scholarly journals Characterization of EIAV env Quasispecies during Long-Term Passage In Vitro: Gradual Loss of Pathogenicity

Viruses ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 380
Author(s):  
Cong Liu ◽  
Xue-Feng Wang ◽  
Yan Wang ◽  
Jie Chen ◽  
Zhaohua Zhong ◽  
...  
Keyword(s):  
Virulent Strain ◽  
Population Level ◽  
Glycosylation Site ◽  
Attenuated Vaccine ◽  
Gradual Loss ◽  
Single Genome ◽  
Attenuation Mechanism ◽  
Passage Number ◽  
Quasispecies Evolution

As the only widely used live lentiviral vaccine, the equine infectious anima virus (EIAV) attenuated vaccine was developed by in vitro passaging of a virulent strain for 121 generations. In our previous study, we observed that the attenuated vaccine was gradually selected under increased environmental pressure at the population level (termed a quasispecies). To further elucidate the potential correlation between viral quasispecies evolution and pathogenesis, a systematic study was performed by sequencing env using several methods. Some key mutations were identified within Env, and we observed that increased percentages of these mutations were accompanied by an increased passage number and attenuated virulence. Phylogenetic analysis revealed that env mutations related to the loss of virulence might have occurred evolutionarily. Among these mutations, deletion of amino acid 236 in the V4 region of Env resulted in the loss of one N-glycosylation site that was crucial for virulence. Notably, the 236-deleted sequence represented a “vaccine-specific” mutation that was also found in wild EIAVLN40 strains based on single genome amplification (SGA) analysis. Therefore, our results suggest that the EIAV attenuated vaccine may originate from a branch of quasispecies of EIAVLN40. Generally, the presented results may increase our understanding of the attenuation mechanism of the EIAV vaccine and provide more information about the evolution of other lentiviruses.

scholarly journals Borrelia burgdorferi Needs Chemotaxis To Establish Infection in Mammals and To Accomplish Its Enzootic Cycle

2012 ◽  
Vol 80 (7) ◽  
pp. 2485-2492 ◽  
Author(s):  
Ching Wooen Sze ◽  
Kai Zhang ◽  
Toru Kariu ◽  
Utpal Pal ◽  
Chunhao Li
Keyword(s):  
Borrelia Burgdorferi ◽  
Capillary Tube ◽  
Virulent Strain ◽  
Gene Encoding ◽  
Content Type ◽  
Immunodeficient Mice ◽  
Passage Number ◽  
Low Passage ◽  
Enzootic Cycle

ABSTRACTBorrelia burgdorferi, the causative agent of Lyme disease, can be recovered from different organs of infected animals and patients, indicating that the spirochete is very invasive. Motility and chemotaxis contribute to the invasiveness ofB. burgdorferiand play important roles in the process of the disease. Recent reports have shown that motility is required for establishing infection in mammals. However, the role of chemotaxis in virulence remains elusive. Our previous studies showed thatcheA2, a gene encoding a histidine kinase, is essential for the chemotaxis ofB. burgdorferi. In this report, thecheA2gene was inactivated in a low-passage-number virulent strain ofB. burgdorferi. In vitroanalyses (microscopic observations, computer-based bacterial tracking analysis, swarm plate assays, and capillary tube assays) showed that thecheA2mutant failed to reverse and constantly ran in one direction; the mutant was nonchemotactic to attractants. Mouse needle infection studies showed that thecheA2mutant failed to infect either immunocompetent or immunodeficient mice and was quickly eliminated from the initial inoculation sites. Tick-mouse infection studies revealed that although the mutant was able to survive in ticks, it failed to establish a new infection in mice via tick bites. The altered phenotypes were completely restored when the mutant was complemented. Collectively, these data demonstrate thatB. burgdorferineeds chemotaxis to establish mammalian infection and to accomplish its natural enzootic cycle.

In Vitro, In Silico and Ex Vivo Studies of Dihydroartemisinin Derivatives as Antitubercular Agents

2019 ◽  
Vol 19 (8) ◽  
pp. 633-644 ◽  
Author(s):  
Komal Kalani ◽  
Sarfaraz Alam ◽  
Vinita Chaturvedi ◽  
Shyam Singh ◽  
Feroz Khan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
In Silico ◽  
Ex Vivo ◽  
Virulent Strain ◽  
Infection Model ◽  
Mouse Bone Marrow ◽  
Significant Activity ◽  
Macrophage Infection ◽  
In Silico Studies

