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Cancer Biology & Therapy 2019Caprine Herpesvirus type 1 (CpHV-1) is a species-specific herpes virus able to induce apoptosis in several biological systems. In the present study we aimed to...
Caprine Herpesvirus type 1 (CpHV-1) is a species-specific herpes virus able to induce apoptosis in several biological systems. In the present study we aimed to investigate the ability of CpHV-1 to reduce cells viability, to replicate and to cause cell death also in human cancer cell lines. We tested the CpHV-1 effects on HEL-299, Vero, MDA-MB-468, HeLa, U2OS, PC3, A549 and K562 neoplastic cell lines and on MDBK cells. Firstly, we evaluated the effect of CpHV-1 infection on cell viability by MTT assay and our data showed that CpHV-1 can induce a marked cytopathic effect (CPE) in most of cell lines tested, except for HEL-299, Vero and K562 cells. The reduction of cell viability was associated with a significant increase of viral production. We next investigated if CpHV-1 was able to induce cell death and so through western blotting analysis we evaluated cleaved caspase 3, LC3II and p62 protein levels after infection. Caspase 3 activation was detected in MDBK cells and, even if at different times p.i., also in MDA-MB-468, U2OS, and PC3 cell lines, while LC3II increase and concomitant p62 protein reduction were observed only in U2OS, and A549 cells, no significant alteration of these proteins was observed in the other cell lines tested. Finally, to confirm virus ability to trigger apoptosis we performed an Annexin-V apoptosis test after 24 h p.i. Although we need to further explore mechanisms underlying CpHV-1 treatment, this study could serve as the basis for the development of new treatment options aiming to fight several cancer types.
Topics: Animals; Apoptosis; Autophagy; Cattle; Cell Line, Tumor; Cell Survival; Chlorocebus aethiops; Humans; Neoplasms; Oncolytic Virotherapy; Oncolytic Viruses; Toxicity Tests; Varicellovirus; Vero Cells
PubMed: 30409104
DOI: 10.1080/15384047.2018.1504722 -
BMC Veterinary Research Mar 2024Feline herpesvirus type 1 (FHV) and Feline calicivirus (FCV) are the primary co-infecting pathogens that cause upper respiratory tract disease in cats. However, there...
BACKGROUND
Feline herpesvirus type 1 (FHV) and Feline calicivirus (FCV) are the primary co-infecting pathogens that cause upper respiratory tract disease in cats. However, there are currently no visual detection assays available for on-site testing. Here, we develop an ultrasensitive and visual detection method based on dual recombinase polymerase amplification (dRPA) reaction and the hybrid Cas12a/Cas13a trans-cleavage activities in a one-tube reaction system, referred to as one-tube dRPA-Cas12a/Cas13a assay.
RESULTS
The recombinant plasmid DNAs, crRNAs, and RPA oligonucleotides targeting the FCV ORF1 gene and FHV-1 TK gene were meticulously prepared. Subsequently, dual RPA reactions were performed followed by screening of essential reaction components for hybrid CRISPR-Cas12a (targeting the FHV-1 TK gene) and CRISPR-Cas13a (targeting the FCV ORF1 gene) trans-cleavage reaction. As a result, we successfully established an ultra-sensitive and visually detectable method for simultaneous detection of FCV and FHV-1 nucleic acids using dRPA and CRISPR/Cas-powered technology in one-tube reaction system. Visual readouts were displayed using either a fluorescence detector (Fluor-based assay) or lateral flow dipsticks (LDF-based assay). As expected, this optimized assay exhibited high specificity towards only FHV-1 and FCV without cross-reactivity with other feline pathogens while achieving accurate detection for both targets with limit of detection at 2.4 × 10 copies/μL for the FHV-1 TK gene and 5.5 copies/μL for the FCV ORF1 gene, respectively. Furthermore, field detection was conducted using the dRPA-Cas12a/Cas13a assay and the reference real-time PCR methods for 56 clinical samples collected from cats with URTD. Comparatively, the results of Fluor-based assay were in exceptional concordance with the reference real-time PCR methods, resulting in high sensitivity (100% for both FHV-1 and FCV), specificity (100% for both FHV-1 and FCV), as well as consistency (Kappa values were 1.00 for FHV-1 and FCV). However, several discordant results for FHV-1 detection were observed by LDF-based assay, which suggests its prudent use and interpretaion for clinical detection. In spite of this, incorporating dRPA-Cas12a/Cas13a assay and visual readouts will facilitate rapid and accurate detection of FHV-1 and FCV in resource-limited settings.
