scholarly journals Detection of pseudorabies virus antibody in swine oral fluid using a serum whole-virus indirect ELISA

2020 ◽  
Vol 32 (4) ◽  
pp. 535-541
Author(s):  
Ting-Yu Cheng ◽  
Alexandra Buckley ◽  
Albert Van Geelen ◽  
Kelly Lager ◽  
Alexandra Henao-Díaz ◽  
...  
Keyword(s):  
Oral Fluid ◽  
Pseudorabies Virus ◽  
Negative Control ◽  
Wild Type Virus ◽  
Wild Type ◽  
Serum Samples ◽  
Anamnestic Response ◽  
Treatment Groups ◽  
Oral Fluids ◽  
Area Under Roc Curve

We evaluated the detection of pseudorabies virus (PRV) antibodies in swine oral fluid. Oral fluid and serum samples were obtained from 40 pigs allocated to 4 treatment groups (10 pigs/group): negative control (NC); wild-type PRV inoculation (PRV 3CR Ossabaw; hereafter PRV); PRV vaccination (Ingelvac Aujeszky MLV; Boehringer Ingelheim; hereafter MLV); and PRV vaccination followed by PRV inoculation at 21 d post-vaccination (MLV-PRV). Using a serum PRV whole-virus indirect IgG ELISA (Idexx Laboratories) adapted to the oral fluid matrix, PRV antibody was detected in oral fluid samples from treatment groups PRV, MLV, and MLV-PRV in a pattern similar to serum. Vaccination alone produced a low oral fluid antibody response (groups MLV and MLV-PRV), but a strong anamnestic response was observed following challenge with wild-type virus (group PRV). Analyses of the oral fluid PRV indirect IgG ELISA results showed good binary diagnostic performance (area under ROC curve = 93%) and excellent assay repeatability (intra-class correlation coefficient = 99.3%). The demonstrable presence of PRV antibodies in swine oral fluids suggests the possible use of oral fluids in pseudorabies surveillance.

scholarly journals Analysis of the Complete Genome Sequence of a Novel, Pseudorabies Virus Strain Isolated in Southeast Europe

2019 ◽  
Vol 2019 ◽  
pp. 1-12
Author(s):  
Zsolt Csabai ◽  
Dóra Tombácz ◽  
Zoltán Deim ◽  
Michael Snyder ◽  
Zsolt Boldogkői
Keyword(s):  
Single Molecule ◽  
Base Composition ◽  
Pseudorabies Virus ◽  
Point Mutations ◽  
Wild Type Virus ◽  
Economic Losses ◽  
Wild Type ◽  
Southeast Europe ◽  
Long Read ◽  
National Programs

Background. Pseudorabies virus (PRV) is the causative agent of Aujeszky’s disease giving rise to significant economic losses worldwide. Many countries have implemented national programs for the eradication of this virus. In this study, long-read sequencing was used to determine the nucleotide sequence of the genome of a novel PRV strain (PRV-MdBio) isolated in Serbia.Results. In this study, a novel PRV strain was isolated and characterized. PRV-MdBio was found to exhibit similar growth properties to those of another wild-type PRV, the strain Kaplan. Single-molecule real-time (SMRT) sequencing has revealed that the new strain differs significantly in base composition even from strain Kaplan, to which it otherwise exhibits the highest similarity. We compared the genetic composition of PRV-MdBio to strain Kaplan and the China reference strain Ea and obtained that radical base replacements were the most common point mutations preceding conservative and silent mutations. We also found that the adaptation of PRV to cell culture does not lead to any tendentious genetic alteration in the viral genome.Conclusion. PRV-MdBio is a wild-type virus, which differs in base composition from other PRV strains to a relatively large extent.

scholarly journals Role of Pseudorabies Virus Us3 Protein Kinase during Neuronal Infection

2006 ◽  
Vol 80 (13) ◽  
pp. 6387-6398 ◽  
Author(s):  
L. M. Olsen ◽  
T. H. Ch'ng ◽  
J. P. Card ◽  
L. W. Enquist
Keyword(s):  
Protein Kinase ◽  
Pseudorabies Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Spread Of Infection ◽  
Viral Components ◽  
Axonal Targeting ◽  
Null Mutants