Introduction: As a part of our drug discovery program for anti-tubercular agents, dihydroartemisinin (DHA-1) was screened against Mtb H37Rv, which showed moderate anti-tubercular activity (>25.0 µg/mL). These results prompted us to carry out the chemical transformation of DHA-1 into various derivatives and study their antitubercular potential. Materials and Methods: DHA-1 was semi-synthetically converted into four new acyl derivatives (DHA-1A – DHA-1D) and in-vitro evaluated for their anti-tubercular potential against Mycobacterium tuberculosis H37Rv virulent strain. The derivatives, DHA-1C (12-O-(4-nitro) benzoyl; MIC 12.5 µg/mL) and DHA-1D (12-O-chloro acetyl; MIC 3.12µg/mL) showed significant activity against the pathogen. Results: In silico studies of the most active derivative (DHA-1D) showed interaction with ARG448 inhibiting the mycobacterium enzymes. Additionally, it showed no cytotoxicity towards the Vero C1008 cells and Mouse bone marrow derived macrophages. Conclusion: DHA-1D killed 62% intracellular M. tuberculosis in Mouse bone marrow macrophage infection model. To the best of our knowledge, this is the first-ever report on the antitubercular potential of dihydroartemisinin and its derivatives. Since dihydroartemisinin is widely used as an antimalarial drug; these results may be of great help in anti-tubercular drug development from a very common, inexpensive, and non-toxic natural product.

scholarly journals Identification of Putative Virulence Genes by DNA Methylation Studies in the Cereal Pathogen Fusarium graminearum

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1192
Author(s):  
Francesco Tini ◽  
Giovanni Beccari ◽  
Gianpiero Marconi ◽  
Andrea Porceddu ◽  
Micheal Sulyok ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Fusarium Graminearum ◽  
Environmental Changes ◽  
Virulent Strain ◽  
Fungal Virulence ◽  
Wheat Seedlings ◽  
Wide Range ◽  
Conidia Production ◽  
Natural Hosts

DNA methylation mediates organisms’ adaptations to environmental changes in a wide range of species. We investigated if a such a strategy is also adopted by Fusarium graminearum in regulating virulence toward its natural hosts. A virulent strain of this fungus was consecutively sub-cultured for 50 times (once a week) on potato dextrose agar. To assess the effect of subculturing on virulence, wheat seedlings and heads (cv. A416) were inoculated with subcultures (SC) 1, 23, and 50. SC50 was also used to re-infect (three times) wheat heads (SC50×3) to restore virulence. In vitro conidia production, colonies growth and secondary metabolites production were also determined for SC1, SC23, SC50, and SC50×3. Seedling stem base and head assays revealed a virulence decline of all subcultures, whereas virulence was restored in SC50×3. The same trend was observed in conidia production. The DNA isolated from SC50 and SC50×3 was subject to a methylation content-sensitive enzyme and double-digest, restriction-site-associated DNA technique (ddRAD-MCSeEd). DNA methylation analysis indicated 1024 genes, whose methylation levels changed in response to the inoculation on a healthy host after subculturing. Several of these genes are already known to be involved in virulence by functional analysis. These results demonstrate that the physiological shifts following sub-culturing have an impact on genomic DNA methylation levels and suggest that the ddRAD-MCSeEd approach can be an important tool for detecting genes potentially related to fungal virulence.

scholarly journals lncRNA Chronos is an aging-induced inhibitor of muscle hypertrophy

2017 ◽  
Vol 216 (11) ◽  
pp. 3497-3507 ◽  
Author(s):  
Ronald L Neppl ◽  
Chia-Ling Wu ◽  
Kenneth Walsh
Keyword(s):  
Skeletal Muscle ◽  
Noncoding Rna ◽  
Muscle Hypertrophy ◽  
Systemic Disease ◽  
Noncoding Rnas ◽  
Rapid Progression ◽  
Gradual Loss ◽  
Epigenetic Modulation

Skeletal muscle exhibits remarkable plasticity in its ability to modulate its mass in response to the physiologic changes associated with functional use, systemic disease, and aging. Although a gradual loss of muscle mass normally occurs with advancing age, its increasingly rapid progression results in sarcopenia in a subset of individuals. The identities of muscle-enriched, long noncoding RNAs that regulate this process are unknown. Here, we identify a long noncoding RNA, named Chronos, whose expression in muscle is positively regulated with advancing age and negatively regulated during Akt1-mediated growth. Inhibition of Chronos induces myofiber hypertrophy both in vitro and in vivo, in part, through the epigenetic modulation of Bmp7 signaling.

