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Virus Engineering and Applications: 2nd Edition
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Virus Engineering and Applications: 2nd Edition

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Microbiology".

Deadline for manuscript submissions: closed (30 June 2024) | Viewed by 17660

Special Issue Editor

Dr. Laura Menotti

E-Mail Website
Guest Editor
Department of Pharmacy and Biotechnology, University of Bologna, 40126 Bologna, Italy
Interests: oncolytic herpes simplex virus; tropism retargeting; cancer receptors; virus engineering; virus arming; virus-mediated transgene expression
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

The recent SARS-CoV-2 pandemic recalled and focused worldwide attention, perception, and awareness on viruses and highlighted the global need to prepare for future emerging threats. At present, this is just the most exposed facet of the virus world. However, viruses are much more. Virus engineering is a well-established discipline, both as a tool and as a research subject, and it is now receiving attention at the intersection with novel expanding omics breakthroughs. Thus, the possibility to retrieve viral sequences from the most diverse matrices expanded the boundaries of the virosphere in an unprecedented way. Virus engineering comes here into play, allowing for the study of the biological properties of viral entities, or just the parts thereof, that have not been isolated as particles, or whose host is not known, with tremendous implications both in ecology and global health. Virus engineering allows for reviving both known extinct or unknown possible pathogens, variants of concern, etc., and designing and devising countermeasures, drugs, or vaccines, with a constant eye on ethics and safety issues. Virus engineering allows for shedding light on virus origin, emergence, and evolution as well. Finally, an ever-growing body of knowledge builds up for increasingly sophisticated viruses engineered as therapeutic platforms themselves, for gene therapy, oncolytic virotherapy, or bio-nanomaterials. In this regard, submissions of original research papers, perspectives, and reviews are welcome for this Special Issue. Topics of interest include, but are not limited to:

  • Basic virology;
  • Viral biotechnology;
  • Virus engineering platforms, toolboxes, and technologies;
  • Viral vaccine platforms;
  • Viral gene therapy;
  • Oncolytic virotherapy and immunovirotherapy;
  • Personalized medicine with virus-based biologicals;
  • Ethics of virus engineering;
  • Virus origin, emergence, and evolution;
  • Viral synthetic biology;
  • Virus metagenomics and the virosphere;
  • Virus-based bio-nanomaterials and bio-nanotechnologies.

Dr. Laura Menotti
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. There is an Article Processing Charge (APC) for publication in this open access journal. For details about the APC please see here. Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • virus engineering
  • oncolytic virotherapy
  • immunovirotherapy
  • viral biotechnology

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Published Papers (10 papers)

