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Pathogenesis of Viral Infections: Implications in the Development of Vaccines and Diagnostic Tools 2.0

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A special issue of Viruses (ISSN 1999-4915). This special issue belongs to the section "Animal Viruses".

Deadline for manuscript submissions: closed (31 December 2023) | Viewed by 14865

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Dr. Llilianne Ganges

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OIE Reference Laboratory for Classical Swine Fever, Institute of Agrifood Research and Technology (IRTA), Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona, 08193 Barcelona, Spain
Interests: virology; viral pathogenesis and immunology; innate and adaptive immunity; virus host interaction; viral evolution; vaccine and diagnostic tools design; viruses in animal health
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Prof. Dr. Jishu Shi

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College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA
Interests: African swine fever (ASF); porcine reproductive and respiratory syndrome (PRRS); classical swine fever virus (CSF); animal vaccine design and evaluation; adjuvant development; diagnostic tool development and evaluation
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

This Special Issue focuses on gathering articles that show the most relevant aspects of viral pathogenesis that constitute novel and strategic targets for the development of vaccines and diagnostic methods of relevant diseases that affect animal health. We will focus on the mechanisms of viral persistence, specifically virus–host interactions, immunology, and immunopathology. The prevention and rapid detection capacity of possible new virus emergencies that may affect animal and human health is one of the great challenges that we must face in the near future. The development of rapid and cost-effective diagnostic methods, as well as new vaccine strategies, will be key tools to overcome these challenges. Fundamental and applied research in immunology and vaccinology will also be collected in this issue.

Dr. Llilianne Ganges
Prof. Dr. Jishu Shi
Guest Editors

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. Viruses is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2600 CHF (Swiss Francs). 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

innate and adaptive immunity
vaccine development
oral vaccination
virus diagnostic
emerging virus
next-generation sequencing
virus variant detection
viruses in animal health

Published Papers (9 papers)

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Research

14 pages, 2853 KiB

Open AccessArticle

Deletion of the EP402R Gene from the Genome of African Swine Fever Vaccine Strain ASFV-G-∆I177L Provides the Potential Capability of Differentiating between Infected and Vaccinated Animals

by Manuel V. Borca, Elizabeth Ramirez-Medina, Nallely Espinoza, Ayushi Rai, Edward Spinard, Lauro Velazquez-Salinas, Alyssa Valladares, Ediane Silva, Leeanna Burton, Amanda Meyers, Jason Clark, Ping Wu, Cyril G. Gay and Douglas P. Gladue

Viruses 2024, 16(3), 376; https://doi.org/10.3390/v16030376 - 28 Feb 2024

Viewed by 1008

Abstract

The African swine fever virus (ASFV) mutant ASFV-G-∆I177L is a safe and efficacious vaccine which induces protection against the challenge of its parental virus, the Georgia 2010 isolate. Although a genetic DIVA (differentiation between infected and vaccinated animals) assay has been developed for this vaccine, still there is not a serological DIVA test for differentiating between animals vaccinated with ASFV-G-∆I177L and those infected with wild-type viruses. In this report, we describe the development of the ASFV-G-∆I177L mutant having deleted the EP402R gene, which encodes for the viral protein responsible for mediating the hemadsorption of swine erythrocytes. The resulting virus, ASFV-G-∆I177L/∆EP402R, does not have a decreased ability to replicates in swine macrophages when compared with the parental ASFV-G-∆I177L. Domestic pigs intramuscularly (IM) inoculated with either 10² or 10⁶ HAD₅₀ of ASFV-G-∆I177L/∆EP402R remained clinically normal, when compared with a group of mock-vaccinated animals, indicating the absence of residual virulence. Interestingly, an infectious virus could not be detected in the blood samples of the ASFV-G-∆I177L/∆EP402R-inoculated animals in either group at any of the time points tested. Furthermore, while all of the mock-inoculated animals presented a quick and lethal clinical form of ASF after the intramuscular inoculation challenge with 10² HAD₅₀ of highly virulent parental field isolate Georgia 2010 (ASFV-G), all of the ASFV-G-∆I177L/∆EP402R-inoculated animals were protected, remaining clinically normal until the end of the observational period. Most of the ASFV-G-∆I177L/∆EP402R-inoculated pigs developed strong virus-specific antibody responses against viral antigens, reaching maximum levels at 28 days post inoculation. Importantly, all of the sera collected at that time point in the ASFV-G-∆I177L/∆EP402R-inoculated pigs did not react in a direct ELISA coated with the recombinant EP402R protein. Conversely, the EP402R protein was readily recognized by the pool of sera from the animals immunized with recombinant live attenuated vaccine candidates ASFV-G-∆I177L, ASFV-G-∆MGF, or ASFV-G-∆9GL/∆UK. Therefore, ASFV-G-∆I177L/∆EP402R is a novel, safe and efficacious candidate with potential to be used as an antigenically DIVA vaccine. Full article

