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Molecular characterization of African swine fever virus isolates originating from outbreaks in the Russian Federation between 2007 and 2011

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Abstract

African swine fever is one of the most important viral diseases of pigs and which caused significant economic damage on the pig production worldwide. Nowadays, it is still present on the African continent, in Transcaucasus countries (TCC), on Island of Sardinia and in Russia. Outbreaks of the disease have been reported in Russia for the last four years, affected especially the Southern Federal District of the country. Since 2010, a new outbreak area has been observed in the Northwestern Federal District.

In order to study the evolution of African swine fever virus (ASFV) isolates, strains were collected in the Russian Federation from 2007 to 2011 and investigated by means of partial sequencing and fragment length polymorphism. In detail, 7 variable regions, namely B646L, E183L, I196L, B602L, I73R/I329R, I78R/I215L and KP86R were investigated. Phylogenetic analyses revealed 100% nucleotide identity of B646L and E183L gene sequences of all examined isolates. All isolates formed one genetic cluster within genotype II. Moreover, no amplified fragment length polymorphism (AFLP) was observed for B602L, I196L, I73R/I329R, and I78R/I215L genes. The flanking primers used to amplify the KP86R gene failed to amplify a product in all the isolates. The obtained data strongly suggests that only one ASFV virus variant caused the outbreaks from 2007 to 2011 in the territory of the Russian Federation.

Introduction

African swine fever (ASF) is among the most dangerous diseases impairing pig production worldwide. ASF is highly contagious and acute forms of the disease have a high rate of mortality that may result in substantial economic losses, due to compulsory pig slaughtering and the international ban on trade of live animals and pig products (Penrith, 2009, Tulman et al., 2009). Epidemiology of ASF confirms that presence of the disease in one area has a potential risk for introduction and further spreading to any directions despite the natural and artificial borders and distance (Wieland et al., 2011). Clinical symptoms of ASF vary considerably, but in general, ASF is well known as a disease with high morbidity and mortality. The disease affects domestic and wild pigs, and can be transmitted through contact as well as via soft ticks of the Ornithodorus complex (Jori and Bastos, 2009, Parker et al., 1969, Plowright et al., 1970). The causative agent is a large enveloped double-stranded DNA virus, sole member of the genus Asfivirus within the Asfarviridae family (Kleiboeker and Scoles, 2001). The viral genome comprises around 170–195 kb, which encode more than 125 different proteins. According to the nucleotide sequence analysis of the variable region of the B646L gene (p72), 22 genotypes can be distinguished (Bastos et al., 2003, Lubisi et al., 2005).

Better phylogenetic resolution between closely related ASFV isolates was reached using other virus genes (E183L (p54), B602L, KP86R, I196L) and intergenic regions (J286L, BtSj, I73R/I329R, I78R/I215L) as the markers for genetic variations (Gallardo et al., 2009, Nix et al., 2006, Rodriguez et al., 1994).

The repeat region next to J268L contains a set of internal repeated sequences composed of 5 types of 200-bp-long tandemly repeated units. These units contain a G-rich core of 10–14 nucleotides surrounded by regions with a high A and T content. The ORF KP86R is cysteine-rich and contains tandem repeats identical to those in the ORF DP86L at the right-hand end of the genome.

Two intergenic region I73R/I329R, I78R/I215L and open reading frame I196L placed 10.6 kbp from the right end of ASFV genome (EcoRI-I fragment) and characterized by presence of terminal repeat sequences (TRS).

The ORF B602L encodes a central region containing twelve-base-pair repeats. The variable region of ORF B602L consists of repeated amino acid tetramers that vary in number and type. The reasons for the variability of the B602L protein are still not clear. The protein has been reported to act as a chaperone involved in assembly of the p72 capsid protein into virions, although B602L protein itself was reported not to be incorporated into virions (Nix et al., 2006).

Until recently, only countries of Sub-Saharan Africa and Sardinia were endemically infected with ASFV. In June 2007, Institute of Animal Health (Pirbright, UK) notified of a new outbreak of ASF in the Republic of Georgia, coherently the outbreaks were registered in Armenia, Azerbaijan and the Russian Federation (Rowlands et al., 2008). Since the first introduction in 2007 and until 2011, ASF outbreaks were regularly detected in different regions of the Russian Federation (Balyshev et al., 2010, Gulenkin et al., 2011, Kurinnov, 2009). From 2008 to 2011, the disease was continuously present in the Southern Federal District. Since 2010, a new outbreak area has been observed in the Northwestern Federal District.

Considering the endemic situation in some territories of Russia over the last four years, there is a clear possibility for genetic changes in ASFV isolates which are obtained from different times, places, and animals.

In this regard, the main goal of our study was to carry out a comparative analysis of ASFV isolates, from different outbreaks of the disease on the territory of Russia since 2007 based on phylogenetic relationship and molecular characteristics.

Section snippets

Virus isolates

16 ASFV positive samples of organs (mainly spleens) obtained from naturally infected wild boars and domestic pigs and stored in the virus collection at the SRI NRIVVaM RAAS were used to study genetic differences between them. The samples for investigation from different ASF outbreaks were provided by the Russian State Veterinary Service and Federal Service for Veterinary and Phytosanitary Surveillance from North Caucasian Federal District, Southern Federal District, Volga Federal District,

Results

To estimate strain variability of ASFV isolates which were obtained from naturally infected wild boars and domestic pigs from different regions of the Russian Federation from 2007 to 2011, seven variable genome regions (B646L, E183L, I196L, B602L, I73R/I329R, I78R/I215L and KP86R) were analysed.

In order to determine the genetic relationship between the obtained isolates and the 22 already identified ASFV genotypes according to nucleotide sequencing of the p72 gene fragment, phylogenetic trees

Discussion

Genetic typing, nucleotide sequencing, and further phylogenetic analyses are worldwide applied the techniques to confirm epidemiological data on a qualitatively different level. It also allows us to estimate virus evolution over time.

Comparison analysis of ASFV genome variable regions containing array of the tandem repeats enables to provide more information about molecular patterns of isolates and allow to separate them in multiple sub-groups even if they were clustered together basis of on

Conclusion

Identical molecular characteristics and genetic patterns of analysed ASF isolates seem to be very likely the result of disease spreading from one common source without an appearance and long-term existing of cylvatic or domestic cycles. Finally, our data obtained based on investigation of variable regions in right (RVR) and central (CVR) part of ASF virus genome allow to conclude single source of origin for all used isolates and absence of genetic changes have occurred in the regions evaluated

Conflict of interest

The authors declare no conflict of interest. None of the authors of this publication has any financial or personal relationships with other people or organizations that could inappropriately influence this work.

Acknowledgements

Authors would like to thank Russian State Veterinary Service and Federal Service for Veterinary and Phytosanitary Surveillance for providing fields samples. We greatly appreciate Dr. Sandra Blome for critically reading this manuscript. Our special thanks to Ilay Titov for an excellent assistance in nucleotide sequencing and all technicians involved in the presented studies.

The present study was supported by Russian Academy of Agricultural Science (RAAS).

Alexander Malogolovkin is a research officer at SRI National Research Institute for Veterinary Virology and Microbiology Russian Academy of Agriculture Science (SRI NRIVVaM RAAS), Pokrov. His research focuses on molecular epidemiology and diagnostic of Classical and African swine fever.

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Alexander Malogolovkin is a research officer at SRI National Research Institute for Veterinary Virology and Microbiology Russian Academy of Agriculture Science (SRI NRIVVaM RAAS), Pokrov. His research focuses on molecular epidemiology and diagnostic of Classical and African swine fever.

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