scholarly journals Assessing the Protective Dose of a Candidate DIVA Vaccine against Classical Swine Fever

Vaccines ◽  
2021 ◽  
Vol 9 (5) ◽  
pp. 483
Author(s):  
Tinka Jelsma ◽  
Jacob Post ◽  
Erwin van den Born ◽  
Ruud Segers ◽  
Jeroen Kortekaas
Keyword(s):  
Infectious Virus ◽  
Clinical Signs ◽  
Classical Swine Fever ◽  
Wild Type ◽  
Challenge Virus ◽  
Virus Particles ◽  
Virus Rna ◽  
Single Vaccination ◽  
Highly Virulent ◽  
Insight Into

Classical swine fever is a highly contagious and deadly disease in swine. The disease can be controlled effectively by vaccination with an attenuated virus known as the “Chinese” (C)-strain. A single vaccination with the C-strain provides complete protection against highly virulent isolates within days after vaccination, making it one of the most efficacious veterinary vaccines ever developed. A disadvantage of the C-strain is that vaccinated animals cannot be serologically differentiated from animals that are infected with wild-type Classical swine fever virus. Previously, a C-strain-based vaccine with a stable deletion in the E2 structural glycoprotein was developed, which allows for differentiation between infected and vaccinated animals (DIVA). The resulting vaccine, which we named C-DIVA, is compatible with a commercial E2 ELISA, modified to render it suitable as a DIVA test. In the present work, three groups of eight piglets were vaccinated with escalating doses of the C-DIVA vaccine and challenged two weeks after vaccination. One group of four unvaccinated piglets served as controls. Piglets were monitored for clinical signs until three weeks after challenge and blood samples were collected to monitor viremia, leukocyte and thrombocyte levels, and antibody responses. The presence of challenge virus RNA in oropharyngeal swabs was investigated to first gain insight into the potential of C-DIVA to prevent shedding. The results demonstrate that a single vaccination with 70 infectious virus particles of C-DIVA protects pigs from the highly virulent Brescia strain.

scholarly journals Analysis of Virus Population Profiles within Pigs Infected with Virulent Classical Swine Fever Viruses: Evidence for Bottlenecks in Transmission but Absence of Tissue-Specific Virus Variants

2020 ◽  
Vol 94 (19) ◽  
Author(s):  
Camille Melissa Johnston ◽  
Ulrik Fahnøe ◽  
Louise Lohse ◽  
Jens Bukh ◽  
Graham J. Belsham ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Population Change ◽  
Virulent Strain ◽  
Severe Disease ◽  
Clinical Signs ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Host Cells ◽  
Virus Population ◽  
Highly Virulent

ABSTRACT Classical swine fever virus (CSFV) contains a specific motif within the E2 glycoprotein that differs between strains of different virulence. In the highly virulent CSFV strain Koslov, this motif comprises residues S763/L764 in the polyprotein. However, L763/P764 represent the predominant alleles in published CSFV genomes. In this study, changes were introduced into the CSFV strain Koslov (here called vKos_SL) to generate modified CSFVs with substitutions at residues 763 and/or 764 (vKos_LL, vKos_SP, and vKos_LP). The properties of these mutant viruses, in comparison to those of vKos_SL, were determined in pigs. Each of the viruses was virulent and induced typical clinical signs of CSF, but the vKos_LP strain produced them significantly earlier. Full-length CSFV cDNA amplicons (12.3 kb) derived from sera of infected pigs were deep sequenced and cloned to reveal the individual haplotypes that contributed to the single-nucleotide polymorphism (SNP) profiles observed in the virus population. The SNP profiles for vKos_SL and vKos_LL displayed low-level heterogeneity across the entire genome, whereas vKos_SP and vKos_LP displayed limited diversity with a few high-frequency SNPs. This indicated that vKos_SL and vKos_LL exhibited a higher level of fitness in the host and more stability at the consensus level, whereas several consensus changes were observed in the vKos_SP and vKos_LP sequences, pointing to adaptation. For each virus, only a subset of the variants present within the virus inoculums were maintained in the infected pigs. No clear tissue-dependent quasispecies differentiation occurred within inoculated pigs; however, clear evidence for transmission bottlenecks to contact animals was observed, with subsequent loss of sequence diversity. IMPORTANCE The surface-exposed E2 protein of classical swine fever virus is required for its interaction with host cells. A short motif within this protein varies between strains of different virulence. The importance of two particular amino acid residues in determining the properties of a highly virulent strain of the virus has been analyzed. Each of the different viruses tested proved highly virulent, but one of them produced earlier, but not more severe, disease. By analyzing the virus genomes present within infected pigs, it was found that the viruses which replicated within inoculated animals were only a subset of those within the virus inoculum. Furthermore, following contact transmission, it was shown that a very restricted set of viruses had transferred between animals. There were no significant differences in the virus populations present in various tissues of the infected animals. These results indicate mechanisms of virus population change during transmission between animals.