(This article belongs to the Special Issue Pathogenesis of Viral Infections: Implications in the Development of Vaccines and Diagnostic Tools 2.0)

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12 pages, 1737 KiB

Open AccessArticle

The Interaction between the DOCK7 Protein and the E2 Protein of Classical Swine Fever Virus Is Not Involved with Viral Replication or Pathogenicity

by Elizabeth Vuono, Elizabeth Ramirez-Medina, Ediane Silva, Keith Berggren, Ayushi Rai, Nallely Espinoza, Manuel V. Borca and Douglas P. Gladue

Viruses 2024, 16(1), 70; https://doi.org/10.3390/v16010070 - 30 Dec 2023

Viewed by 893

Abstract

The classical swine fever virus (CSFV) particle consists of three glycoproteins, all of which have been shown to be important proteins involved in many virus functions, including interaction with several host proteins. One of these proteins, E2, has been shown to be directly involved with adsorption to the host cell and important for virus virulence. Using the yeast two-hybrid system, we have previously shown that CSFV E2 specifically interacts with the (DOCK7) dedicator of cytokinesis, a scaffolding protein. In this report, the interaction between E2 and DOCK7 was evaluated. To confirm the yeast two-hybrid results and to determine that DOCK7 interacts in swine cells with E2, we performed co-immunoprecipitation and proximity ligation assay (PLA). After demonstrating the protein interaction in swine cells, E2 amino acid residues Y65, V283, and T149 were determined to be critical for interaction with Dock7 by using a random mutated library of E2 and a reverse yeast two-hybrid approach. That disruption of these three residues with mutations Y65F, V283D, and T149A abrogated the Dock7-E2 protein interaction. These mutations were then introduced into a recombinant CSFV, E2DOCK7v, by a reverse genomics approach using the highly virulent CSFV Brescia isolate as a backbone. E2DOCKv was shown to have similar growth kinetics in swine primary macrophages and SK6 cell cultures to the parental Brescia strain. Similarly, E2DOCK7v demonstrated a similar level of virulence to the parental Brescia when inoculated in domestic pigs. Animals intranasally inoculated with 10⁵ TCID₅₀ developed a lethal form of clinical disease with virological and hematological kinetics changes indistinguishable from that produced by the parental strain. Therefore, interaction between CSFV E2 and host DOCK7 is not critically involved in the process of virus replication and disease production. Full article

(This article belongs to the Special Issue Pathogenesis of Viral Infections: Implications in the Development of Vaccines and Diagnostic Tools 2.0)

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13 pages, 1923 KiB

Open AccessArticle

Reclassification of ASFV into 7 Biotypes Using Unsupervised Machine Learning

by Mark Dinhobl, Edward Spinard, Nicolas Tesler, Hillary Birtley, Anthony Signore, Aruna Ambagala, Charles Masembe, Manuel V. Borca and Douglas P. Gladue

