Monitoring of Highly Pathogenic Swine Fever viruses (HPSFVs) in

Kazakhstan.

Primary Institute: Branch of “National Center for Biotechnology” in Almaty

Central Reference Laboratory

Project manager: профессор Мамадалиев С.М.

Project Collaborator: Dr. Guillermo R. Risatti

University of Connecticut, USA

Almaty, 2019

Central Reference LaboratoryThe Republic of Kazakhstan

Project objectives:

• Determine current trends of occurrence (prevalence) of HPSFVs indomestic pigs in Kazakhstan.

• Describe virus associated with HPSFVs in different pig populations.

• Genetically identify (partial sequencing) of selected HPSFVs detectedin clinical samples obtained from swine.

Figure 1: Distribution of domestic swine population as percentage of total domestic swine population (~500,000 heads in 2017) in Kazakhstan.

54%

32%

12%

West Kazakhstan

Atyrau

Mangystau

Aktobe

Kyzylorda

South KZ

Zhambyl

Almaty

East Kazakhstan

Karagandy

PavlodarKostanay Akmola

North Kaz.

• The study will assess the prevalence of diseases in the Republic ofKazakhstan with significant consequences for pig health and publichealth, including swine influenza (SI), classical swine fever (CSF),African swine fever (ASF) and reproductive respiratory porcinesyndrome (PRRS).

• Despite the etiological difference of pathogens these swine diseaseshave similar clinical signs which often lead to errors in the diagnosis.As a result, when studying these diseases, it is necessary to have alarge set of differential diagnosis methods and anamnestic datathat allow a correct diagnosis.

Swine influenza (SI)family Orthomyxoviridae,

genus Influenza А

• Swine influenza (SI) is caused by different serotypes of influenza Aviruses (IAV). The three most common viruses that affect pigs globallyare H1N1, H1N2 and H3N2

• Infections with these viruses result in rapid onset of disease that insome cases is characterized by high morbidity and mortality events.Immunity to influenza viruses is often short lived (6 months) and theimmunity profile in breeding herds varies considerably with time.

• SIV do not normally infect humans and it should be noticed thattransmission of SIV to humans is considered sporadic.

Figure 2: Main flyways for wild birds across Asia.

• Of important relevance to this proposal is that Southern Asiaincludes areas within the continent where wintering of wild birdstakes place. Northward migration of wild birds through the threemajor Asian flyways converge during the summer time in Central andNorthern Asia, including a fly pathways over Kazakhstan (Figure 2).Thus, in this way IAVs (influenza A viruses) are moved through thecontinent and eventually intercontinentally.

• Flu isolates belonging to the H3N6, H4N6, H1N1, H10N7, H3N8 andH13N8 subtypes were detected in Kazakhstan, Mongolia and Russiathat are part of the CAF (Central Asia flyway) (Marchenko, et al.,2012). In Kazakhstan samples were collected at the Alakol Lakelocated in the East Kazakhstan Oblast close to the border with China.The array of flu viruses detected in the region were associated withbirds of six orders, including species inhabiting aquatic and dry plainenvironments.

• Overall the information accumulated so far indicates that thenorthern area of the CAF is the source of a variety of low-path andhigh-path flu viruses some of them with the potential to infectdomestic and wild swine populations. Therefore, the location of wildbird flyways over Kazakhstan will be key for defining suitable areasfor sampling swine in the country.

• In summary, due to its geographical location and certain swinebreeding practices (i.e. backyard, free- roaming), and the presence ofwild boars (Figure 3), Kazakhstan may be at the verge of HPSFVs spill-over mainly from Russia and China— Kazakhstan’s largest neighbors.Therefore, Northern and Eastern Kazakhstan are suitable groundsfor surveillance of HPSFs.

Figure 3: wild boar range; native (green) and introduced (blue).

Classic swine fever virus (CSFV)family Togaviridae,

genus Pestivirus

• Virulent CSFV infection in susceptible pigs causes severe andwidespread epidemic pig morbidity and mortality (Risatti and Borca,2016).

• Less virulent CSFV strains or less susceptible pigs suffer decreasedgrowth and productivity.

• CSFV is endemic-to-epidemic in regions of South America, CentralAmerica, and Mexico.

• In Western Europe the disease is endemic in wild boars withoccasional epidemics affecting domestic swine.