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Research

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13 pages, 3777 KiB  
Article
Optimization of PCA Error Correction Conditions to Improve Efficiency of Virus Genome De Novo Synthesis
by Jiazhen Cui, Ao Hu, Xianghua Xiong, Qingyang Wang, Chen Zhu, Zhili Chen, Yuanyuan Lu, Xianzhu Xia, Huipeng Chen and Gang Liu
Int. J. Mol. Sci. 2024, 25(21), 11514; https://doi.org/10.3390/ijms252111514 - 26 Oct 2024
Viewed by 545
Abstract
In recent years, there have been frequent global outbreaks of viral epidemics such as Zika, COVID-19, and monkeypox, which have had a huge impact on human health and society and have also spurred innovation in virus engineering technology. The rise of synthetic virus [...] Read more.
In recent years, there have been frequent global outbreaks of viral epidemics such as Zika, COVID-19, and monkeypox, which have had a huge impact on human health and society and have also spurred innovation in virus engineering technology. The rise of synthetic virus genome technology has provided researchers with a new platform to accelerate vaccine and drug development. Although DNA synthesis technology has made significant progress, the current virus genome synthesis technology still requires the assembly of short oligonucleotides of around 60 bp into kb-level lengths when constructing long segments, a process in which the commonly used polymerase chain reaction assembly (PCA) technology has high error rates and is cumbersome to operate. This study optimized the error correction conditions after PCA assembly, increasing the accuracy of synthesizing 1 kb DNA fragments from 4.2 ± 2.1% before error correction to 31.3 ± 3.1% after two rounds of correction, an improvement of over 6 times. This study provides a more efficient operational process for synthesizing virus genomes from scratch, indicating greater potential for virus engineering in epidemic prevention and control and the field of biomedicine. Full article
(This article belongs to the Special Issue Virus Engineering and Applications: 2nd Edition)
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22 pages, 7955 KiB  
Article
Development and Evaluation of a Shrimp Virus (IHHNV)-Mediated Gene Transfer and Expression System for Shrimps
by Yiwen Tao, Jinwu Wang, Rui Xiao, Qingli Zhang and Huarong Guo
Int. J. Mol. Sci. 2024, 25(16), 8999; https://doi.org/10.3390/ijms25168999 - 19 Aug 2024
Viewed by 707
Abstract
An efficient gene transfer and expression tool is lacking for shrimps and shrimp cells. To solve this, this study has developed a shrimp DNA virus-mediated gene transfer and expression system, consisting of insect Sf9 cells for viral packaging, the shrimp viral vector of [...] Read more.
An efficient gene transfer and expression tool is lacking for shrimps and shrimp cells. To solve this, this study has developed a shrimp DNA virus-mediated gene transfer and expression system, consisting of insect Sf9 cells for viral packaging, the shrimp viral vector of pUC19-IHHNV-PH-GUS and the baculoviral vector of Bacmid or Bacmid-VP28 encoding the shrimp WSSV envelope protein VP28. The pUC19-IHHNV-PH-GUS vector was constructed by assembling the genomic DNA of shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV), which has shortened inverted terminal repeats, into a pUC19 backbone, and then an expression cassette of baculoviral polyhedron (PH) promoter-driven GUS (β-glucuronidase) reporter gene was inserted immediately downstream of IHHNV for proof-of-concept. It was found that the viral vector of pUC19-IHHNV-PH-GUS could be successfully packaged into