Viruses 2024, 16(1), 67; https://doi.org/10.3390/v16010067 - 30 Dec 2023

Viewed by 1588

Abstract

In 2007, an outbreak of African swine fever (ASF), a deadly disease of domestic swine and wild boar caused by the African swine fever virus (ASFV), occurred in Georgia and has since spread globally. Historically, ASFV was classified into 25 different genotypes. However, a newly proposed system recategorized all ASFV isolates into 6 genotypes exclusively using the predicted protein sequences of p72. However, ASFV has a large genome that encodes between 150–200 genes, and classifications using a single gene are insufficient and misleading, as strains encoding an identical p72 often have significant mutations in other areas of the genome. We present here a new classification of ASFV based on comparisons performed considering the entire encoded proteome. A curated database consisting of the protein sequences predicted to be encoded by 220 reannotated ASFV genomes was analyzed for similarity between homologous protein sequences. Weights were applied to the protein identity matrices and averaged to generate a genome-genome identity matrix that was then analyzed by an unsupervised machine learning algorithm, DBSCAN, to separate the genomes into distinct clusters. We conclude that all available ASFV genomes can be classified into 7 distinct biotypes. Full article

(This article belongs to the Special Issue Pathogenesis of Viral Infections: Implications in the Development of Vaccines and Diagnostic Tools 2.0)

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13 pages, 2855 KiB

Open AccessArticle

A Re-Evaluation of African Swine Fever Genotypes Based on p72 Sequences Reveals the Existence of Only Six Distinct p72 Groups

by Edward Spinard, Mark Dinhobl, Nicolas Tesler, Hillary Birtley, Anthony V. Signore, Aruna Ambagala, Charles Masembe, Manuel V. Borca and Douglas P. Gladue

Viruses 2023, 15(11), 2246; https://doi.org/10.3390/v15112246 - 11 Nov 2023

Cited by 10 | Viewed by 3265

Abstract

The African swine fever virus (ASFV) is currently causing a world-wide pandemic of a highly lethal disease in domestic swine and wild boar. Currently, recombinant ASF live-attenuated vaccines based on a genotype II virus strain are commercially available in Vietnam. With 25 reported ASFV genotypes in the literature, it is important to understand the molecular basis and usefulness of ASFV genotyping, as well as the true significance of genotypes in the epidemiology, transmission, evolution, control, and prevention of ASFV. Historically, genotyping of ASFV was used for the epidemiological tracking of the disease and was based on the analysis of small fragments that represent less than 1% of the viral genome. The predominant method for genotyping ASFV relies on the sequencing of a fragment within the gene encoding the structural p72 protein. Genotype assignment has been accomplished through automated phylogenetic trees or by comparing the target sequence to the most closely related genotyped p72 gene. To evaluate its appropriateness for the classification of genotypes by p72, we reanalyzed all available genomic data for ASFV. We conclude that the majority of p72-based genotypes, when initially created, were neither identified under any specific methodological criteria nor correctly compared with the already existing ASFV genotypes. Based on our analysis of the p72 protein sequences, we propose that the current twenty-five genotypes, created exclusively based on the p72 sequence, should be reduced to only six genotypes. To help differentiate between the new and old genotype classification systems, we propose that Arabic numerals (1, 2, 8, 9, 15, and 23) be used instead of the previously used Roman numerals. Furthermore, we discuss the usefulness of genotyping ASFV isolates based only on the p72 gene sequence. Full article

(This article belongs to the Special Issue Pathogenesis of Viral Infections: Implications in the Development of Vaccines and Diagnostic Tools 2.0)

