187 research outputs found
Flow cytometry sorting of cells infected with African swine fever virus
The African swine fever panzootic is continuing to spread, and the number of affected countries and material losses are increasing. In particular, India, Papua New Guinea, Malaysia, Greece and Bhutan joined the list of ASF infected countries in 2020–2021. The disease control is hindered by the lack of commercially available and effective vaccines, which, in its turn, is attributable to the insufficient knowledge of ASF pathogenesis and immune defense against the disease. The use of attenuated virus variants enables a thorough investigation of the factors influencing the virulence of African swine fever virus and the immune response to it. This involves the use of naturally attenuated virus variants, as well as of the variants attenuated by a long-term passaging of the virus in cell cultures. However, virulence heterogeneity characteristic of the ASF virus population, necessitates the additional selection of infected cells for the virus cloning. Conventional culture-based techniques for virus particle cloning are rather time- and labour-consuming; it is therefore appropriate to use flow cytometry cell sorting for the selection and cloning of virus infected cells with a view of selecting homologous virus lineages. The paper presents the results of sorting of African green monkey kidney cells (CV-1) and porcine bone marrow cells infected with African swine fever virus; the cells were sorted into the 96-well culture plates using a MoFlo Astrios EQ cell sorter in order to isolate a population of the virus originating from one infected cell. After the single cell sorting of the infected cell cultures into the 96-well plates, ASF positive cell detection rates in the plate wells were 30% for porcine bone marrow cells and 20% for CV-1
Determination of reproductive properties of virulent and vaccine classical swine fever virus strains in primary and continuous cell cultures
Classical swine fever (CSF) is a highly dangerous porcine disease. CSF outbreaks are annually notified in several countries. Despite the availability of specific prevention tools, the disease spread risk still persists both at country level and at world level. Hence, the disease surveillance and eradication require highly sensitive methods for early diagnosis of the infection and for tests for the virus circulation in the environment. Development of up-to-date diagnostic methods is based on well-established virus cultivation system; therefore, CSF virus reproduction enhancement, tests of new cell lines without endogenous contamination for their possible use are still of current importance. The said study was aimed at testing of primary and continuous cell cultures for their susceptibility to classical swine fever virus (vaccine virus strains and some field virus isolates recovered in the Russian Federation) and detection of the virus reproduction dynamics with real-time polymerase chain reaction with fluorescent hybridization probes used for detection. Virus replication intensity in primary and continuous cell cultures was also analyzed. The CSF virus was found incapable of replicating in some cell cultures without its preliminary adaptation. Primary porcine and lamb testicle cell cultures grown in minimal essential medium supplemented with 10% normal CSFV-negative porcine serum instead of fetal bovine serum were shown to be useful for the virus accumulation, both for vaccine strains and field isolates. Cultivation parameters and optimal minimal essential medium composition contributing to the 4–10-fold increase in the virus accumulation both in primary and continuous cell cultures were determined
African swine fever in the Primorsky Krai: disease situation and molecular and biological properties of the isolate recovered from a wild boar long bone
It is necessary to continue the analysis of the situation and molecular and biological properties of the current African swine fever virus isolates, recovered in the Russian border territories to cover the following tasks: eradication of African swine fever; development of effective disease surveillance and control programs; search for promising genome markers for the vaccine development; implementation of the differentiation strategy between vaccinated and non-vaccinated animals; and clustering of the isolates. The post-hoc analysis of some ASF epidemiological data and comparative genetic analysis of isolates circulating in the Far East Federal District suggested the agent introduction and spread routes, as well as the seasonality of the infection occurrence in the Primorsky Krai. It was established, that