CONCLUSIONS
The one-tube dRPA-Cas12a/Cas13a assay enables simultaneously ultrasensitive and visual detection of FHV-1 and FCV with user-friendly modality, providing unparalleled convenience for FHV-1 and FCV co-infection surveillance and decision-making of URTD management.
Topics: Cats; Animals; Calicivirus, Feline; Herpesviridae; Recombinases; CRISPR-Cas Systems; Varicellovirus
PubMed: 38493286
DOI: 10.1186/s12917-024-03953-9 -
Infection and Immunity Sep 1976Systematic studies on the replication of varicella-zoster virus in infected human fetal diploid lung cells have defined more optimal conditions for infection and...
Systematic studies on the replication of varicella-zoster virus in infected human fetal diploid lung cells have defined more optimal conditions for infection and harvesting of cultures and have led to the production of cell-free virus preparations with infectivity titers of greater than or equal to 10(6) plaque-forming units per ml. The highest yields of cell-free virus were obtained by (i) sonic treatment of the cellular phase of cultures inoculated with trypsin-dispersed infected cells at ratios of 1 infected cell to 6 to 10 uninfected cells in the monolayer and (ii) harvesting cells after 24 to 36 h of incubation at 36 degrees C. At this time the cultures showed minimal viral cytopathic effect. Spread of infectivity occurred much more rapidly in cultures inoculated with whole infected cells than in those infected with cell-free virus. Complement-fixing antigens with improved titers of greater than or equal to 1:128 were prepared from varicella-zoster virus-infected cell cultures in the same manner as cell-free virus, but harvested after 3 to 4 days of incubation when the cultures showed an advanced cytopathic effect.
Topics: Antigens, Viral; Cell-Free System; Cytopathogenic Effect, Viral; Edetic Acid; Glass; Herpesvirus 3, Human; Sonication; Temperature; Trypsin; Virus Cultivation
PubMed: 184051
DOI: 10.1128/iai.14.3.709-715.1976 -
Journal of Feline Medicine and Surgery Oct 2019The aim of this study was to conduct a comprehensive assessment of feline infectious upper respiratory tract infection (URTI) and disease (URTD) in Australian cats.
OBJECTIVES
The aim of this study was to conduct a comprehensive assessment of feline infectious upper respiratory tract infection (URTI) and disease (URTD) in Australian cats.
METHODS
Laboratory data demonstrating URTI from feline URTD multiplex PCR panel (feline herpesvirus 1 [FHV-1], feline calicivirus [FCV], and H1N1 influenza) submissions in Australia (2013-2015) were obtained. For comparison, reports of feline URTD during the same time period were sourced from a voluntary companion animal disease surveillance system.
RESULTS
A total of 3126 samples were submitted for testing; 1533 (49%) were positive. Of these, the most commonly detected agents were (21.5%) and FCV (16.0%) alone, followed by FCV and (13.4%) together as a respiratory infection complex, then FHV-1 (7.0%) alone. During the study period, there were 262 reports of 320 clinical feline URTD cases. Most cases (69%) were reported from New South Wales, <1 year of age (41%) and equally distributed between the sexes. Infection was more common in entire cats (69%) and most cases (55%) involved domestic shorthair cats. Of the 90 reports that had a known vaccination status, 63 had a vaccination history, 40 of which were recently vaccinated. Most (72%) feline URTD cases recovered from clinical disease. Both feline URTI and URTD were more common during winter months.
CONCLUSIONS AND RELEVANCE
Feline URTI and URTD cause substantial impact in Australia, being most commonly associated with and FCV infection. This information can be used by veterinarians to educate clients about prevention and management of this important infectious disease of cats.
Topics: Animals; Australia; Caliciviridae; Caliciviridae Infections; Calicivirus, Feline; Cat Diseases; Cats; Chlamydophila; Herpesviridae; Herpesviridae Infections; Picornaviridae Infections; Respiratory Tract Infections; Risk Factors; Varicellovirus
PubMed: 30465616
DOI: 10.1177/1098612X18813248 -
Journal of Veterinary Diagnostic... Jul 2018A 2-y-old female Grant's zebra ( Equus quagga [ burchellii] boehmi) was presented with a clinical history of depression, anorexia, and weakness of 1-wk duration....
