Transcript Отчет о научно-исследовательской работе

Reference Laboratory for African Swine Fever
Topic: «Investigation of the pathogenic factors of ASF,
circulating in the Russian Federation, improving of methods of
ASF diagnosis» in 2013
Supervisor
D.b.s. prof. Gruzdev K.N.
www.arriah.ru
тeл./факс: 8(4922)-26-18-67; 45-37-96.
History of the Laboratory
The Reference Laboratory for African swine fever was
established on August 1, 2012.
Currently, the Laboratory is a unit of the FGBI
“ARRIAH” and is a part of its Laboratory for livestock
animal disease diagnosis.
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Laboratory staff
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Head of the Laboratory, PhD. A.S. Igolkin
Chief Researcher, Doc. of sci. N.N. Vlasova
Leading Researcher, PhD. A.A. Shevtsov
Senior Researcher, PhD. O.S. Puzankova
Research Officer, PhD. V.L. Gavrilova
Junior Researchers, PhD. S.G. Remyga and PhD. A.S. Pershin
Leading Veterinarian, A.A. Varentsova
Leading Biologists, I.V. Shevchenko and I.Yu. Zhukov
Laboratory Technicians, V.M. Zaynutdinova, N.I.Kamaeva,
T.V.Klochkova
• Total: 13 staff-members
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Laboratory Tasks
• Research activities;
• Analysis of ASF risks and ASF situation dynamics in
the territories of the Russian Federation and
Customs Union;
• Scientific justification of the ASF monitoring plans
approved by the Rosselkhoznadzor;
• Performance of official tasks for ASF monitoring and
diagnosis;
• Preparation of regulatory and methodological
documents on ASF in accordance with the
international standards;
• Carrying out of laboratory proficiency tests;
• Participation in international scientific projects;
• Training and educational activities.
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Research Activities
• After the research project “Investigation of
pathogenicity factors of ASF agent circulating in the
territory of the Russian Federation and ASF diagnostic
method improvement” within the main component
“African swine fever spread prevention and eradication
in the territory of the Russian Federation” of the
Governmental Program on agricultural development
and agricultural, raw material and food market
regulation for 2013-2020 was approved by the
Rosselkhoznadzor on April 30, 2013 the researches
comprising 6 stages have been performed according to
the schedule.
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Goals of the laboratory
Laboratory crew are currently working on the topic
"Investigation of the pathogenic factors of African
swine fever (ASF)", circulating in the Russian
Federation, improving of methods of ASF diagnosis in
the Federal program "Development of agriculture
and agricultural production, food and primary
products markets regulation in 2013-2020 years".
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Stage 1 Analysis of existing international methods and
approaches used for investigation of ASF virus
variability and for setting-up of ASF virus variability
criteria
Stage 2 Recovery of at least 5 isolates of ASF virus
Stage 3 Examination of recovered ASFV isolates for their
cultural and biological properties including their
virulence, contagiousness for domestic pigs
Stage 4 Examination of the recovered ASFV isolates for their
molecular-biological properties
Stage 5 Optimization of the isolate storage conditions
including selection of optimal stabilizing media and
freeze-drying protocol
Stage 6 Development and improvement of ASF diagnostic
methods
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Results of the ASF virus isolation in porcine alveolar macrophage
(PAM) cell culture
Virus titre (lg HAD
units/g )
No.
