Project Objectives. The aim of the project is to study the genetic variability of PRRS viruses in different regions of the Republic of Kazakhstan. To achieve this goal it is necessary to implement the following tasks: collect samples from animals in large pig farms, backyards and wild boars from different regions of Kazakhstan, isolate the virus, perform viral typing, sequencing and phylogenetic analysis. Implementation of the project will allow to solve and elucidate numerous basic and applied research problems connected with this infection, for researchers and practitioners in agricultural sector of Kazakhstan. These results will expand the knowledge of the biological characteristics of the causative agent, its genetics and epizootology, laboratory diagnosis and prevention of the disease. The project tasks will be carried out in close contact with a collaborator through joint research, discussions on the results and joint publications. The collaborator will aid in determining sampling plans, providing sample workflow protocols and hands-on laboratory guidance, and assist in genetic analysis of results, along with help in publications and presentations.
Impact of the proposed project on the progress in the given field of science. During the project implementation PRRSV monitoring will be conducted in different regions of Kazakhstan, currently circulating strains will be isolated, and novel data on their genetic variability and viral variant introduction or evolution will be obtained. The project results will provide the scientific basis for accurate diagnostic tools(nucleic acid based diagnostic assays) and effective preventative countermeasures (vaccines) to minimize the effects of the disease in RoK and the region. A lower incidence of PRRS would not only improve the economics of meat production, but also potentially lessen the likelihood of an emerging zoonotic infection arising from this highly adaptable and genetically plastic virus.
Project Participants Competence. Scientists participating in the project have extensive experience in the collecting and analyzing of the results of research. The existing publications and presentations at international conferences, which can be found in the press and on the Internet sites of organizations – participants confirm this. Project manager Mamadaliev S.M. - Doctor of Veterinary Sciences, is one of the leading virologists in Kazakhstan, more than 20 years he was in charge of one of the largest research institutes in the country. He studied the biological characteristics of many pathogens, pathogenesis of diseases (including the PRRS causative agent), developed and introduced in veterinary practice new means of prevention and control methods for many diseases of wild and domestic animals. Main Executor, Zholdybaeva EV - PhD, is a Head of laboratory, an expert in the field of molecular biology of viruses, population genetics and genomic research. She is the author of 42 scientific articles, 6 author’s certificates for the development of PCR diagnostic test kits. Dr. Zholdybaeva is a winner of scholarship of L. Orbeli, Russia (2001). She was awarded the Certificate of Merit by the Ministry of Education and Science for her contribution to the development of science in Kazakhstan (2009). She is a Winner of the MES and the World Bank for the project "Technology Commercialization" (2011). Dr.Zholdybaeva is a Scholar of the state scientific scholarship for talented scientists (2012-2013). Main Executor, Kopochenya A.A. is an expert in epizootology, monitoring of pig diseases, including PRRS, biological characterization of viruses. He is theauthor of 30 scientific articles, has 8 author’s certificates for various strains of viruses. He was awarded the Certificate of Merit by the Ministry of Education and Science for his contribution to the development of science in Kazakhstan (2010). Main Executor, Dmitrovskiy A.M. - Doctor of Medicine, Professor, is one of the leading experts in the field of infectious pathology in Kazakhstan. He works in the field of zoonotic infections for about 40 years, an expert in the field of epidemiology, laboratory diagnostics and analytics. Main Executor, Kopochenya M.A. is a specialist in the field of epidemiology, monitoring, analysis of risks of dangerous viral infections, including PRRS. She has published several scientific papers on PRRS. Scientific Advisor, Muhanov K.K. - Deputy Director, Doctor of Veterinary Sciences, professor, is an expert in the field of animals’ epidemiology (epizootiology).
Expected Results and their Application. The obtained results of the project will provide: reliable data on the PRRSV distribution in different regions of Kazakhstan; epidemiological characteristics of the spread of infection; isolation of KZ PRRSV variant strains$; PRRSV genetic variability in the regions of our country, and their phylogenetic relatedness to other known strains; the genetic characteristics of PRRSV strains isolated in Kazakhstan.
The above mentioned results will allow for the first time to characterize the PRRSV strains circulating in Kazakhstan. This will enable the development of accurate diagnostic tools and effective preventive measures.
Meeting ISTC Goals and Objectives. As the project participants are former ??weapon scientists, and the project itself has peaceful aims and purposes, it fully complies with the ISTC goals and objectives. Also, meeting of ISTC goals and objectives is achieved through the planned involvement of scientists from a participating organization in the world scientific community, and by providing generated data through publications and participation at international conferences and seminars.
Scope of Activities. In the framework of the project the following activities will be carried out: formulation of a sampling plan in targeted regions of KZ; field sample collection travel to several different regions of the country, to collect samples from animals in large pig farms, private backyards and from wild boars; virus isolation in sensitive biological systems; - primary viral identification using rRT-PCR; typing, sequencing, genetic mapping of strains; phylogenetic analysis of PRRSV isolates.
Role of Foreign Collaborators/Partners. MRIGlobal has agreed to be a collaborator on this project. MRIGlobal scientists will participate in formulating sample plans, typing, sequencing, genetic mapping and phylogenetic analysis of PRRSV isolates, as well as joint publications of the project results in leading international journals and presentation in scientific meetings.
Technical Approach and Methodology. The work will include: sample collection activities in relevant regions of the country; along with samples relevant epidemiological data will be obtained; collection and transportation of blood samples and other putative pathological tissues from animals; isolation of PRRSV strains; identification of the infectious agent; PRRSV typing; sequencing of PRRSV isolates; phylogenetic analysis.
In the course of the project implementation there will be several field expedition to regions of Kazakhstan, to conduct epizootological survey of large pig farms, private backyards and reserves of the region. A questionnaire will be generated that specifies the origin of sampled animals, their disease symptoms, and farm conditions. Field material (blood and pathological material) will be collected for detailed molecular analysis.
Those collected samples will be initially identified by rRT-PCR using commercial kits; positive samples will be further enriched to increase the viral titer in sensitive cell lines (MARC-145) . Samples (either plaque purified or crude tissue or cell supernatants) will be preserved for immediate and downstream analysis.
For sequencing and genetic mapping of PRRSV isolates, viral RNA will be extracted from concentrated preparations. Using rRT-PCR employing primers homologous to loci common to all known PRRSV, whole genome amplification (using Phi 29 polymerase) will be undertaken in individual samples to provide sufficient quantity of cDNA to use for restriction analysis, PCR, and sequencing. The whole genome preparations will be directly used, in singleplex reactions for cycle sequencing. Phylogenetic analysis of the elucidated PRRSV genetic data will be the final stage of the study in the project.
Measuring the success of the project tasks achievement will be based on validation of generated sequence data using a comparison with existing PRRSV sequence data available in the open databases, and peer review of the results.