• CSFV is also widespread in Asia mainly in China (Luo et al., 2014) and South East Asia (figure 4).

Figure 4: Global distribution of CSF based on country reports to the OIE 2016 (World Animal Health Organization)(Source: “Current Policy Needs in the Effective Management in TADs in the Asia-Pacific Region -OIE Perspectives-”Trans-Boundary Animal Disease Workshop, July 2016, Manila, ThePhilippines).

• ASFV induces a well characterized, economically important and acute,clinically severe viral hemorrhagic fever of domestic and wild pigs(Risatti et al., 2015).

• Thus, differential diagnostics is important since ASFV has not beenreported in most countries in Asia with the exception of the Asianregion of the Russian Federation (Figure 5).

• As on August 2018 China has reported outbreaks of ASF in different regions of the country (Normile, 2018).

African swine fever virus (ASFV)family Iridoviridae

Figure 5: Progression of ASF outbreaks in a period of 5 years [from: African swine fever (ASF): Five ears around Europe” 2013 Vet. Microbiol. 165(1-2): 45-60].

• PRRSV, was detected in early 2006 in the People’s Republic of China, following anoutbreak in which 2 million pigs were affected and 400,000 died (Tian et al.,2007).

• The disease in domestic swine is characterized by high fever (41°C), and highmorbidity (50%-100%), and high mortality (20%-100%) rates for pigs of all ages.

• The virus originated from a type 2 PRRSV that was already circulating in China.

• New HP-PRRSVs continue to emerge and highly pathogenic forms seem to behighly prevalent in China (Tian et al., 2007; Liu et al., 2017; Chen et al., 2018). Thevirus has now been detected in China, Vietnam, the Laos People’s DemocraticRepublic, Thailand, Cambodia, Russia and India.

• Prevalence of PRRSV in Kazakhstan has not been assessed. Clinically the diseaseresembles CSF or ASF thus a confirmatory laboratory diagnostic test is required.

Porcine reproductive and respiratory syndrome virus (PRRSV)genus Arterivirus,

family Arteriviridae

Figure 6: Global distribution of PRRSV.

Selection of region for sample collection from pigs during this project

• A study was conducted on the prevalence of the disease amongvarious groups of animals, which made it possible to determine thecurrent prevalence of highly pathogenic viral fever in pigs in twopriority regions of Kazakhstan.

• These regions are characterized as regions with marshy (rivers,swamps, lakes) and forest landscapes, and which are located in closeproximity to the border regions of Russia and / or China (Fig. 7 and 8).

Figure 7: Sampling area in North Kazakhstan Oblast in the. Petropavl area. This area is bordering with Russia could be considered a priority area The region encompasses lakes, and farming areas and major roads that go across the international border, where wild and domestic animals (swine and waterfowl) might be at close proximity and a risk of spill-over of diseases into domestic swine is possible

Figure 8: Sampling area in Eastern Kazakhstan. The selected area in East Kazakhstan Oblast including the Semey region close to the border with Russia and Lake Zaysan close to the border with China is considered at high-risk for swine disease. The Lake Alakol region in the Almaty Oblast is close to the international border with China will also be considered for surveillance program. These areas in Eastern Kazakhstan support a lot of movement particularly across the border with China.

Plan for sample collection from pigs in each region

If such an opportunity presents itself, samples from wild pigs can also be used. Samples will becollected by four field teams.

Sample collection areas

North Kazakhstan and Pavlodar regions

East Kazakhstan and Almaty region

Total number of samples for each season

Blood 400 400 800

Serum 400 400 800

Nasal swabs 400 400 800

Research methods/Approaches

• Sanger sequencing: specific areas/segments within the genomes of SIV/IAV (matrix, H andN genes), CSFV (NS5B, E2, 5’UTR), ASFV (p54, p72, B602L) and PRRSV (NS2, GP5) will beamplified.

• Next-Generation Sequencing (NGS). Partial genome sequencing of detected viruses willbe conducted by using a MinION (Oxford Nanopore technology) once the scope ofpositives to test becomes apparent. Genes of greatest interest will be specifically targetedfor sequencing. The affordable technology is based on a device that can be used for DNAand RNA sequencing and is handled from a laptop computer. The system can produce 10-20 gigabites of DNA sequence data.