ABSTRACT The pseudorabies virus (PRV) Us3 gene is conserved among the alphaherpesviruses and encodes a serine/threonine protein kinase that is not required for growth in standard cell lines. In this report, we used a compartmented culture system to investigate the role of PRV Us3 in viral replication in neurons, in spread from neurons to PK15 cells, and in axon-mediated spread of infection. We also examined the role of Us3 in neuroinvasion and virulence in rodents. Us3 null mutants produce about 10-fold less infectious virus from neurons than wild-type virus and have no discernible phenotypes for axonal targeting of viral components in cultured peripheral nervous system neurons. After eye infection in rodents, Us3 null mutants were slightly attenuated for virulence, with a delayed onset of symptoms compared to the wild type or a Us3 null revertant. While initially delayed, the symptoms increased in severity until they approximated those of the wild-type virus. Us3 null mutants were neuroinvasive, spreading in both efferent and afferent circuits innervating eye tissues.

scholarly journals UL54-Null Pseudorabies Virus Is Attenuated in Mice but Productively Infects Cells in Culture

2006 ◽  
Vol 80 (2) ◽  
pp. 769-784 ◽  
Author(s):  
Jennifer A. Schwartz ◽  
Elizabeth E. Brittle ◽  
Ashley E. Reynolds ◽  
Lynn W. Enquist ◽  
Saul J. Silverstein
Keyword(s):  
Protein Synthesis ◽  
Pseudorabies Virus ◽  
Host Protein ◽  
Wild Type Virus ◽  
Cell Protein ◽  
Transcriptional Level ◽  
Dependent Manner ◽  
Type Virus ◽  
Wild Type ◽  
In Cells

ABSTRACT The pseudorabies virus (PRV) UL54 homologs are important multifunctional proteins with roles in shutoff of host protein synthesis, transactivation of virus and cellular genes, and regulation of splicing and translation. Here we describe the first genetic characterization of UL54. We constructed UL54 null mutations in a PRV bacterial artificial chromosome using sugar suicide and λRed allele exchange systems. Surprisingly, UL54 is dispensable for growth in tissue culture but exhibits a small-plaque phenotype that can be complemented in trans by both the herpes simplex virus type 1 ICP27 and varicella-zoster virus open reading frame 4 proteins. Deletion of UL54 in the virus vJSΔ54 had no effect on the ability of the virus to shut off host cell protein synthesis but did affect virus gene expression. The glycoprotein gC accumulated to lower levels in cells infected with vJSΔ54 compared to those infected with wild-type virus, while gK levels were undetectable. Other late gene products, gB, gE, and Us9, accumulated to higher levels than those seen in cells infected with wild-type virus in a multiplicity-dependent manner. DNA replication is also reduced in cells infected with vJSΔ54. UL54 appears to regulate UL53 and UL52 at the transcriptional level as their respective RNAs are decreased in cells infected with vJSΔ54. Interestingly, vJSΔ54 is highly attenuated in a mouse model of PRV infection. Animals infected with vJSΔ54 survive twice as long as animals infected with wild-type virus, and this results in delayed accumulation of virus-specific antigens in skin, dorsal root ganglia, and spinal cord tissues.

scholarly journals A Spike protein-based subunit SARS-CoV-2 vaccine for pets: safety, immunogenicity, and protective efficacy in juvenile cats

2021 ◽  
Author(s):  
Kairat Tabynov ◽  
Madiana Orynbassar ◽  
Leila Yelchibayeva ◽  
Nurkeldi Turebekov ◽  
Toktassyn Yerubayev ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Neutralizing Antibodies ◽  
Large Scale ◽  
Protective Efficacy ◽  
Spike Protein ◽  
Wild Type Virus ◽  
Upper Respiratory Tract ◽  
Wild Type ◽  
Serum Samples ◽  
Virus Shedding