IHHNV-like infective virions in the Sf9 cells, and the gene transfer efficiency of this system was evaluated and verified in three systems of Sf9 cells, shrimp hemolymph cells and tissues of infected shrimps, but the GUS expression could only be detected in cases where the viral vector was co-transfected or co-infected with a baculovirus of Bacmid or Bacmid-VP28 due to the Bacmid-dependence of the PH promoter. Moreover, the packaging and infection efficiencies could be significantly improved when Bacmid-VP28 was used instead of Bacmid. Full article
(This article belongs to the Special Issue Virus Engineering and Applications: 2nd Edition)
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20 pages, 5700 KiB  
Article
High-Throughput Determination of Infectious Virus Titers by Kinetic Measurement of Infection-Induced Changes in Cell Morphology
by Dominik Hotter, Marco Kunzelmann, Franziska Kiefer, Chiara Leukhardt, Carolin Fackler, Stefan Jäger and Johannes Solzin
Int. J. Mol. Sci. 2024, 25(15), 8076; https://doi.org/10.3390/ijms25158076 - 24 Jul 2024
Viewed by 1157
Abstract
Infectivity assays are the key analytical technology for the development and manufacturing of virus-based therapeutics. Here, we introduce a novel assay format that utilizes label-free bright-field images to determine the kinetics of infection-dependent changes in cell morphology. In particular, cell rounding is directly [...] Read more.
Infectivity assays are the key analytical technology for the development and manufacturing of virus-based therapeutics. Here, we introduce a novel assay format that utilizes label-free bright-field images to determine the kinetics of infection-dependent changes in cell morphology. In particular, cell rounding is directly proportional to the amount of infectious virus applied, enabling rapid determination of viral titers in relation to a standard curve. Our kinetic infectious virus titer (KIT) assay is stability-indicating and, due to its sensitive readout method, provides results within 24 h post-infection. Compared to traditional infectivity assays, which depend on a single readout of an infection endpoint, cumulated analysis of kinetic data by a fit model results in precise results (CV < 20%) based on only three wells per sample. This approach allows for a high throughput with ~400 samples processed by a single operator per week. We demonstrate the applicability of the KIT assay for the genetically engineered oncolytic VSV-GP, Newcastle disease virus (NDV), and parapoxvirus ovis (ORFV), but it can potentially be extended to a wide range of viruses that induce morphological changes upon infection. The versatility of this assay, combined with its independence from specific instruments or software, makes it a promising solution to overcome the analytical bottleneck in infectivity assays within the pharmaceutical industry and as a routine method in academic research. Full article
(This article belongs to the Special Issue Virus Engineering and Applications: 2nd Edition)
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10 pages, 1775 KiB  
Article
Antibody Binding Captures High Energy State of an Antigen: The Case of Nsp1 SARS-CoV-2 as Revealed by Hydrogen–Deuterium Exchange Mass Spectrometry
by Ravi Kant, Nawneet Mishra and Michael L. Gross
Int. J. Mol. Sci. 2023, 24(24), 17342; https://doi.org/10.3390/ijms242417342 - 11 Dec 2023
Cited by 1 | Viewed by 1116
Abstract
We describe an investigation using structural mass spectrometry (MS) of the impact of two antibodies, 15497 and 15498, binding the highly flexible SARS-CoV-2 Nsp1 protein. We determined the epitopes and paratopes involved in the antibody–protein interactions by using hydrogen–deuterium exchange MS (HDX-MS). Notably, [...] Read more.