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16 pages, 3913 KiB

Open AccessArticle

A Cell-Adapted Live-Attenuated Vaccine Candidate Protects Pigs against the Homologous Strain VNUA-ASFV-05L1, a Representative Strain of the Contemporary Pandemic African Swine Fever Virus

by Quang Lam Truong, Lihua Wang, Tuan Anh Nguyen, Hoa Thi Nguyen, Son Danh Tran, Anh Thi Vu, Anh Dao Le, Van Giap Nguyen, Phuong Thi Hoang, Yen Thi Nguyen, Thi Luyen Le, Thang Nguyen Van, Thi My Le Huynh, Huong Thi Lan Lai, Rachel Madera, Yuzhen Li, Jishu Shi and Lan Thi Nguyen

Viruses 2023, 15(10), 2089; https://doi.org/10.3390/v15102089 - 13 Oct 2023

Cited by 2 | Viewed by 1846

Abstract

African swine fever (ASF) is a lethal and highly contagious transboundary animal disease with the potential for rapid international spread. Currently, there is no ASF vaccine commercially available. All infected animals must be isolated and culled immediately upon the confirmation of the presence of the virus. Studies leading to the rational development of protective ASF vaccines are urgently needed. Here, we generated a safe and efficacious live-attenuated vaccine (LAV) VNUA-ASFV-LAVL2 by serially passaging a field isolate (VNUA-ASFV-05L1, genotype II) in porcine alveolar macrophages (PAMs, 65 passages) and an immortalized porcine alveolar macrophage cell line (3D4/21, 55 passages). VNUA-ASFV-LAVL2 can efficiently replicate in both PAMs and 3D4/21 cells. It provides 100% protection, even with the low dose of 10² HAD₅₀, to the vaccinated pigs against the challenge of contemporary pandemic ASFV field isolate. Pigs vaccinated with this LAV in a dose range of 10² to 10⁵ HAD₅₀ remained clinically healthy during both the 28-day observation period of immunization and the 28-day observation period of challenge. VNUA-ASFV-LAVL2 was eliminated from blood by 28 days post-inoculation (DPI), and from feces or oral fluids by 17 DPI. Although the vaccine strain in serum remained a safe and attenuated phenotype after five passages in swine, a reversion-to-virulence study using blood or tissue homogenates at peak viremia will be conducted in the future. ASFV-specific IgG antibodies and significant cellular immunity were detected in vaccinated pigs before the ASFV challenge. These results indicate that the VNUA-ASFV-LAVL2 strain is a safe and efficacious LAV against the genotype II ASFV strain responsible for current ASF outbreaks in Asia. Full article

(This article belongs to the Special Issue Pathogenesis of Viral Infections: Implications in the Development of Vaccines and Diagnostic Tools 2.0)

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10 pages, 2105 KiB

Open AccessArticle

African Swine Fever Vaccine Candidate ASFV-G-ΔI177L Produced in the Swine Macrophage-Derived Cell Line IPKM Remains Genetically Stable and Protective against Homologous Virulent Challenge

by Manuel V. Borca, Ayushi Rai, Nallely Espinoza, Elizabeth Ramirez-Medina, Edward Spinard, Lauro Velazquez-Salinas, Alyssa Valladares, Ediane Silva, Leeanna Burton, Amanda Meyers, Cyril G. Gay and Douglas P. Gladue