A 2-y-old female Grant's zebra ( Equus quagga [ burchellii] boehmi) was presented with a clinical history of depression, anorexia, and weakness of 1-wk duration. Postmortem examination identified ulcers on the tongue and palate; a large abscess adjacent to the larynx; left lung consolidation; mild swelling, darkening, and congestion of the liver with accentuation of the lobular pattern; and edema and congestion of the distal small and large intestines. Histologic examination identified necrotizing bronchopneumonia, necrotizing hepatitis, nephritis, and enterocolitis. Eosinophilic intranuclear inclusions were detected in syncytial cells and degenerate bronchial epithelium in the lungs and in some hepatocytes associated with necrotic foci. Bacterial cultures of the lung, liver, and laryngeal abscess failed to detect any significant pathogen. Lung and liver tested positive for equine herpesvirus with neuropathogenic marker by real-time PCR. Subsequently, equine herpesvirus was isolated in tissue culture, and the entire viral DNA polymerase gene (ORF30) was sequenced. The zebra lung isolate had a very close nucleotide and amino acid sequence identity to equid alphaherpesvirus 9 (EHV-9; 99.6% and 99.8%, respectively) in contrast to the neuropathogenic T953 strain of EHV-1 (94.7% and 96.6%, respectively). Although zebras are considered the natural host for EHV-9, we document an unusual acute systemic, fatal EHV-9 infection in a 2-y-old Grant's zebra.
Topics: Amino Acid Sequence; Animals; Base Sequence; DNA, Viral; Equidae; Female; Herpesviridae Infections; Varicellovirus; Viral Proteins
PubMed: 29648506
DOI: 10.1177/1040638718767722 -
Cell Cycle (Georgetown, Tex.) Dec 2019Although several effective treatments exist against virus (VZV), resistant strains have emerged and the treatment is usually not definite and may have various undesired...
Although several effective treatments exist against virus (VZV), resistant strains have emerged and the treatment is usually not definite and may have various undesired side effects. Thus, alternative treatment options are necessary. Here we studied the inhibitory effects of natural polysaccharides (PSs) obtained from renewable sources, varied by their structure and charge, on VZV infection , using a plaque assay. In terms of selectivity indices, almost all the tested PSs were very active; in the order of > > > > against VZV compared to Acyclovir as a reference drug and exhibited dose-dependent behavior. Our results, which showed a strong inhibition of VZV infection when the cells were treated with only at the time of infection or only post infection may indicate a multistep inhibitory effect. It seems that may block different stages of the virus replication cycle including early steps such as absorption and penetration to the host cells and also late steps after the penetration into the host cells. These results are part of an on-going research that highlights the PSs as potential novel nontoxic candidates that can be used against VZV, and contributes to the elucidation of their mode of action.
Topics: Acyclovir; Antiviral Agents; Cell Line; Dose-Response Relationship, Drug; Herpesvirus 3, Human; Humans; Polysaccharides; Varicella Zoster Virus Infection; Virus Replication
PubMed: 31724465
DOI: 10.1080/15384101.2019.1691363 -
Current Microbiology Jan 2011Lipid rafts are special microdomains in the plasma membrane. They are enriched in sphingolipids and cholesterol, playing critical roles in many biological processes. The...
Lipid rafts are special microdomains in the plasma membrane. They are enriched in sphingolipids and cholesterol, playing critical roles in many biological processes. The purpose of this study is to analyze the requirement of cholesterol, a crucial component of lipid rafts for cell infection by pseudorabies virus (PrV). Cholesterol of plasma membrane or viral envelope was depleted with methyl-beta-cyclodextrin (MβCD), and the infectivity of three strains of PrV was determined with plaque assays. The effect of adding cholesterol to MβCD-treated cells and viruses on cell infection was analyzed. Furthermore, effect of post-adsorption cholesterol depletion on PrV infection was investigated. We show that cholesterol depletion of either the plasma membrane or the viral membrane by MβCD significantly impaired the infectivity of PrV strains Kaplan, Becker, and Bartha K-61. The virus was shown to have lower cholesterol content and to respond to lower MβCD concentrations. Exogenous cholesterol added to either MβCD-treated cells or virions partially restored the virus infectivity. Optimal PrV infection requires cholesterol in viral and plasma membranes.