• Sample processing: RNA and DNA will be extracted from collected samples usingcommercial kits. Oxford Nanopore chemistry will be used for library preparation andsequencing including Direct cDNA Sequencing Kit, Direct RNA Sequencing Kit and RapidSequencing Kit.

• Sequencing data processing: the MinION device is controlled by MinKNOW software.MinKNOW carries out several core tasks: data acquisition; real-time analysis and feedback;base-calling; data streaming; device control including selecting the run parameters; sampleidentification and tracking.

Results

Figure 9. The areas where samples were collected at the first stage of theexpedition are indicated by gray color.

The results of the ELISA analysis of samples from the North Kazakhstan (NK) region

№ Sample collection locationnumber of

samplesnumber of

samplesnumber of

samplesPRRS

positiveInfluenza A positive

CSF positive

ASF positive

Blood Serum Swabs

1NK, Magzhan Zhumabayev district, Zolotaya Niva village 7 7 7 0 0 0 0

2NK, Magzhan Zhumabayev district, Vozvyshenka village 6 4 6 0 3 0 0

3NK, Magzhan Zhumabayev district, Sovetskoe village 14 17 10 0 14 0 0

4 NK, Esil district, Tarangul village 19 19 19 0 8 10 0

5NK, Gabit Musrepov district, Urozhainoe village 5 5 5 1 0 0 0

6NK, Shalakyn district, Gorodninskoevillage, collective farm ”Ayna" 29 29 16 0 6 0 0

7NK, Taiynshy district, Krasnaya polyanavillage 31 31 31 0 3 0 0

8 NK, Taiynshy district, Chkalovo village 9 9 21 0 0 0 0

9NK, Taiynshycity, collective farm"AgroFirma" 0 45 0 0 0 11 0

Total number in North Kazakhstan 120 166 115 1 34 21 0

% of positive 1 20 13 0

The results of the ELISA analysis of samples from Pavlodar region

№ Sample collection locationnumber of

samplesnumber of

samplesnumber of

samplesPRRS

positiveInfluenza A positive

CSF positive

ASF positive

Blood Serum Swabs

1 Pavlodar reg., Pavlodarskoe vil. 40 22 17 34 0

2 Pavlodar reg., Chernoyarka vil. 41 22 28 31 0

3 Pavlodar reg., Lvovka vil. 10 0 2 0 0

4 Pavlodar reg., Zhelezinka vil. 15 0 15 11 0

5 Pavlodar reg., Leninskiy vil. 20 1 20 17 0

Total number in Pavlodar region 0 126 0 45 82 93 0

% of positive 36 65 74 0

The results of the ELISA analysis of samples from Karagandy region

№ Sample collection locationnumber of

samplesnumber of

samplesnumber of

samplesPRRS

positive

Influenza A

positive

CSF positive

ASF positive

Blood Serum Swabs

1

Karagandy region, Buhar Zhyraudistrict, ”Doskei” village, collective farm ”Medeu" 77 94 0 77 38 80 0