ASFV Isolate
Origin
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ASFV RU 123080 S/12/1
Domestic pig
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3
ASFV RU 123080 S/12/2
ASFV RU 190651 B/13
Domestic pig
Dead wild boar
< 1.0
2.0
4
ASFV RU 69601-B/13/1
Shot wild boar
5.0
5
ASFV RU 36775-S/13/1
Domestic pig
5.0
6
ASFV RU 36775-S/13/2
Domestic pig
4.5
7
8
ASFV RU 33830-B/13/1
ASFV RU 33830-B/13/2
Dead wild boar
Dead wild boar
6.0
5.0
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ASFV RU 67119-S/13/1
Domestic pig
5.0
10
11
ASFV RU 67119-S/13/2
ASFV RU 69315-B/13/1
Domestic pig
Shot 1-year-old wild boar
5.0
4.5
12
ASFV RU 69315-B/13/2
Shot 3-year-old wild boar
4.0
1.5
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Examination of ASF virus (Russian isolates) replication
in continuous cell cultures
ASFV Isolate
A4C2
SPEV
PPK
ASFV RU 190651
B/13
ASFV RU 69315B/13/
ASFV RU 67119S/13
ASFV RU 67119S/13
ASFV RU 69315B/13
ASFV RU 69315B/13
CPE
Hem
adsorp
tion
CPE
Hem
adsorpti
on
CPE
Hem
adsorptio
n
2p*
+
3p
+
3p
-
1p
+
1p
+
3p
-
2p
+
3p
+
3p
-
2p
+
1p
+
1p
-
1p
+
3p
+
3p
-
1p
+
3p
+
3p
-
* number of passages
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Thermometry history of pigs intranasally infected with different
ASFV isolates at a dose of 5000 HAD
42,5
died 9
42
died 9
41,5
died 12
died 9
41
died 18
died 9
died 21
40,5
died 9
40
died 11
39,5
died 12
39
38,5
1
2
3
4
5
ASFV RU 69601-B/13
ASFV RU 69315-B/13
6
7
8
9
10
11
12
ASFV RU 36775-S/13
ASFV Stavropol 01/08
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14
15
16
17
18
19
ASFV RU 67119-S/13
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Thermometry history of pigs intranasally infected with different
ASFV isolates at a dose of 50 HAD
42,5
died 14
42
died 9
41,5
died 20
slaughter 13
slaughter 21
died 12
41
died 8
40,5
slaughter14
40
died 6
39,5
39
38,5
38
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2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20
ASFV RU 69601-B/13
ASFV RU 67119-S/13
ASFV Stavropol 01/08
ASFV RU 36775-S/13
ASFV RU 69315-B/13
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Thermometry history of intact pigs at their keeping in contact
with animals infected with different ASFV isolates
42,5
slaughter 14
42
died 14
slaughter 14
41,5
died 19
slaughter 14
41
40,5
40
slaughter 21
39,5
39
38,5
38
1
2
3
4
5
ASFV RU 69601-B/13
ASFV RU 69315-B/13
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7
8
9 10 11 12 13 14 15 16 17 18 19 20 21
ASFV RU 36775-S/13
ASFV RU 67119-S/13
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Disease dynamic on pigs
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Layout diagram of genes selected for gene library on the fullsize ASF virus genome
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• Gene library of immunologically relevant ASFV
proteins was established and analysis of
nucleotide sequences of 8 genes of Krasnodar
06/12 isolate was performed.
• DNA pyrosequencing of ASFV RU 69601-B/13
isolate and comparative analysis of changes
demonstrated that in comparison with Georgia
2007/1 isolate there were 52 singular insertions,
12 deletions and 8 substitutions in the genome of
ASFV RU 69601-B/13 isolate, which amounts to
~0.0327% of changes against 189 398
nucleotides.
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arrows indicate changes in genes, ASFV isolate ASFV RU 69601-B/13.
Dixon, L.K., Abrams C.C., Bowick G., Goatley L.C., Kay-Jackson P.C., Chapman D., Liverani E., Nix R., Silk R., Zhang F. (2004) African swine fever 16
virus
proteins involved in evading host defence systems. / Vet. Immunol Immunopathol. 100:117-134.
Further investigations
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The following is planned for further investigation of ASFV pathogenicity and
variability basics:
Isolation, comparative studies of biological properties of ASFV as well as
development of new tools for diagnosis and specific prevention
Formation and reinforcement of ASFV strain collection
Determination of genome changes in ASFV isolates having different biological
properties
Improvement of methods for early detection of ASFV antibodies in blood sera
of infected animals
Development of methods for ASFV indication in feed and soil
Examination of chances for ASFV transmission with tick species inhabiting the
territory of the Russian Federation
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International cooperation on ASF
• Development of cooperation with OIE and FAO ASF
Reference Laboratories, training courses for
researchers;
• Collaborative work on examination of biological
properties of ASFV circulating in the RF;
• Participation in programs dealing with the issues of
prevention of transboundary swine disease spread
along the border of Russia with neighboring
countries;
• Participation in International workshops and
conferences
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Research and advisory support is needed for
productive operation of the ASF Reference Laboratory
in the FGBI “ARRIAH”:
• Genome sequencing (from sample preparation to analysis and
computer processing of data)
• Recombinant proteins and antibodies
• Electronic microscopy
• Serological diagnosis – enzyme-linked immunosorbent assay,
immunoblotting, dot-blot analysis, immunofluorescence test,
haemadsorption, etc.
• Cytofluorometry
• Entomology of ticks – ASFV vectors
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Conclusion
•The study of culture-biological properties of ASF
virus isolates collected in the territory of the Russian
Federation in 2013, allowed to discover the variation
of pathogenicity towards reduction of virulence and
increase of the disease duration.
• Long-read pyrosequencing genome ASFV RU 69601B/13 isolate revealed nucleotide changes in 3 genes
responsible for the virulence of ASF virus.
THANK YOU FOR YOUR ATTENTION!
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