Topics: Animals; Cell Line; Cell Membrane; Chlorocebus aethiops; Cholesterol; Cricetinae; Herpesvirus 1, Suid; Viral Plaque Assay; Virus Internalization
PubMed: 20625735
DOI: 10.1007/s00284-010-9700-8 -
Virology Journal Jan 2022Equine herpesvirus-8 (EHV-8) is one of the most economically significant viruses that infect mammals of the genus Equus worldwide, which cause severe respiratory...
BACKGROUND
Equine herpesvirus-8 (EHV-8) is one of the most economically significant viruses that infect mammals of the genus Equus worldwide, which cause severe respiratory diseases and abortion in horses. However, there is no report of abortion caused by EHV-8 in donkeys.
CASE PRESENTATION
The present case report is about a 4-year-old donkey having an abortion and showing a serious respiratory issue on the 296th day of pregnancy. Bacteriological and molecular tests were used to screen possible bacterial/viral pathogens to detect the etiological agent. Salmonella abortus equi, EHV-1, EHV-4, and EAV were all negative in the current study. EHV-8, on the other hand, was the only agent that was isolated and identified.
CONCLUSIONS
This was for the first time that EHV-8 had been isolated from a donkey in China. EHV-8 infection can cause abortion in donkeys; therefore, veterinarians and breeders should be aware of it.
Topics: Animals; China; Equidae; Female; Herpesviridae Infections; Herpesvirus 1, Equid; Horse Diseases; Horses; Pregnancy; Varicellovirus
PubMed: 34991640
DOI: 10.1186/s12985-021-01738-2 -
PLoS Pathogens May 2008Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented...
Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP) plays an essential role in MHC class I-restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL 49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1), pseudorabies virus (PRV), and equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are characterized as members of a novel class of viral immune evasion proteins. These UL 49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL 49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL 49.5 proteins block TAP as well, these data indicate that UL 49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL 49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL 49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL 49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL 49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL 49.5. Taken together, these results classify the UL 49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms.
Topics: ATP-Binding Cassette Transporters; Animals; Antigen Presentation; Cattle; Cell Line, Tumor; Cell Survival; Dogs; Herpesvirus 1, Bovine; Herpesvirus 1, Equid; Herpesvirus 1, Suid; Horses; Humans; Protein Transport; Recombination, Genetic; Swine; Transduction, Genetic; Varicellovirus; Viral Envelope Proteins
PubMed: 18516302
DOI: 10.1371/journal.ppat.1000080 -
Virology Journal Sep 2013Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus that infects humans and results in chickenpox and herpes zoster. A number of VZV genes remain functionally...
BACKGROUND
Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus that infects humans and results in chickenpox and herpes zoster. A number of VZV genes remain functionally uncharacterized and since VZV is an obligate human pathogen, rigorous evaluation of VZV mutants in vivo remains challenging. Simian varicella virus (SVV) is homologous to VZV and SVV infection of rhesus macaques (RM) closely mimics VZV infection of humans. Recently the SVV genome was cloned as a bacterial artificial chromosome (BAC) and BAC-derived SVV displayed similar replication kinetics as wild-type (WT) SVV in vitro.
METHODS
RMs were infected with BAC-derived SVV or WT SVV at 4x10(5) PFU intrabronchially (N=8, 4 per group, sex and age matched). We collected whole blood (PBMC) and bronchoalveolar lavage (BAL) at various days post-infection (dpi) and sensory ganglia during latent infection (>84 dpi) at necropsy and compared disease progression, viral replication, immune response and the establishment of latency.
RESULTS
Viral replication kinetics and magnitude in bronchoalveolar lavage cells and whole blood as well as rash severity and duration were similar in RMs infected with SVV BAC or WT SVV. Moreover, SVV-specific B and T cell responses were comparable between BAC and WT-infected animals. Lastly, we measured viral DNA in sensory ganglia from both cohorts of infected RMs during latent infection.
CONCLUSIONS
SVV BAC is as pathogenic and immunogenic as WT SVV in vivo. Thus, the SVV BAC genetic system combined with the rhesus macaque animal model can further our understanding of viral ORFs important for VZV pathogenesis and the development of second-generation vaccines.
Topics: Animals; Blood; Bronchoalveolar Lavage Fluid; Chickenpox; Chromosomes, Artificial, Bacterial; Disease Models, Animal; Ganglia, Sensory; Macaca mulatta; Varicellovirus; Virus Latency
PubMed: 24010815
DOI: 10.1186/1743-422X-10-278