prevalence in reptiles sampled in the UK and related studies

Arenaviruses are enveloped RNA viruses with a genome of two segments of single stranded RNA which is classified as ambisense in its coding strategy. Arenaviruses within the Reptarenavirus genus infect many species of snake and are associated with inclusion body disease (IBD). Reptarenavirus infection of humans has not been reported. In this thesis molecular techniques were utilized for RNA analysis and gene amplification by reverse transcription PCR (RT-PCR) in order to find evidence of reptarenavirus sequences in reptilian samples. Total RNAs were isolated from frozen specimens of snakes and other reptiles followed by an evaluation of RNA integrity. Degenerate primers of ribosomal mitochondrial and reptarenavirus genes were used for quality control of the RNA and for identification of virus sequence respectively, and then the results were confirmed by Sanger sequencing. The mitochondrial RNA was successfully amplified, whereas no reptarenavirus sequences were found in the samples screened. Spiking experiments determined that the cut-off for detection was less than 108 copies of virus RNA per sample. Although no evidence for the circulation of reptarenaviruses was found the study serves as a guide for reptarenavirus investigations using the RT-PCR technique in cases where new samples are suspected of having been infected with an existing or novel reptarenavirus. In addition, a related study investigating the role of replication organelles in the pathogenicity of infectious bronchitis virus (IBV) was performed using Transmission Electron Microscopy (TEM) of infected cells. Further, an assessment was made of the use of the arenavirus L sequence to look for related sequences associated with currently unknown viruses in the TSA database. Several matches were found which, along with a reasoned approach to their phyla, was used to provide a likelihood score of their zoonotic potential

Key Virus-Host Interactions Required For Arenavirus Particle Assembly And Release

Viruses are infectious agents that must infect the cells of living organisms in order to reproduce. They have relatively simple genomes which encode few proteins but can compensate for their simplicity by hijacking components of their cellular hosts. Arenaviruses, a family of zoonotic viruses carried by rodents, encode only 4 proteins. One of these proteins, Z, is responsible for several functions during the virus life cycle including driving the formation and release of new virus particles at the plasma membrane of infected cells. Relatively little is known about how this viral protein is regulated or the complement of host proteins it engages in order to produce new virus particles or augment Z\u27s other functions. To address this gap in knowledge, mass spectrometry was used to identify phosphorylation sites in the Old World arenavirus, lymphocytic choriomeningitis virus (LCMV) Z protein. Phosphorylation sites were identified at serine 41 (S41) and tyrosine 88 (Y88). Functional studies using recombinant (r)LCMV containing mutations at these phosphorylation sites revealed that both were important for the production of defective interfering (DI) particles. DI particles are replication-incompetent virus particles that interfere with the production of infectious virus and mitigate its cytopathic effect. While a mutation that mimics phosphorylation at S41 reduced LCMV\u27s ability to produce both infectious and DI particles, this mutation had a much stronger impact on DI particles. Production of DI particles in Y88-mutant rLCMV was drastically reduced while the impact on infectious virus was minimal. Y88 lies within a type of viral late domain (PPXY) also found in matrix proteins of several disparate virus families where it has been shown to drive infectious virus release by recruiting the membrane scission machinery of the cellular endosomal sorting complex required for transport (ESCRT). Inhibition of the ESCRT pathway drastically reduced LCMV DI particle but not infectious virus release indicating that Z\u27s PPXY late domain and the cellular ESCRT complex are required specifically for the production of DI particles. Mass spectrometry was also used to identify host protein partners of Z as well as the host proteins recruited into virus particles for the New World arenavirus, Junin (JUNV). ESCRT complex proteins were enriched in JUNV virus-like particles (VLPs) and bona fide virions. In contrast to LCMV, inhibition of the ESCRT complex resulted in significantly less infectious JUNV release. This indicates that the ultimate role of ESCRT engagement by the Old World arenavirus, LCMV, differs from that of New World, JUNV. This work represents the first demonstration that a viral protein motif and the host machinery it engages selectively drive DI particle production independently of infectious virus. It also suggests that host cell kinases can dynamically regulate the production of DI particles through phosphorylation of Z. Finally, the late domain-mutant rLCMV generated in these studies represents the first LCMV strain known to produce undetectable levels of DI particles which provides the opportunity to assess the impact that a loss of DI particles has on the ability of LCMV to establish or maintain a persistent infection