scholarly journals The Influenza A Virus M2 Cytoplasmic Tail Is Required for Infectious Virus Production and Efficient Genome Packaging

2005 ◽  
Vol 79 (6) ◽  
pp. 3595-3605 ◽  
Author(s):  
Matthew F. McCown ◽  
Andrew Pekosz
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Infectious Virus ◽  
Virus Production ◽  
Cytoplasmic Tail ◽  
Host Cells ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virus Particles

ABSTRACT The M2 integral membrane protein encoded by influenza A virus possesses an ion channel activity that is required for efficient virus entry into host cells. The role of the M2 protein cytoplasmic tail in virus replication was examined by generating influenza A viruses encoding M2 proteins with truncated C termini. Deletion of 28 amino acids (M2Stop70) resulted in a virus that produced fourfold-fewer particles but >1,000-fold-fewer infectious particles than wild-type virus. Expression of the full-length M2 protein in trans restored the replication of the M2 truncated virus. Although the M2Stop70 virus particles were similar to wild-type virus in morphology, the M2Stop70 virions contained reduced amounts of viral nucleoprotein and genomic RNA, indicating a defect in vRNP packaging. The data presented indicate the M2 cytoplasmic tail plays a role in infectious virus production by coordinating the efficient packaging of genome segments into influenza virus particles.

scholarly journals Ubiquitination of the Cytoplasmic Domain of Influenza A Virus M2 Protein is Crucial for Production of Infectious Virus Particles

2017 ◽  
pp. JVI.01972-17 ◽  
Author(s):  
Wen-Chi Su ◽  
Wen-Ya Yu ◽  
Shih-Han Huang ◽  
Michael M.C. Lai
Keyword(s):  
Virus Replication ◽  
Influenza A ◽  
Functional Role ◽  
Infectious Virus ◽  
Virus Production ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virus Particles

Virus replication is mediated by interactions between virus and host. Here, we demonstrate that influenza A virus membrane protein 2 (M2) can be ubiquitinated. The lysine residue at position 78, which is located in the cytoplasmic domain of M2, is essential for M2 ubiquitination. An M2-K78R (Lys78→Arg78) mutant, which produces ubiquitination-deficient M2, showed a severe defect in production of infectious virus particles. M2-K78R mutant progeny contained more HA proteins, less viral RNAs and less internal viral proteins, including M1 and NP, than the wild-type virus. Furthermore, most of the M2-K78R mutant viral particles lacked viral ribonucleoproteins upon examination under electron microscopy and exhibited slightly lower densities. We also found that mutant M2 colocalized with M1 protein to a lesser extent than for wild-type virus. These findings may account for the reduced incorporation of viral ribonucleoprotein into virions. By blocking the second round of virus infection, we showed that the M2 ubiquitination-defective mutant exhibited normal level of virus replication during the first round of infection, thereby proving that M2 ubiquitination is involved in the virus production step. Finally, we found that M2-K78R mutant virus induced autophagy and apoptosis earlier than wild-type virus. Collectively, these results suggest that M2 ubiquitination plays an important role in infectious virus production by coordinating efficient packaging of the viral genome into virus particles and timing of viral-induced cell death.IMPORTANCEAnnual epidemics and recurring pandemics of influenza viruses represent a very high global health and economic burden. Influenza virus M2 protein has been extensively studied for its important roles in virus replication, particularly in viral entry and release. Rimantadine, one of the most commonly used antiviral drugs, binds to the channel lumen near the N-terminus of M2 proteins. However, viruses resistant to Rimantadine have emerged. M2 undergoes several posttranslational modifications, such as phosphorylation and palmitoylation. Here, we reveal that ubiquitination mediates the functional role of M2. A ubiquitination-deficient M2 mutant predominately produced virus particles either lacking viral ribonucleoproteins or containing smaller amounts of internal viral components, resulting in lower infectivity. Our findings offer insights into the mechanism of influenza virus morphogenesis, particularly the functional role of M1-M2 interactions in viral particle assembly, and can be applied to the development of new influenza therapies.