Viruses 2023, 15(10), 2064; https://doi.org/10.3390/v15102064 - 08 Oct 2023

Cited by 2 | Viewed by 1057

Abstract

ASFV vaccine candidate ASFV-G-ΔI177L has been shown to be highly efficacious in inducing protection against challenges with the parental virus, the Georgia 2010 isolate, as well as against field strains isolated from Vietnam. ASFV-G-ΔI177L has been shown to produce protection even when used at low doses (10² HAD₅₀) and shows no residual virulence even when administered at high doses (10⁶ HAD₅₀) or evaluated for a relatively long period of time (6 months). ASFV-G-ΔI177L stocks can only be massively produced in primary cell macrophages. Alternatively, its modified version (ASFV-G-ΔI177L/ΔLVR) grows in a swine-derived cell line (PIPEC), acquiring significant genomic modifications. We present here the development of ASFV-G-ΔI177L stocks in a swine macrophage cell line, IPKM, and its protective efficacy when evaluated in domestic pigs. Successive passing of ASFV-G-ΔI177L in IPKM cells produces minimal genomic changes. Interestingly, a stock of ASFV-G-ΔI177L obtained after 10 passages (ASFV-G-ΔI177Lp10) in IPKM cells showed very small genomic changes when compared with the original virus stock. ASFV-G-ΔI177Lp10 conserves similar growth kinetics in primary swine macrophage cultures than the original parental virus ASFV-G-ΔI177L. Pigs infected with 10³ HAD₅₀ of ASFV-G-ΔI177Lp10 developed a strong virus-specific antibody response and were completely protected against the challenge with the parental virulent field isolate Georgia 2010. Therefore, IPKM cells could be an effective alternative for the production of ASFV vaccine stocks for those vaccine candidates exclusively growing in swine macrophages. Full article

(This article belongs to the Special Issue Pathogenesis of Viral Infections: Implications in the Development of Vaccines and Diagnostic Tools 2.0)

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18 pages, 2578 KiB

Open AccessArticle

Generation and Efficacy of Two Chimeric Viruses Derived from GPE⁻ Vaccine Strain as Classical Swine Fever Vaccine Candidates

by Loc Tan Huynh, Norikazu Isoda, Lim Yik Hew, Saho Ogino, Yume Mimura, Maya Kobayashi, Taksoo Kim, Tatsuya Nishi, Katsuhiko Fukai, Takahiro Hiono and Yoshihiro Sakoda

Viruses 2023, 15(7), 1587; https://doi.org/10.3390/v15071587 - 20 Jul 2023

Cited by 3 | Viewed by 1554

Abstract

A previous study proved that vGPE⁻ mainly maintains the properties of classical swine fever (CSF) virus, which is comparable to the GPE⁻ vaccine seed and is a potentially valuable backbone for developing a CSF marker vaccine. Chimeric viruses were constructed based on an infectious cDNA clone derived from the live attenuated GPE⁻ vaccine strain as novel CSF vaccine candidates that potentially meet the concept of differentiating infected from vaccinated animals (DIVA) by substituting the glycoprotein E^rns of the GPE⁻ vaccine strain with the corresponding region of non-CSF pestiviruses, either pronghorn antelope pestivirus (PAPeV) or Phocoena pestivirus (PhoPeV). High viral growth and genetic stability after serial passages of the chimeric viruses, namely vGPE⁻/PAPeV E^rns and vGPE⁻/PhoPeV E^rns, were confirmed in vitro. In vivo investigation revealed that two chimeric viruses had comparable immunogenicity and safety profiles to the vGPE⁻ vaccine strain. Vaccination at a dose of 10^4.0 TCID₅₀ with either vGPE⁻/PAPeV E^rns or vGPE⁻/PhoPeV E^rns conferred complete protection for pigs against the CSF virus challenge in the early stage of immunization. In conclusion, the characteristics of vGPE⁻/PAPeV E^rns and vGPE⁻/PhoPeV E^rns affirmed their properties, as the vGPE⁻ vaccine strain, positioning them as ideal candidates for future development of a CSF marker vaccine. Full article

(This article belongs to the Special Issue Pathogenesis of Viral Infections: Implications in the Development of Vaccines and Diagnostic Tools 2.0)

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14 pages, 6843 KiB

Open AccessEditor’s ChoiceArticle

A Candidate Antigen of the Recombinant Membrane Protein Derived from the Porcine Deltacoronavirus Synthetic Gene to Detect Seropositive Pigs

by Francisco Jesus Castañeda-Montes, José Luis Cerriteño-Sánchez, María Azucena Castañeda-Montes, Julieta Sandra Cuevas-Romero and Susana Mendoza-Elvira