We describe an investigation using structural mass spectrometry (MS) of the impact of two antibodies, 15497 and 15498, binding the highly flexible SARS-CoV-2 Nsp1 protein. We determined the epitopes and paratopes involved in the antibody–protein interactions by using hydrogen–deuterium exchange MS (HDX-MS). Notably, the Fab (Fragment antigen binding) for antibody 15498 captured a high energy form of the antigen exhibiting significant conformational changes that added flexibility over most of the Nsp1 protein. The Fab for antibody 15497, however, showed usual antigen binding behavior, revealing local changes presumably including the binding site. These findings illustrate an unusual antibody effect on an antigen and are consistent with the dynamic nature of the Nsp1 protein. Our studies suggest that this interaction capitalizes on the high flexibility of Nsp1 to undergo conformational change and be trapped in a higher energy state by binding with a specific antibody. Full article
(This article belongs to the Special Issue Virus Engineering and Applications: 2nd Edition)
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14 pages, 2931 KiB  
Article
Anti-Tumor Immunogenicity of the Oncolytic Virus CF33-hNIS-antiPDL1 against Ex Vivo Peritoneal Cells from Gastric Cancer Patients
by Zhifang Zhang, Annie Yang, Shyambabu Chaurasiya, Anthony K. Park, Sang-In Kim, Jianming Lu, Hannah Valencia, Yuman Fong and Yanghee Woo
Int. J. Mol. Sci. 2023, 24(18), 14189; https://doi.org/10.3390/ijms241814189 - 16 Sep 2023
Cited by 1 | Viewed by 2636
Abstract
We studied the immunotherapeutic potential of CF33-hNIS-antiPDL1 oncolytic virus (OV) against gastric cancer with peritoneal metastasis (GCPM). We collected fresh malignant ascites (MA) or peritoneal washings (PW) during routine paracenteses and diagnostic laparoscopies from GC patients (n = 27). Cells were analyzed for [...] Read more.
We studied the immunotherapeutic potential of CF33-hNIS-antiPDL1 oncolytic virus (OV) against gastric cancer with peritoneal metastasis (GCPM). We collected fresh malignant ascites (MA) or peritoneal washings (PW) during routine paracenteses and diagnostic laparoscopies from GC patients (n = 27). Cells were analyzed for cancer cell markers and T cells, or treated with PBS, CF33-GFP, or CF33-hNIS-antiPDL1 (MOI = 3). We analyzed infectivity, replication, cytotoxicity, CD107α upregulation of CD8+ and CD4+ T cells, CD274 (PD-L1) blockade of cancer cells by virus-encoded anti-PD-L1 scFv, and the release of growth factors and cytokines. We observed higher CD45−/large-size cells and lower CD8+ T cell percentages in MA than PW. CD45−/large-size cells were morphologically malignant and expressed CD274 (PD-L1), CD252 (OX40L), and EGFR. CD4+ and CD8+ T cells did not express cell surface exhaustion markers. Virus infection and replication increased cancer cell death at 15 h and 48 h. CF33-hNIS-antiPDL1 treatment produced functional anti-PD-L1 scFv, which blocked surface PD-L1 binding of live cancer cells and increased CD8+CD107α+ and CD4+CD107α+ T cell percentages while decreasing EGF, PDGF, soluble anti-PD-L1, and IL-10. CF33-OVs infect, replicate in, express functional proteins, and kill ex vivo GCPM cells with immune-activating effects. CF33-hNIS-antiPDL1 displays real potential for intraperitoneal GCPM therapy. Full article
(This article belongs to the Special Issue Virus Engineering and Applications: 2nd Edition)
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Review