scholarly journals Mutant Human Cytomegalovirus Lacking the Immediate-Early TRS1 Coding Region Exhibits a Late Defect

2002 ◽  
Vol 76 (23) ◽  
pp. 12290-12299 ◽  
Author(s):  
Catherine A. Blankenship ◽  
Thomas Shenk
Keyword(s):  
Human Cytomegalovirus ◽  
Infectious Virus ◽  
Human Fibroblasts ◽  
Reporter Genes ◽  
Immediate Early ◽  
Viral Gene ◽  
Wild Type ◽  
Viral Mrnas ◽  
Virus Particles ◽  
Infected Cells

ABSTRACT The human cytomegalovirus IRS1 and TRS1 open reading frames encode immediate-early proteins with identical N-terminal domains and divergent C-terminal regions. Both proteins have been shown previously to activate reporter genes in transfection assays in cooperation with other viral gene products. We have constructed two viruses carrying substitution mutations within either the IRS1 or TRS1 open reading frame. ADsubIRS1 failed to produce the related IRS1 and IRS1263 proteins, but it replicated with normal kinetics to produce a wild-type yield in human fibroblasts. The addition in trans of the IRS1263 protein, which antagonizes the ability of IRS1 and TRS1 proteins to activate reporter genes, did not inhibit the growth of the mutant virus. ADsubTRS1 failed to produce the TRS1 protein, and it generated an ∼200-fold-reduced yield of infectious virus in comparison to its wild-type parent. Viral DNA accumulated normally, as did a set of viral mRNAs that were monitored in ADsubTRS1-infected cells. However, two tegument proteins were partially mislocalized and infectious virus particles did not accumulate to normal levels within ADsubTRS1-infected cells. Further, infectious ADsubTRS1 particles sedimented abnormally in a glycerol-tartrate gradient, indicating that the structure of the mutant particles is aberrant. Our analysis of the ADsubTRS1 phenotype indicates that the TRS1 protein is required, either directly or indirectly, for efficient assembly of virus particles.

scholarly journals Virion-Independent Transfer of Replication-Competent Hepatitis C Virus RNA between Permissive Cells

2014 ◽  
Vol 89 (5) ◽  
pp. 2956-2961 ◽  
Author(s):  
Andrea Longatti ◽  
Bryan Boyd ◽  
Francis V. Chisari
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Infectious Virus ◽  
Immune Escape ◽  
Hcv Rna ◽  
Virus Particles ◽  
Viral Spread ◽  
Virus Rna ◽  
Huh7 Cells

In this study, we show that replication-competent subgenomic hepatitis C virus (HCV) RNA can be transferred to permissive Huh7 cells, leading to the establishment of viral RNA replication. Further, we show that these events are mediated by exosomes rather than infectious virus particles. If similar events occurin vivo, this could represent a novel, albeit inefficient, mechanism of viral spread and immune escape.