Abstract Whereas multiple vaccine types have been developed to curb the spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) among humans, there are very few vaccines being developed for animals including pets. To combat the threat of human-to-animal, animal-to-animal and animal-to-human transmission and the generation of new virus variants, we developed a subunit SARS-CoV-2 vaccine which is based on recombinant spike protein extracellular domain expressed in insect cells then formulated with appropriate adjuvants. Sixteen 8-12-week-old outbred female and male kittens (n=4/group) were randomly assigned into four treatment groups: Group 1, Antigen alone; Group 2, Sepivac SWE™ adjuvant; Group 3, aluminum hydroxide adjuvant; Group 4, PBS administered control animals. All animals were vaccinated twice at day 0 and 14, intramuscularly in a volume of 0.5 mL (Groups 1-3: 5 µg of Spike protein). On days 0 and 28 serum samples were collected to evaluate anti-spike IgG, inhibition of spike binding to angiotensin-converting enzyme 2 (ACE-2), neutralizing antibodies to Wuhan-01 SARS-CoV-2 D614G (wild-type) and Delta variant viruses, and whole blood for hematology studies. At day 28, all groups were challenged with SARS-CoV-2 wild-type virus 106 TCID50 intranasally. On day 31, tissue samples (lung, heart, and nasal turbinates) were collected for histology, viral RNA detection and virus titration. Parameters evaluated in this study included safety, immunogenicity, and protection from infection with wild-type SARS-CoV-2 virus. After two immunizations, both vaccines induced high titers of serum anti-spike IgG, ACE-2 binding inhibitory and neutralizing antibodies against both wild-type and Delta variant virus in the juvenile cats. Both subunit vaccines provided protection of juvenile cats against virus shedding from the upper respiratory tract, and against viral replication in the lower respiratory tract and hearts. These promising data warrant ongoing evaluation of the vaccine’s ability to protect cats against SARS-CoV-2 Delta variant and in particular to prevent transmission of the infection to naïve cats, before proceeding with large-scale field trials.

scholarly journals Characterisation of SARS-CoV-2 Lentiviral Pseudotypes and Correlation between Pseudotype-Based Neutralisation Assays and Live Virus-Based Micro Neutralisation Assays

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 1011 ◽  
Author(s):  
Inesa Hyseni ◽  
Eleonora Molesti ◽  
Linda Benincasa ◽  
Pietro Piu ◽  
Elisa Casa ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Spike Protein ◽  
Wild Type Virus ◽  
Wild Type ◽  
Serum Samples ◽  
Live Virus ◽  
Rapid Spread ◽  
Recent Outbreak ◽  
Human Sera ◽  
Novel Coronavirus

The recent outbreak of a novel Coronavirus (SARS-CoV-2) and its rapid spread across the continents has generated an urgent need for assays to detect the neutralising activity of human sera or human monoclonal antibodies against SARS-CoV-2 spike protein and to evaluate the serological immunity in humans. Since the accessibility of live virus microneutralisation (MN) assays with SARS-CoV-2 is limited and requires enhanced bio-containment, the approach based on “pseudotyping” can be considered a useful complement to other serological assays. After fully characterising lentiviral pseudotypes bearing the SARS-CoV-2 spike protein, we employed them in pseudotype-based neutralisation assays in order to profile the neutralising activity of human serum samples from an Italian sero-epidemiological study. The results obtained with pseudotype-based neutralisation assays mirrored those obtained when the same panel of sera was tested against the wild type virus, showing an evident convergence of the pseudotype-based neutralisation and MN results. The overall results lead to the conclusion that the pseudotype-based neutralisation assay is a valid alternative to using the wild-type strain, and although this system needs to be optimised and standardised, it can not only complement the classical serological methods, but also allows serological assessments to be made when other methods cannot be employed, especially in a human pandemic context.

scholarly journals Influence of Tegument Proteins of Pseudorabies Virus on Neuroinvasion and Transneuronal Spread in the Nervous System of Adult Mice after Intranasal Inoculation

2004 ◽  
Vol 78 (6) ◽  
pp. 2956-2966 ◽  
Author(s):  
Robert Klopfleisch ◽  
Jens P. Teifke ◽  
Walter Fuchs ◽  
Martina Kopp ◽  
Barbara G. Klupp ◽  
...  
Keyword(s):  
Cell Culture ◽  
Nervous System ◽  
Pseudorabies Virus ◽  
Single Cells ◽  
Wild Type Virus ◽  
Wild Type ◽  
Survival Times ◽  
Intranasal Infection ◽  
Tegument Proteins ◽  
Adult Mice

