Reference Laboratory for African Swine Fever Topic Investigation

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. 2

Laboratory staff Head of the Laboratory, Ph. D. A. S. Igolkin Chief Researcher, Doc. of sci. N. N. Vlasova Leading Researcher, Ph. D. A. A. Shevtsov Senior Researcher, Ph. D. O. S. Puzankova Research Officer, Ph. D. V. L. Gavrilova Junior Researchers, Ph. D. S. G. Remyga and Ph. D. 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 • • • 3

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. 4

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. 5

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". 6

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 7

Results of the ASF virus isolation in porcine alveolar macrophage (PAM) cell culture No. ASFV Isolate Origin Virus titre (lg HAD units/g ) 1 ASFV RU 123080 S/12/1 Domestic pig 2 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 6. 0 5. 0 9 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 8

Examination of ASF virus (Russian isolates) replication in continuous cell cultures ASFV Isolate A 4 C 2 SPEV PPK CPE ASFV RU 190651 B/13 ASFV RU 69315 B/13/ ASFV RU 67119 S/13 ASFV RU 69315 B/13 Hem adsorp tion CPE Hem adsorptio n 2 p* + 3 p - 1 p + 3 p - 2 p + 1 p - 1 p + 3 p - * number of passages 9

Thermometry history of pigs intranasally infected with different ASFV isolates at a dose of 5000 HAD 10

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 slaughter 14 40 died 6 39, 5 39 38, 5 38 1 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 11

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 6 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 12

Disease dynamic on pigs 13

Layout diagram of genes selected for gene library on the fullsize ASF virus genome 14

• Gene library of immunologically relevant ASFV proteins was established 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. 15

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 • • • 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 17

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 18

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 21

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 69601 B/13 isolate revealed nucleotide changes in 3 genes responsible for the virulence of ASF virus.

THANK YOU FOR YOUR ATTENTION! 22