scholarly journals Herpes Simplex Virus Type 1 Glycoprotein B Requires a Cysteine Residue at Position 633 for Folding, Processing, and Incorporation into Mature Infectious Virus Particles

1998 ◽  
Vol 72 (6) ◽  
pp. 4940-4949 ◽  
Author(s):  
Sylvie Laquerre ◽  
Dina B. Anderson ◽  
Rafaela Argnani ◽  
Joseph C. Glorioso
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Infectious Virus ◽  
Disulfide Bridge ◽  
Cysteine Residue ◽  
Wild Type ◽  
Virus Particles ◽  
Simplex Virus ◽  
Mature Virus

ABSTRACT Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) resides in the virus envelope in an oligomeric form and plays an essential role in virus entry into susceptible host cells. The oligomerizing domain is a movable element consisting of amino acids 626 to 653 in the gB external domain. This domain contains a single cysteine residue at position 633 (Cys-633) that is predicted to form an intramolecular disulfide bridge with Cys-596. In this study, we examined gB oligomerization, processing, and incorporation into mature virus during infection by two mutant viruses in which either the gB Cys-633 [KgB(C633S)] or both Cys-633 and Cys-596 [KgB(C596S/C633S)] residues were mutated to serine. The result of immunofluorescence studies and analyses of released virus particles showed that the mutant gB molecules were not transported to the cell surface or incorporated into mature virus envelopes and thus infectious virus was not produced. Immunoprecipitation studies revealed that the mutant gB molecules were in an oligomeric configuration and that these mutants produced hetero-oligomers with a truncated form of gB consisting of residues 1 to 43 and 595 to 904, the latter containing the oligomerization domain. Pulse-chase experiments in combination with endoglycosidase H treatment determined that the mutant molecules were improperly processed, having been retained in the endoplasmic reticulum (ER). Coimmunoprecipitation experiments revealed that the cysteine mutations resulted in gB misfolding and retention by the molecular chaperones calnexin, calreticulin, and Grp78 in the ER. The altered conformation of the gB mutant glycoproteins was directly detected by a reduction in monoclonal antibody recognition of two previously defined distinct antigenic sites located within residues 381 to 441 and 595 to 737. The misfolded molecules were not transported to the cell surface as hetero-oligomers with wild-type gB, suggesting that the conformational change could not be corrected by intermolecular interactions with the wild-type molecule. Together, these experiments confirmed that a disulfide bridge involving Cys-633 and Cys-596 is not essential for oligomerization but rather is required for proper folding and maintenance of a gB domain essential to complete posttranslational modification, transport, and incorporation into mature virus particles.

Survival of classic swine fever virus in hams made from the meat of pigs vaccinated with the PAV-250 strain and unvaccinated pigs

2019 ◽  
Vol 10 (3) ◽  
pp. 536-551
Author(s):  
Heidi Amezcua Hempel ◽  
María Salud Rubio Lozano ◽  
Eliseo Manuel Hernández Baumgarten ◽  
Pablo Correa Girón † ◽  
Oscar Torres Ángeles ◽  
...  
Keyword(s):  
Reference Strain ◽  
Clinical Signs ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Direct Immunofluorescence ◽  
Biometric Analysis ◽  
Treatment Groups ◽  
Group A ◽  
Group B ◽  
Group D

The study was to determine the presence of Classical Swine Fever virus (CSFv), in the meat of vaccinated pigs with the PAV-250 strain and then challenged using the same strain. Five treatment groups were established (each with four pigs). Group A: Pigs thatwere fed with processed hams from negative animals; Group B: Pigs that were fed with processed hams from commercial pigs inoculated with the ALD (reference strain) (titre of 104.0/ml); Group C: Pigs fed with processed hams from pigs infected with the virulent ALD strain (titre of 102.5/ml); Group D: Pigs fed with processed hams from pigs vaccinated with the PAV-250 strain and challenged with the ALD strain (titre of 101.1/ml); and Group E: Pigs fed with processed hams from pigs vaccinated with two doses of the PAV-250 strain and challenged with the ALD strain (negative). Blood samples were taken at d 1, 5, 10, 15 and 20 for biometric analysis. Groups B, C and D manifested clinical signs of CSFv: 40 °C temperature, anorexia, paralysis, vomiting, diarrhea, tremor, hirsute hair and cyanosis. Pigs were slaughtered and necropsies performed to identify lesions in tissues. Results of direct immunofluorescence testing of tissues were positive and the virus was recovered. Under these study conditions, it was found that CSFv resisted the cooking method at 68 °C for 40 min in hams from unvaccinated pigs, and that the virus was able to transmit the disease to healthy unvaccinated pigs, whereas the hams from the vaccinated animals did not transmit the virus.