Using the host immune response to hemorrhagic fever Viruses to understand pathogenesis and improve diagnostics

Hemorrhagic fever viruses cause severe infections characterized by a hyperactive immune response that often leads to multiorgan failure and death. Current diagnostic tests are based on detecting viral proteins and nucleic acids in the blood. These are late-stage events during infection, which makes it impossible to perform a diagnosis before they are present in the collected sample. In this thesis, I explore an alternative approach using the transcriptional changes of circulating immune cells during the early stages of infection to identify unique markers of viral infection. The main advantage of this method is that it can be used to identify highly pathogenic viruses before standard detection methods become effective. I initially used RNA sequencing data to compare the host patterns of expression of macaques infected with either Lassa virus or Marburg virus, two related hemorrhagic fever viruses. I identified a set of genes that quickly become upregulated after a viral infection and remain highly expressed throughout the entire disease course, irrespective of the specific virus that caused the infection. I was also able to identify a set of biomarker genes that follow unique patterns of expression depending on the type of infection. I used an independent dataset to validate the potential of these genes to be used as biomarkers of infection. Additionally, I compared these results to the patterns of expression of macaques infected with Ebola virus, looking at multiple experimental conditions, tissues and routes of infection. Finally, I validated the host patterns of expression using two independently generated datasets corresponding to infection by different strains of arenaviruses and filoviruses. Studying the host immune response has the potential to improve the diagnosis of viral hemorrhagic fevers and other diseases. It can also accelerate our efforts to understand the underlying molecular mechanisms that lead to pathogenesis and severe disease

Molecular evolution of Azagny virus, a newfound hantavirus harbored by the West African pygmy shrew (Crocidura obscurior) in Côte d'Ivoire

<p>Abstract</p> <p>Background</p> <p>Tanganya virus (TGNV), the only shrew-associated hantavirus reported to date from sub-Saharan Africa, is harbored by the Therese's shrew (<it>Crocidura theresae</it>), and is phylogenetically distinct from Thottapalayam virus (TPMV) in the Asian house shrew (<it>Suncus murinus</it>) and Imjin virus (MJNV) in the Ussuri white-toothed shrew (<it>Crocidura lasiura</it>). The existence of myriad soricid-borne hantaviruses in Eurasia and North America would predict the presence of additional hantaviruses in sub-Saharan Africa, where multiple shrew lineages have evolved and diversified.</p> <p>Methods</p> <p>Lung tissues, collected in RNAlater^®, from 39 Buettikofer's shrews (<it>Crocidura buettikoferi</it>), 5 Jouvenet's shrews (<it>Crocidura jouvenetae</it>), 9 West African pygmy shrews (<it>Crocidura obscurior</it>) and 21 African giant shrews (<it>Crocidura olivieri</it>) captured in Côte d'Ivoire during 2009, were systematically examined for hantavirus RNA by RT-PCR.</p> <p>Results</p> <p>A genetically distinct hantavirus, designated Azagny virus (AZGV), was detected in the West African pygmy shrew. Phylogenetic analysis of the S, M and L segments, using maximum-likelihood and Bayesian methods, under the GTR+I+Γ model of evolution, showed that AZGV shared a common ancestry with TGNV and was more closely related to hantaviruses harbored by soricine shrews than to TPMV and MJNV. That is, AZGV in the West African pygmy shrew, like TGNV in the Therese's shrew, did not form a monophyletic group with TPMV and MJNV, which were deeply divergent and basal to other rodent- and soricomorph-borne hantaviruses. Ancestral distributions of each hantavirus lineage, reconstructed using Mesquite 2.74, suggested that the common ancestor of all hantaviruses was most likely of Eurasian, not African, origin.</p> <p>Conclusions</p> <p>Genome-wide analysis of many more hantaviruses from sub-Saharan Africa are required to better understand how the biogeographic origin and radiation of African shrews might have contributed to, or have resulted from, the evolution of hantaviruses.</p