ABSTRACT Pseudorabies virus (PrV) is a neurotropic alphaherpesvirus that, after intranasal infection of adult mice, enters peripheral neurons and propagates to the central nervous system. In recent years we have analyzed the contribution of virus-encoded glycoproteins to neuroinvasion and transneuronal spread (reviewed in T. C. Mettenleiter, Virus Res. 92:197-206, 2003). We now extend our studies to analyze the role of tegument proteins in these processes. To this end, PrV mutants unable to express the UL11, UL37, UL46, UL47, and UL48 tegument proteins, as well as the corresponding rescued viruses, were intranasally instilled into 6- to 8-week-old CD1 strain mice. First, mean survival times were determined which showed that mice infected with the UL46 deletion mutant succumbed to the disease as early as wild-type PrV-infected animals. Survival times increased in the order: PrV-ΔUL47-, PrV-ΔUL11-, and PrV-ΔUL48-infected animals, a finding which parallels the growth phenotype of these viruses in cell culture. In contrast, none of the PrV-ΔUL37-infected animals died. Upon closer histological examination, all viruses except PrV-ΔUL37 were able to infect the nasal cavity and propagate to first- and second-order neurons as shown by two-color immunofluorescence. However, neuroinvasion was delayed in PrV-ΔUL47, PrV-ΔUL11, and PrV-ΔUL48, a finding that correlated with the extended survival times. Surprisingly, whereas PrV-ΔUL48 and PrV-ΔUL37 replicated to similar titers in cell culture which were ∼500-fold lower than those of wild-type virus, after intranasal infection of mice PrV-ΔUL48 was able to infect areas of the brain like wild-type PrV, although only after a considerably longer time period. In contrast, PrV-ΔUL37 was not able to enter neurons and was restricted to the infection of single cells in the nasal respiratory epithelium. Thus, our data demonstrate the importance of herpesviral tegument proteins in neuronal infection and show a different contribution of tegument proteins to the neuroinvasion phenotype of a neurotropic alphaherpesvirus.

scholarly journals LB7. Ad26.COV2.S-Elicted Neutralizing Activities Against SARS-CoV-2 Variants of Concern in Phase 1/2a and Phase 3 Clinical Trials

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S804-S805
Author(s):  
Mathieu Le Gars ◽  
Jerald Sadoff ◽  
Mandy Jongeneelen ◽  
Dirk Heerwegh ◽  
Georgi Shukarev ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Single Dose ◽  
Phase 1 ◽  
Geometric Mean ◽  
Wild Type Virus ◽  
Original Strain ◽  
Wild Type ◽  
Serum Samples ◽  
Phase 3 ◽  
Neutralizing Activity