Suggestive Evidence for an Oncorna-Virus-Specific DNA Polymerase from C-Type Particles of Bovine Leukosis

1974 ◽  
Vol 29 (1-2) ◽  
pp. 72-75 ◽  
Author(s):  
B. Dietzschold ◽  
O.R. Kaaden ◽  
S. Ueberschaer ◽  
F. Weiland ◽  
O. C. Straub
Keyword(s):  
Dna Polymerase ◽  
Polymerase Activity ◽  
Friend Virus ◽  
Leukaemia Virus ◽  
Virus Particles ◽  
Virus Rna ◽  
Feline Leukaemia Virus ◽  
Bovine Leukosis ◽  
Bovine Lymphocytes ◽  
Viral Preparation

Abstract Typical C-type oncorna virus particles as shown by electron microscopy have been purified from the supernatant of cultured lymphocytes from bovine leukosis. In the purified C-particle fraction a DNA-polymerase activity was detected. Using several synthetic RNA-or DNA-homopolymers and 70S Friend virus RNA the template response of this bovine leukosis cell particle DNA polymerase was compared with those of feline leukaemia virus DNA polymerase and DNA polymerase from normal bovine lymphocytes. The DNA polymerase detected in the viral preparation of bovine leukosis is suggested to be an oncorna-virus-specific enzyme.

scholarly journals Salicylic Acid Accumulation Controlled by LSD1 Is Essential in Triggering Cell Death in Response to Abiotic Stress

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 962
Author(s):  
Maciej Jerzy Bernacki ◽  
Anna Rusaczonek ◽  
Weronika Czarnocka ◽  
Stanisław Karpiński
Keyword(s):  
Cell Death ◽  
Salicylic Acid ◽  
Abiotic Stress ◽  
Wild Type ◽  
Pr Genes ◽  
Genes Expression ◽  
Salicylic Acid Accumulation ◽  
Cell Death Phenotype ◽  
Insight Into

Salicylic acid (SA) is well known hormonal molecule involved in cell death regulation. In response to a broad range of environmental factors (e.g., high light, UV, pathogens attack), plants accumulate SA, which participates in cell death induction and spread in some foliar cells. LESION SIMULATING DISEASE 1 (LSD1) is one of the best-known cell death regulators in Arabidopsis thaliana. The lsd1 mutant, lacking functional LSD1 protein, accumulates SA and is conditionally susceptible to many biotic and abiotic stresses. In order to get more insight into the role of LSD1-dependent regulation of SA accumulation during cell death, we crossed the lsd1 with the sid2 mutant, caring mutation in ISOCHORISMATE SYNTHASE 1(ICS1) gene and having deregulated SA synthesis, and with plants expressing the bacterial nahG gene and thus decomposing SA to catechol. In response to UV A+B irradiation, the lsd1 mutant exhibited clear cell death phenotype, which was reversed in lsd1/sid2 and lsd1/NahG plants. The expression of PR-genes and the H2O2 content in UV-treated lsd1 were significantly higher when compared with the wild type. In contrast, lsd1/sid2 and lsd1/NahG plants demonstrated comparability with the wild-type level of PR-genes expression and H2O2. Our results demonstrate that SA accumulation is crucial for triggering cell death in lsd1, while the reduction of excessive SA accumulation may lead to a greater tolerance toward abiotic stress.

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