scholarly journals Modelling the population-level protection conferred by COVID-19 vaccination

2021 ◽  
Author(s):  
Pranesh Padmanabhan ◽  
Rajat Desikan ◽  
Narendra M Dixit
Keyword(s):  
Mathematical Model ◽  
Severe Acute Respiratory Syndrome ◽  
Dose Response ◽  
Neutralizing Antibodies ◽  
Population Level ◽  
Response Curves ◽  
Post Vaccination ◽  
Dose Response Curves ◽  
Vaccine Availability

Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines work predominantly by eliciting neutralizing antibodies (NAbs), how the protection they confer depends on the NAb response to vaccination is unclear. Here, we collated and analysed in vitro dose-response curves of >70 NAbs and constructed a landscape defining the spectrum of neutralization efficiencies of NAbs elicited. We mimicked responses of individuals by sampling NAb subsets of known sizes from the landscape and found that they recapitulated responses of convalescent patients. Combining individual responses with a mathematical model of within-host SARS-CoV-2 infection post-vaccination, we predicted how the population-level protection conferred would increase with the NAb response to vaccination. Our predictions captured the outcomes of vaccination trials. Our formalism may help optimize vaccination protocols, given limited vaccine availability.

scholarly journals Identification of a Novel Virulence Factor in Recombinant Pneumonia Virus of Mice

2007 ◽  
Vol 81 (17) ◽  
pp. 9490-9501 ◽  
Author(s):  
Christine D. Krempl ◽  
Anna Wnekowicz ◽  
Elaine W. Lamirande ◽  
Giw Nayebagha ◽  
Peter L. Collins ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Virulent Strain ◽  
Consensus Sequence ◽  
Cytoplasmic Tail ◽  
Syncytial Virus

ABSTRACT Pneumonia virus of mice (PVM) is a murine relative of human respiratory syncytial virus (HRSV). Here we developed a reverse genetics system for PVM based on a consensus sequence for virulent strain 15. Recombinant PVM and a version engineered to express green fluorescent protein replicated as efficiently as the biological parent in vitro but were 4- and 12.5-fold attenuated in vivo, respectively. The G proteins of HRSV and PVM have been suggested to contribute to viral pathogenesis, but this had not been possible to study in a defined manner in a fully permissive host. As a first step, we evaluated recombinant mutants bearing a deletion of the entire G gene (ΔG) or expressing a G protein lacking its cytoplasmic tail (Gt). Both G mutants replicated as efficiently in vitro as their recombinant parent, but both were nonpathogenic in mice at doses that would otherwise be lethal. We could not detect replication of the ΔG mutant in mice, indicating that its attenuation is based on a severe reduction in the virus load. In contrast, the Gt mutant appeared to replicate as efficiently in mice as its recombinant parent. Thus, the reduction in virulence associated with the Gt mutant could not be accounted for by a reduction in viral replication. These results identified the cytoplasmic tail of G as a virulence factor whose effect is not mediated solely by the viral load. In addition to its intrinsic interest, a recombinant virus that replicates with wild-type-like efficiency but does not cause disease defines optimal properties for vaccine development.

scholarly journals The alpha subunit of the Saccharomyces cerevisiae oligosaccharyltransferase complex is essential for vegetative growth of yeast and is homologous to mammalian ribophorin I.

1995 ◽  
Vol 128 (4) ◽  
pp. 525-536 ◽  
Author(s):  
S Silberstein ◽  
P G Collins ◽  
D J Kelleher ◽  
P J Rapiejko ◽  
R Gilmore
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Vegetative Growth ◽  
Functional Characterization ◽  
Glycosylation Site ◽  
Alpha Subunit ◽  
Protein Sequence Analysis ◽  
Exogenous Lipid ◽  
Microsomal Membranes ◽  
High Mannose

Oligosaccharyltransferase mediates the transfer of a preassembled high mannose oligosaccharide from a lipid-linked oligosaccharide donor to consensus glycosylation acceptor sites in newly synthesized proteins in the lumen of the rough endoplasmic reticulum. The Saccharomyces cerevisiae oligosaccharyltransferase is an oligomeric complex composed of six nonidentical subunits (alpha-zeta), two of which are glycoproteins (alpha and beta). The beta and delta subunits of the oligosaccharyltransferase are encoded by the WBP1 and SWP1 genes. Here we describe the functional characterization of the OST1 gene that encodes the alpha subunit of the oligosaccharyltransferase. Protein sequence analysis revealed a significant sequence identity between the Saccharomyces cerevisiae Ost1 protein and ribophorin I, a previously identified subunit of the mammalian oligosaccharyltransferase. A disruption of the OST1 locus was not tolerated in haploid yeast showing that expression of the Ost1 protein is essential for vegetative growth of yeast. An analysis of a series of conditional ost1 mutants demonstrated that defects in the Ost1 protein cause pleiotropic underglycosylation of soluble and membrane-bound glycoproteins at both the permissive and restrictive growth temperatures. Microsomal membranes isolated from ost1 mutant yeast showed marked reductions in the in vitro transfer of high mannose oligosaccharide from exogenous lipid-linked oligosaccharide to a glycosylation site acceptor tripeptide. Microsomal membranes isolated from the ost1 mutants contained elevated amounts of the Kar2 stress-response protein.