Abstract Background In a Phase 3 trial, the Janssen COVID-19 vaccine, Ad26.COV2.S, showed robust efficacy against severe–critical COVID-19 in countries where different SARS-CoV-2 variants were circulating. We evaluated Ad26.COV2.S-elicited antibody neutralizing activity against variants of concern (VOC) B.1.1.7 (Alpha), B.1.351 (Beta), and B.1.617.2 (Delta) in sera from participants in clinical trials following a single dose of Ad26.COV2.S. Methods Neutralizing activities of Ad26.COV2.S (given at a dose level of 5 x 1010 viral particles [vp]) against VOC were assessed by wild-type virus neutralizing (wtVNA) and pseudovirion neutralization (psVNA) assays in sera from participants in Phase 1/2a and Phase 3 clinical trials, respectively. Geometric mean titers (GMTs) were determined at Days 29 and 71 after vaccination. Results In serum samples from Phase 1/2a participants (n = 6), at Day 29 after 1 dose of Ad26.COV2.S, wtVNA titers against VOC were lower than for the original strain (GMT = 573), with GMT = 65, 14, and 15 for Alpha, Beta, and Delta, respectively, representing 8.8-, 40.9-, and 37.7-fold decreases. By Day 71 after vaccination (n = 14), fold differences between the original strain (GMT = 375) and VOC (GMT = 113, 27, and 28) were smaller (3.3-, 13.9-, and 13.4-fold) than at Day 29, suggestive of B-cell maturation (Figure 1). Day 71 titers against the Delta variant were maintained for at least 8 months following a single dose of Ad26.COV2.S (5 x 1010 vp). In serum samples from Phase 3 participants (n = 8), psVNA titers against VOC were lower than the original strain at Day 71 after vaccination, with the lowest titers observed for the Beta variant (3.6-fold decrease vs original strain). Smaller reductions in Nab titers for VOC were observed in the psVNA assay compared to wtVNA. Figure 1. Neutralization of B.1.1.7 (Alpha), B.1.351 (Beta), and B.1.617.2 (Delta) lineages in serum samples from participants who received Ad26.COV2.S. n = 6 samples at Day 29 and n = 14 (n = 14 for Alpha and Beta; n = 6 for Delta, comprising the same 6 participants at Day 29) samples at Day 71 after vaccination with a single dose of Ad26.COV2.S (5 x 10^10 vp dose level) were analyzed in wild-type virus neutralization assays against the SARS-CoV-2 Victoria strain (D614, black dots), the B.1.1.7 (Alpha; green dots) the B.1.351 (Beta; blue dots), and the B.1.617.2 (Delta; purple dots) lineages. Dots represent the IC50 (inhibitory concentration) titers per participant. Geometric mean titers (GMTs) and fold decrease in neutralizing activity between the original Victoria strain and each lineage are shown. Conclusion Ad26.COV2.S-elicited serum neutralizing activity against VOC showed an overall decrease in titers relative to the original strain that was largest for the Beta variant, even though vaccine efficacy against severe–critical COVID-19 was maintained in countries where these variants were circulating versus in countries where they were not circulating. Over time, titers against variants increased, suggesting B-cell affinity maturation leading to increasing coverage of VOC. Disclosures Mathieu Le Gars, n/a, Johnson & Johnson (Employee, Shareholder) Jerald Sadoff, MD, Johnson & Johnson (Employee, Shareholder) Mandy Jongeneelen, n/a, Johnson & Johnson (Employee, Shareholder) Dirk Heerwegh, n/a, Janssen Research and Development (Employee) Georgi Shukarev, MD, Janssen (Employee) Carla Truyers, n/a, Janssen Research and Development (Employee) Anne Marit de Groot, n/a, Johnson & Johnson (Employee) Gert Scheper, n/a, Johnson & Johnson (Employee, Shareholder) Jenny Hendriks, n/a, Johnson & Johnson (Employee, Shareholder) Boerries Brandenburg, n/a, Johnson & Johnson (Employee, Shareholder) Frank Struyf, n/a, Johnson & Johnson (Employee, Shareholder) Johan Van Hoof, n/a, Johnson & Johnson (Employee, Shareholder) Macaya Douoguih, MD, MPH, Janssen (Employee) Hanneke Schuitemaker, PhD, Johnson & Johnson (Employee, Shareholder)

Assessment of Anti-Asparaginase (ASNase) Antibodies (Ab) and ASNase Activity after Suspected Clinical Allergy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1878-1878 ◽  
Author(s):  
Prakash N. Tiwari ◽  
Monica Zielinski ◽  
John J. Quinn ◽  
Stuart E. Siegel ◽  
Paul S. Gaynon ◽  
...  
Keyword(s):  
Lymphoblastic Leukemia ◽  
Negative Control ◽  
Serum Samples ◽  
Anamnestic Response ◽  
Cutaneous Manifestations ◽  
Female Ratio ◽  
Clinical Reaction ◽  
Clinical Allergy ◽  
Increased Risk ◽  
Male Female Ratio