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17 pages, 2755 KiB  
Review
Electron Tomography as a Tool to Study SARS-CoV-2 Morphology
by Hong Wu, Yoshihiko Fujioka, Shoichi Sakaguchi, Youichi Suzuki and Takashi Nakano
Int. J. Mol. Sci. 2024, 25(21), 11762; https://doi.org/10.3390/ijms252111762 - 1 Nov 2024
Viewed by 523
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel betacoronavirus, is the causative agent of COVID-19, which has caused economic and social disruption worldwide. To date, many drugs and vaccines have been developed for the treatment and prevention of COVID-19 and have effectively [...] Read more.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel betacoronavirus, is the causative agent of COVID-19, which has caused economic and social disruption worldwide. To date, many drugs and vaccines have been developed for the treatment and prevention of COVID-19 and have effectively controlled the global epidemic of SARS-CoV-2. However, SARS-CoV-2 is highly mutable, leading to the emergence of new variants that may counteract current therapeutic measures. Electron microscopy (EM) is a valuable technique for obtaining ultrastructural information about the intracellular process of virus replication. In particular, EM allows us to visualize the morphological and subcellular changes during virion formation, which would provide a promising avenue for the development of antiviral agents effective against new SARS-CoV-2 variants. In this review, we present our recent findings using transmission electron microscopy (TEM) combined with electron tomography (ET) to reveal the morphologically distinct types of SARS-CoV-2 particles, demonstrating that TEM and ET are valuable tools for visually understanding the maturation status of SARS-CoV-2 in infected cells. This review also discusses the application of EM analysis to the evaluation of genetically engineered RNA viruses. Full article
(This article belongs to the Special Issue Virus Engineering and Applications: 2nd Edition)
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22 pages, 3451 KiB  
Review
Molecular Engineering of Virus Tropism
by Bo He, Belinda Wilson, Shih-Heng Chen, Kedar Sharma, Erica Scappini, Molly Cook, Robert Petrovich and Negin P. Martin
Int. J. Mol. Sci. 2024, 25(20), 11094; https://doi.org/10.3390/ijms252011094 - 15 Oct 2024
Viewed by 1235
Abstract
Engineered viral vectors designed to deliver genetic material to specific targets offer significant potential for disease treatment, safer vaccine development, and the creation of novel biochemical research tools. Viral tropism, the specificity of a virus for infecting a particular host, is often modified [...] Read more.
Engineered viral vectors designed to deliver genetic material to specific targets offer significant potential for disease treatment, safer vaccine development, and the creation of novel biochemical research tools. Viral tropism, the specificity of a virus for infecting a particular host, is often modified in recombinant viruses to achieve precise delivery, minimize off-target effects, enhance transduction efficiency, and improve safety. Key factors influencing tropism include surface protein interactions between the virus and host-cell, the availability of host-cell machinery for viral replication, and the host immune response. This review explores current strategies for modifying the tropism of recombinant viruses by altering their surface proteins. We provide an overview of recent advancements in targeting non-enveloped viruses (adenovirus and adeno-associated virus) and enveloped viruses (retro/lentivirus, Rabies, Vesicular Stomatitis Virus, and Herpesvirus) to specific cell types. Additionally, we discuss approaches, such as rational design, directed evolution, and in silico and machine learning-based methods, for generating novel AAV variants with the desired tropism and the use of chimeric envelope proteins for pseudotyping enveloped viruses. Finally, we highlight the applications of these advancements and discuss the challenges and future directions in engineering viral tropism. Full article
(This article belongs to the Special Issue Virus Engineering and Applications: 2nd Edition)
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20 pages, 798 KiB  
Review
Optimizing Pancreatic Cancer Therapy: The Promise of Immune Stimulatory Oncolytic Viruses
by Shivani Thoidingjam, Aseem Rai Bhatnagar, Sushmitha Sriramulu, Farzan Siddiqui and Shyam Nyati
Int. J. Mol. Sci. 2024, 25(18), 9912; https://doi.org/10.3390/ijms25189912 - 13 Sep 2024
Cited by 1 | Viewed by 1545
Abstract
Pancreatic cancer presents formidable challenges due to rapid progression and resistance to conventional treatments. Oncolytic viruses (OVs) selectively infect cancer cells and cause cancer cells to lyse, releasing molecules that can be identified by the host’s immune system. Moreover, OV can carry immune-stimulatory [...] Read more.
Pancreatic cancer presents formidable challenges due to rapid progression and resistance to conventional treatments. Oncolytic viruses (OVs) selectively infect cancer cells and cause cancer cells to lyse, releasing molecules that can be identified by the host’s immune system. Moreover, OV can carry immune-stimulatory payloads such as interleukin-12, which when delivered locally can enhance immune system-mediated tumor killing. OVs are very well tolerated by cancer patients due to their ability to selectively target tumors without affecting surrounding normal tissues. OVs have recently been combined with other therapies, including chemotherapy and immunotherapy, to improve clinical outcomes. Several OVs including adenovirus, herpes simplex viruses (HSVs), vaccinia virus, parvovirus, reovirus, and measles virus have been evaluated in preclinical and clinical settings for the treatment of pancreatic cancer. We evaluated the safety and tolerability of a replication-competent oncolytic adenoviral vector carrying two suicide genes (thymidine kinase, TK; and