scholarly journals INFECTION INDUCED BY ORAL ADMINISTRATION OF ATTENUATED POLIOVIRUS TO PERSONS POSSESSING HOMOTYPIC ANTIBODY

1957 ◽  
Vol 106 (1) ◽  
pp. 159-177 ◽  
Author(s):  
Dorothy M. Horstmann ◽  
J. R. Paul ◽  
J. L. Melnick ◽  
Joyce V. Deutsch
Keyword(s):  
Tissue Culture ◽  
Neutralizing Antibodies ◽  
Close Contact ◽  
Virulent Strain ◽  
The Other ◽  
Clear Correlation ◽  
Type Iii ◽  
Virus Dose ◽  
Virus Excretion

Four of five individuals possessing homotypic antibody in titers of 8 to 64 were infected on being fed Type III (Leon KP-34) poliovirus attenuated by Sabin by passage through tissue culture. None of the infected subjects or controls showed any evidence of illness which could be attributed to virus infection. There was no evidence of spread of infection to any of the control adult wardmates of the experimental subjects, although the two groups were in close contact: none of the controls excreted virus, none showed any antibody shift. One control who had no Type III antibodies at the start of the experiment was still antibody-negative on the 63rd day of the experiment. Three of the four individuals who became infected had naturally acquired-Type III antibodies; the other had antibodies induced by formalinized vaccine. Virus excretion in the stool was of short duration (7 to 13 days) in the three with natural antibodies, and lasted at least 6 weeks after feeding in the vaccinated child. Virus in the throat was detected only in the two persons receiving the larger virus dose (107.5 TCD50). In them it was present in small amounts between the 2nd and 6th day after feeding. No virus was detected in the blood of any of the infected individuals. The antibody responses of the four infected individuals were variable. There was no clear correlation with virus dosage, amount of virus excretion in the stools, or presence of virus in the throat. Only the child whose neutralizing antibodies were "Salk" vaccine induced showed a marked CF response. The virus excreted by two of the individuals who became infected, as tested in the 2nd tissue culture passage by monkey inoculation, was slightly more neurotropic than the virus which was ingested. Virus excreted by one of these individuals behaved as a virulent strain when tested by the in vitro plaque virulence test, while that isolated from the other had the characteristics of an attenuated strain in this test.

scholarly journals THE MULTIPLICATION OF TUBERCLE BACILLI WITHIN NORMAL PHAGOCYTES IN TISSUE CULTURE

1952 ◽  
Vol 96 (2) ◽  
pp. 137-150 ◽  
Author(s):  
Emanuel Suter
Keyword(s):  
Tissue Culture ◽  
Culture Medium ◽  
Mononuclear Cells ◽  
Virulent Strain ◽  
Host Cells ◽  
Avirulent Strain ◽  
Glass Slides ◽  
Intracellular Multiplication ◽  
Cultivation In Vitro

A technique has been described for the cultivation in vitro of normal mononuclear cells on glass slides in a liquid medium. Under these conditions the monocytes transformed into macrophages which proliferated as in ordinary tissue culture. These cultures of monocytes could be infected with tubercle bacilli. The numbers of stainable tubercle bacilli within the monocytes increased steadily in cultures infected with virulent or attenuated strains. Evidence is given to support the view that this increase in numbers of bacilli was due to intracellular multiplication. There was no evidence of intracellular bacillary multiplication in cultures infected with an avirulent strain. Tubercle bacilli multiplying within phagocytes in vitro exert a damaging effect upon the host cells. The damage was most obvious in cells infected with a virulent strain. Tubercle bacilli within phagocytes were protected against the bacteriostatic effect of streptomycin added in a concentration of 5 γ per ml. of culture medium. This permitted the use of streptomycin in infected cultures to prevent extracellular multiplication of the bacilli.

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