two ASFV subgenotypes (IGR-I и IGR-II), differentiated by intergenic region I73R/I329L, circulated in the region under study during the first months post infection. Analysis of biological properties of ASFV/Primorsky 19/WB-6723 isolate recovered from the long bone of a dead wild boar in the Primorsky Krai suggested that the isolate is highly virulent, able to cause peracute to subacute disease and up to 100% mortality among infected animals. The incubation period and duration of the disease course in experimentally infected pigs were 4–6 and 3–5 days post infection, respectively. The ASFV genome was detected in blood samples collected from infected pigs on 5–8 days post infection by real-time polymerase chain reaction. Specific antibodies in blood samples were not detected. The need in further research of molecular and biological properties of current ASFV isolates was reaffirmed. To prevent the continuation of the epizooty and deterioration of the current situation the approaches to the disease surveillance and control need to be modified
Techniques of blood sampling for detection of African swine fever virus in wild boar and domestic pigs in the field conditions
It is thought that due to the high virulence of the African swine fever virus its circulation in the Russian Federation is accompanied by a low seroprevalence. However taking into account a long-term ASF unfavourable situation, the introduction of the virus into the wild boar population, and the occurrence of attenuated viral variants, the significance of serological testing aimed at the detection of viral antibodies is increasing. To collect field samples of biological material from animals for molecular genetic, virological, and serological tests, filter paper, as well as swabs, can be used. The specificity and sensitivity of enzyme-linked immunosorbent assay when testing blood absorbed by filter paper are worse than those shown when testing sera, but they allow effective detection of African swine fever virus antibodies. It was demonstrated that blood absorbed on filter paper can be used for the immunoblot analysis, but the optimum performance could be achieved when the immunoperoxidase technique in combination with samples, taken by swabs was used. When comparing results of enzyme-linked immunosorbent assay performed on sera collected from domestic pigs (infected with ASFV isolates Antonovo 07/14 and Sobinka 07/15), and blood from ear veins absorbed on filter paper the sensitivity was 88.9%, specificity – 90.6%. However, the use of the immunoperoxidase technique for testing blood from swabs showed 100% coincidence with ELISA, while testing of sera with immunoperoxidase technique was superior to ELISA in sensitivity. This means blood sampling using swabs may be recommended for tests after proper validation. This technique can be especially useful for collecting data about infected wild boars because effective eradication strategies are impossible without such data
Measurements of , K, p and spectra in proton-proton interactions at 20, 31, 40, 80 and 158 GeV/c with the NA61/SHINE spectrometer at the CERN SPS
Measurements of inclusive spectra and mean multiplicities of ,
K, p and produced in inelastic p+p interactions at
incident projectile momenta of 20, 31, 40, 80 and 158 GeV/c ( 6.3,
7.7, 8.8, 12.3 and 17.3 GeV, respectively) were performed at the CERN Super
Proton Synchrotron using the large acceptance NA61/SHINE hadron spectrometer.
Spectra are presented as function of rapidity and transverse momentum and are
compared to predictions of current models. The measurements serve as the
baseline in the NA61/SHINE study of the properties of the onset of
deconfinement and search for the critical point of strongly interacting matter
NA61/SHINE facility at the CERN SPS: beams and detector system
NA61/SHINE (SPS Heavy Ion and Neutrino Experiment) is a multi-purpose
experimental facility to study hadron production in hadron-proton,
hadron-nucleus and nucleus-nucleus collisions at the CERN Super Proton
Synchrotron. It recorded the first physics data with hadron beams in 2009 and
with ion beams (secondary 7Be beams) in 2011.
NA61/SHINE has greatly profited from the long development of the CERN proton
and ion sources and the accelerator chain as well as the H2 beamline of the
CERN North Area. The latter has recently been modified to also serve as a
fragment separator as needed to produce the Be beams for NA61/SHINE. Numerous
components of the NA61/SHINE set-up were inherited from its predecessors, in
particular, the last one, the NA49 experiment. Important new detectors and
upgrades of the legacy equipment were introduced by the NA61/SHINE
Collaboration.