cytosine deaminase, CD) and human interleukin-12 (hIL12) in metastatic pancreatic cancer patients in a phase 1 trial. This vector was found to be safe and well-tolerated at the highest doses tested without causing any significant adverse events (SAEs). Moreover, long-term follow-up studies indicated an increase in the overall survival (OS) in subjects receiving the highest dose of the OV. Our encouraging long-term survival data provide hope for patients with advanced pancreatic cancer, a disease that has not seen a meaningful increase in OS in the last five decades. In this review article, we highlight several preclinical and clinical studies and discuss future directions for optimizing OV therapy in pancreatic cancer. We envision OV-based gene therapy to be a game changer in the near future with the advent of newer generation OVs that have higher specificity and selectivity combined with personalized treatment plans developed under AI guidance. Full article
(This article belongs to the Special Issue Virus Engineering and Applications: 2nd Edition)
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24 pages, 870 KiB  
Review
Scanning the Horizon for Environmental Applications of Genetically Modified Viruses Reveals Challenges for Their Environmental Risk Assessment
by Michael F. Eckerstorfer, Marion Dolezel, Marianne Miklau, Anita Greiter, Andreas Heissenberger and Margret Engelhard
Int. J. Mol. Sci. 2024, 25(3), 1507; https://doi.org/10.3390/ijms25031507 - 25 Jan 2024
Cited by 2 | Viewed by 2155
Abstract
The release of novel genetically modified (GM) virus applications into the environment for agricultural, veterinary, and nature-conservation purposes poses a number of significant challenges for risk assessors and regulatory authorities. Continuous efforts to scan the horizon for emerging applications are needed to gain [...] Read more.
The release of novel genetically modified (GM) virus applications into the environment for agricultural, veterinary, and nature-conservation purposes poses a number of significant challenges for risk assessors and regulatory authorities. Continuous efforts to scan the horizon for emerging applications are needed to gain an overview of new GM virus applications. In addition, appropriate approaches for risk assessment and management have to be developed. These approaches need to address pertinent challenges, in particular with regard to the environmental release of GM virus applications with a high probability for transmission and spreading, including transboundary movements and a high potential to result in adverse environmental effects. However, the current preparedness at the EU and international level to assess such GM virus application is limited. This study addresses some of the challenges associated with the current situation, firstly, by conducting a horizon scan to identify emerging GM virus applications with relevance for the environment. Secondly, outstanding issues regarding the environmental risk assessment (ERA) of GM virus applications are identified based on an evaluation of case study examples. Specifically, the limited scientific information available for the ERA of some applications and the lack of detailed and appropriate guidance for ERA are discussed. Furthermore, considerations are provided for future work that is needed to establish adequate risk assessment and management approaches. Full article
(This article belongs to the Special Issue Virus Engineering and Applications: 2nd Edition)
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34 pages, 3079 KiB  
Review
Oncolytic Virotherapy: A New Paradigm in Cancer Immunotherapy
by Simona Ruxandra Volovat, Dragos Viorel Scripcariu, Ingrid Andrada Vasilache, Cati Raluca Stolniceanu, Constantin Volovat, Iolanda Georgiana Augustin, Cristian Constantin Volovat, Madalina-Raluca Ostafe, Slevoacă-Grigore Andreea-Voichița, Toni Bejusca-Vieriu, Cristian Virgil Lungulescu, Daniel Sur and Diana Boboc
Int. J. Mol. Sci. 2024, 25(2), 1180; https://doi.org/10.3390/ijms25021180 - 18 Jan 2024
Cited by 9 | Viewed by 4895
Abstract
Oncolytic viruses (OVs) are emerging as potential treatment options for cancer. Natural and genetically engineered viruses exhibit various antitumor mechanisms. OVs act by direct cytolysis, the potentiation of the immune system through antigen release, and the activation of inflammatory responses or indirectly by [...] Read more.
Oncolytic viruses (OVs) are emerging as potential treatment options for cancer. Natural and genetically engineered viruses exhibit various antitumor mechanisms. OVs act by direct cytolysis, the potentiation of the immune system through antigen release, and the activation of inflammatory responses or indirectly by interference with different types of elements in the tumor microenvironment, modification of energy metabolism in tumor cells, and antiangiogenic action. The action of OVs is pleiotropic, and they show varied interactions with the host and tumor cells. An important impediment in oncolytic virotherapy is the journey of the virus into the tumor cells and the possibility of its binding to different biological and nonbiological vectors. OVs have been demonstrated to eliminate cancer cells that are resistant to standard treatments in many clinical trials for various cancers (melanoma, lung, and hepatic); however, there are several elements of resistance to the action of viruses per se. Therefore, it is necessary to evaluate the combination of OVs with other standard treatment modalities, such as chemotherapy, immunotherapy, targeted therapies, and cellular therapies, to increase the response rate. This review provides a comprehensive update on OVs, their use in oncolytic virotherapy, and the future prospects of this therapy alongside the standard therapies currently used in cancer treatment. Full article
(This article belongs to the Special Issue Virus Engineering and Applications: 2nd Edition)
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