Viruses 2023, 15(5), 1049; https://doi.org/10.3390/v15051049 - 25 Apr 2023

Cited by 1 | Viewed by 1518

Abstract

Porcine deltacoronavirus (PDCoV) is an emergent swine coronavirus which infects cells from the small intestine and induces watery diarrhea, vomiting and dehydration, causing mortality in piglets (>40%). The aim of this study was to evaluate the antigenicity and immunogenicity of the recombinant membrane protein (M) of PDCoV (rM-PDCoV), which was developed from a synthetic gene obtained after an in silico analysis with a group of 138 GenBank sequences. A 3D model and phylogenetic analysis confirmed the highly conserved M protein structure. Therefore, the synthetic gene was successfully cloned in a pETSUMO vector and transformed in E. coli BL21 (DE3). The rM-PDCoV was confirmed by SDS-PAGE and Western blot with ~37.7 kDa. The rM-PDCoV immunogenicity was evaluated in immunized (BLAB/c) mice and iELISA. The data showed increased antibodies from 7 days until 28 days (p < 0.001). The rM-PDCoV antigenicity was analyzed using pig sera samples from three states located in “El Bajío” Mexico and positive sera were determined. Our results show that PDCoV has continued circulating on pig farms in Mexico since the first report in 2019; therefore, the impact of PDCoV on the swine industry could be higher than reported in other studies. Full article

(This article belongs to the Special Issue Pathogenesis of Viral Infections: Implications in the Development of Vaccines and Diagnostic Tools 2.0)

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12 pages, 4191 KiB

Open AccessArticle

Development of Porcine Monoclonal Antibodies with In Vitro Neutralizing Activity against Classical Swine Fever Virus from C-Strain E2-Specific Single B Cells

by Lihua Wang, Rachel Madera, Yuzhen Li, Douglas P. Gladue, Manuel V. Borca, Michael T. McIntosh and Jishu Shi

Viruses 2023, 15(4), 863; https://doi.org/10.3390/v15040863 - 28 Mar 2023

Cited by 1 | Viewed by 1468

Abstract

Neutralizing antibodies (nAbs) can be used before or after infection to prevent or treat viral diseases. However, there are few efficacious nAbs against classical swine fever virus (CSFV) that have been produced, especially the porcine-originated nAbs. In this study, we generated three porcine monoclonal antibodies (mAbs) with in vitro neutralizing activity against CSFV, aiming to facilitate the development of passive antibody vaccines or antiviral drugs against CSFV that offer the advantages of stability and low immunogenicity. Pigs were immunized with the C-strain E2 (CE2) subunit vaccine, KNB-E2. At 42 days post vaccination (DPV), CE2-specific single B cells were isolated via fluorescent-activated cell sorting (FACS) baited by Alexa Fluor™ 647-labeled CE2 (positive), goat anti-porcine IgG (H + L)-FITC antibody (positive), PE mouse anti-pig CD3ε (negative) and PE mouse anti-pig CD8a (negative). The full coding region of IgG heavy (H) chains and light (L) chains was amplified by reverse transcription-polymerase chain reaction (RT-PCR). Overall, we obtained 3 IgG H chains, 9 kappa L chains and 36 lambda L chains, which include three paired chains (two H + κ and one H + λ). CE2-specific mAbs were successfully expressed in 293T cells with the three paired chains. The mAbs exhibit potent neutralizing activity against CSFVs. They can protect ST cells from infections in vitro with potent IC₅₀ values from 14.43 µg/mL to 25.98 µg/mL for the CSFV C-strain, and 27.66 µg/mL to 42.61 µg/mL for the CSFV Alfort strain. This study is the first report to describe the amplification of whole-porcine IgG genes from single B cells of KNB-E2-vaccinated pig. The method is versatile, sensitive, and reliable. The generated natural porcine nAbs can be used to develop long-acting and low-immunogenicity passive antibody vaccine or anti-CSFV agents for CSF control and prevention. Full article

(This article belongs to the Special Issue Pathogenesis of Viral Infections: Implications in the Development of Vaccines and Diagnostic Tools 2.0)

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