This paper describes the state of the NA61/SHINE facility - the beams and the
detector system - before the CERN Long Shutdown I, which started in March 2013
Measurements of , , , and proton production in proton-carbon interactions at 31 GeV/ with the NA61/SHINE spectrometer at the CERN SPS
Measurements of hadron production in p+C interactions at 31 GeV/c are
performed using the NA61/ SHINE spectrometer at the CERN SPS. The analysis is
based on the full set of data collected in 2009 using a graphite target with a
thickness of 4% of a nuclear interaction length. Inelastic and production cross
sections as well as spectra of , , p, and are
measured with high precision. These measurements are essential for improved
calculations of the initial neutrino fluxes in the T2K long-baseline neutrino
oscillation experiment in Japan. A comparison of the NA61/SHINE measurements
with predictions of several hadroproduction models is presented.Comment: v1 corresponds to the preprint CERN-PH-EP-2015-278; v2 matches the
final published versio
Pion emission from the T2K replica target: method, results and application
The T2K long-baseline neutrino oscillation experiment in Japan needs precise
predictions of the initial neutrino flux. The highest precision can be reached
based on detailed measurements of hadron emission from the same target as used
by T2K exposed to a proton beam of the same kinetic energy of 30 GeV. The
corresponding data were recorded in 2007-2010 by the NA61/SHINE experiment at
the CERN SPS using a replica of the T2K graphite target. In this paper details
of the experiment, data taking, data analysis method and results from the 2007
pilot run are presented. Furthermore, the application of the NA61/SHINE
measurements to the predictions of the T2K initial neutrino flux is described
and discussed.Comment: updated version as published by NIM
Measurement of negatively charged pion spectra in inelastic p+p interactions at = 20, 31, 40, 80 and 158 GeV/c
We present experimental results on inclusive spectra and mean multiplicities
of negatively charged pions produced in inelastic p+p interactions at incident
projectile momenta of 20, 31, 40, 80 and 158 GeV/c ( 6.3, 7.7,
8.8, 12.3 and 17.3 GeV, respectively). The measurements were performed using
the large acceptance NA61/SHINE hadron spectrometer at the CERN Super Proton
Synchrotron.
Two-dimensional spectra are determined in terms of rapidity and transverse
momentum. Their properties such as the width of rapidity distributions and the
inverse slope parameter of transverse mass spectra are extracted and their
collision energy dependences are presented. The results on inelastic p+p
interactions are compared with the corresponding data on central Pb+Pb
collisions measured by the NA49 experiment at the CERN SPS.
The results presented in this paper are part of the NA61/SHINE ion program
devoted to the study of the properties of the onset of deconfinement and search
for the critical point of strongly interacting matter. They are required for
interpretation of results on nucleus-nucleus and proton-nucleus collisions.Comment: Numerical results available at: https://edms.cern.ch/document/1314605
Updates in v3: Updated version, as accepted for publicatio
Multiplicity and transverse momentum fluctuations in inelastic proton-proton interactions at the CERN Super Proton Synchrotron
Measurements of multiplicity and transverse momentum fluctuations of charged
particles were performed in inelastic p+p interactions at 20, 31, 40, 80 and
158 GeV/c beam momentum. Results for the scaled variance of the multiplicity
distribution and for three strongly intensive measures of multiplicity and
transverse momentum fluctuations \$\Delta[P_{T},N]\$, \$\Sigma[P_{T},N]\$ and
\$\Phi_{p_T}\$ are presented. For the first time the results on fluctuations
are fully corrected for experimental biases. The results on multiplicity and
transverse momentum fluctuations significantly deviate from expectations for
the independent particle production. They also depend on charges of selected
hadrons. The string-resonance Monte Carlo models EPOS and UrQMD do not describe
the data. The scaled variance of multiplicity fluctuations is significantly
higher in inelastic p+p interactions than in central Pb+Pb collisions measured
by NA49 at the same energy per nucleon. This is in qualitative disagreement
with the predictions of the Wounded Nucleon Model. Within the statistical
framework the enhanced multiplicity fluctuations in inelastic p+p interactions
can be interpreted as due to event-by-event fluctuations of the fireball energy
and/or volume.Comment: 18 pages, 12 figure
- …