Abstract Background: ASNase is a critical treatment component in childhood acute lymphoblastic leukemia (ALL). As ASNase is a bacterial protein, production of anti-ASNase Ab, often associated with clinical allergy, poses a frequent problem during ASNase therapy. Sixty percent of higher risk ALL patients, who received native ASNase in induction, were Ab(+) by 5 months into therapy (Panosyan et al., 2004). Moreover, 22%–29% of these patients had no clinical allergy (silent hypersensitivity) and were at increased risk for relapse. Patients and Methods: Physicians from 13 institutions submitted samples for 48 children in first remission, aged 14 to 252 months, on various SR or HR ALL protocols with suspected ASNase allergy. The male/female ratio was 1.4. The serum samples were assessed for ASNase activity and anti-ASNase Ab as reported earlier (Avramis, et al., Blood 2002). Ab titer is expressed as the numeric ratio relative to an Ab negative control. Results: Allergic symptoms ranged from localized skin rash and swelling to respiratory difficulties and/or anaphylaxis. Neutralizing anti-PEG-ASNase Ab’s were seen in 41 patients (85%). The results were examined with respect to age, gender, ASNase formulation received, time of post-ASNase administration, and the physician’s clinical observations. Higher Ab ratios > 1.1 correlated with low or no ASNase activity post-PEG-ASNase (46/48 patients) or post-Native ASNase dosing (2 patients). Fourteen patients had cutaneous manifestations (rash, hives, urticaria). All had neutralizing Ab and no enzymatic activity. Nine had higher Ab ratios and 5 had lower Ab ratios. Eight additional patients had localized or generalized swelling; all had higher Ab ratios. Similarly, over the entire CCG-1961 study, 526/1100 patients had a clinical reaction; 476/526 had higher Ab ratios (90%). Conclusions: We found neutralizing Ab in 85% of patients with apparent clinical ASNase allergy. Neutralizing Ab was correlated with lower or absent ASNase activity. Lower Ab ratios may be associated with earlier time points in the anamnestic response. In the remaining 15% of patients with an apparent clinical reaction, we found no Ab and substantial ASNase activity. Monitoring ASNase activity and Ab may be useful for guiding ASNase therapy. Patients with no Ab and substantial activity, might be rechallenged with the same product despite an apparent allergic reaction.

scholarly journals Detection of antibodies to Pseudomonas aeruginosa in serum and oral fluid from patients with cystic fibrosis

2007 ◽  
Vol 56 (5) ◽  
pp. 670-674 ◽  
Author(s):  
Abbie M. Weisner ◽  
Henrik Chart ◽  
Andrew Bush ◽  
Jane C. Davies ◽  
Tyrone L. Pitt
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Oral Fluid ◽  
Specific Antigen ◽  
Serum Antibodies ◽  
Serum Samples ◽  
Core Oligosaccharide ◽  
Weak Reaction ◽  
Bacterial Surface ◽  
Oral Fluids

Cystic fibrosis (CF) patients who are chronically infected with Pseudomonas aeruginosa make serum antibodies to bacterial surface LPS as well as other pseudomonas antigens. This study investigated the feasibility of using oral fluid samples for the detection of pseudomonas antibodies in CF patients and compared these results with corresponding serum antibodies. Most strains of P. aeruginosa produce two forms of LPS molecule, termed A-band (described as a common antigen) and B-band (O-serotype-specific antigen), apparently bound to a common core oligosaccharide moiety. A-band LPS was demonstrated in 45 out of 49 clinical isolates of P. aeruginosa by SDS-PAGE and immunoblotting with a specific antibody. Oral fluids were collected from 17 adult CF patients, all of whom were sputum culture positive for P. aeruginosa (13 also provided serum samples), 11 primary ciliary dyskinesia (PCD) patients and 37 healthy volunteers. Antibodies to A-band LPS were detected by immunoblotting in all of the CF patients’ oral fluids but 10 of the volunteer samples gave weak reactions with immunoblotting. Six of the PCD patients gave a weak reaction with A-band antibodies and only one demonstrated antibodies to core LPS. In a quantitative ELISA, 15 of the 17 CF patients’ oral fluids were shown to contain antibodies to A-band LPS, whilst none of the volunteer samples contained antibodies to A-band LPS. All serum samples from the CF patients were positive by both methods. Thus this is a sensitive procedure for the detection of antibodies to A-band LPS of P. aeruginosa in oral fluid and serum from patients with CF.

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