Total number in Karagandy region. 77 94 0 77 38 80 0

% of positive 82 40 85 0

The results of the ELISA analysis of samples from East Kazakhstan (EK) region

№ Sample collection locationnumber of

samplesnumber of

samplesnumber of

samplesPRRS

positiveInfluenza A positive

CSF positive

ASF positive

Blood Serum Swabs

1 EK, Vostochnoe vil. 57 52 0 45 0

2EK, Leshoz vil., collective farm”Rodnichok" 30 30 28 0 0

3 EK, Parygino vil. 5 1 0 0 0

4 EK, Donskoe vil. 10 10 4 0 0

5 EK, Teplyi kluych vil. 2 0 2 0 0

6 EK, Landmn vil. 10 10 10 4 1 0 0

7 EK, Urdjar city. 19 19 19 0 0 0 0

8 EK, Mirolyubovka vil. 5 5 7 0 0 0 0

9 EK, Vozdvizhenka vil. 4 4 4 0 0 0 0

10 EK, Preobrazhenka vil. 6 6 6 0 0 0 0Total number in East Kazakhstan 0 148 0 97 35 45 0

% of positive 66 24 30 0

The results of the ELISA analysis of samples from Almaty region

№ Sample collection locationnumber of

samplesnumber of

samplesnumber of

samplesPRRS

positiveInfluenza A positive

CSF positive

ASF positive

Blood Serum Swabs1 Almaty reg., Tekeli city 5 5 6 0 0 0 0

2 Almaty reg., Aksu district, Kapal vil. 17 17 18 4 0 0 03 Almaty reg., Sarkand city 6 6 6 0 0 0 0

4Almaty reg., Sarkand district, Karaultobe vil. 6 6 6 0 1 0 0

5 Almaty reg., Sarkand district, Koilyk vil. 6 6 6 0 0 0 0

6Almaty reg., Sarkand district, Sokolovkavil. 7 7 8 0 0 0 0

7 Almaty reg., Alakol district, Akzhar vil. 2 2 3 0 0 0 0

8 Almaty reg., Alakol district, Konyr vil. 10 10 10 0 0 0 0

9Almaty reg., Alakol district, Kabanbayvil. 2 2 2 0 0 0 0

10 Almaty reg., Alakol district, Terekti vil 10 10 10 0 0 0 0Total number in Almaty region 71 71 75 4 1 0 0

% of positive 6 1 0 0

Conclusion on the KZ-35 project

• As a result of the study of collected samples for the detection of pathogensof CSF, ASF, SI and PRRS the following were established :

1. No ASF pathogen was detected in all tested samples.2. The causative agent of CSF was found in samples from the North

Kazakhstan region, in those farms where pigs are vaccinated against thisinfection.

3. Antibodies against PRRS infection were also detected in samplescollected from the North Kazakhstan and Karaganda regions, where alsosows are routinely vaccinated.

4. Antibodies against swine influenza were detected within 20 to 65% of allsamples collected from the North Kazakhstan, Pavlodar, East Kazakhstan,Karaganda regions.

5. Farms in these areas do not use any prophylactic drugs and vaccinesagainst swine inflienza.

6. The routine collection and testing/study of samples in these areas willcontinue.

KZ-35 project manager and staffMamadaliyev S.M., professor, Doctor of veterinary science

Zhigailov A.V., PhD

Dmitrovsky A.M., professor, Doctor of medical science

Nizkorodova A.S, Ph.D

Skiba Y.A., Ph.D Maltseva E.R., PhD

Ismagulova G.A., Ph.D Neupokoyeva A.S., MSc in Biotecnhology

Berdygulova Z.A., PhD Kuatbekova S., MSc in Veterinary

Ostapchuk E.O., PhD Naizabayeva D.A., MSc in Biotecnhology

Perfilyeva Y.V., Ph.D Bissenbay A.O., MSc in Biomedicaland Molecular sciences research

Kuznetsova T.V., MSc in Veterinary

International scientific projects on the study of biosafety problems and infections in the Republic of Kazakhstan:

1. Project KZ-31 CBR: Rickettsia & Plague. Institute: M. AIKIMBAYEV KAZAKH SCIENTIFICCENTER OF QUARANTINE AND ZOONOTIC DISEASES (KSCQZD)

2. Project KZ-32 CBR: Bluetongue and brucellosis. Institute: Research Institute for BiologicalSafety Problems (RIBSP)

3. Project KZ-33 CBR: MERS. Institute: Research Institute for Biological Safety Problems (RIBSP)Project TAP-08: Saiga. Institute: Research Institute for Biological Safety Problems (RIBSP)

4. Project TAP-10: TBEV. Institute: Scientific and Practical Center for Sanitary andEpidemiological Expertise and Monitoring.

5. Project TAP-11: Hantavirus. Institute: Ural anti-plague station.

6. Project TAP-12 Bioinformatics: Newcastle disease virus. Institute: Research Institute forBiological Safety Problems (RIBSP)

7. ISTC Project 53: Strengthening the national legal framework and providing specializedtraining in biosafety and biosecurity in the countries of Central Asia.

8. CDC Project: Study of rickettsia genotypes circulating in Pavlodar region of Kazakhstan.

Conclusions

• The work on ensuring biosafety of dangerous and highly dangerousinfections in the Republic of Kazakhstan is carried out both throughthe Ministry of Health and the Ministry of Agriculture, as well as inthe framework of state and international projects.

• A network of reference laboratories for monitoring dangerous andршпрдн dangerous infections in the Republic of Kazakhstan has beenestablished and is functioning.

• A legislative and legal base has been created for ensuring biosafety inthe Republic of Kazakhstan, and this base is constantly beingimproved and many articles fully harmonize with internationalstandards..