Biological properties of African swine fever virus Odintsovo 02/14 isolate and its genome analysis
Abstract
We performed analysis of the biological properties of African swine fever virus (ASF) isolate Odintsovo 02/14.
Domestic pigs were inoculated with 50 (low) or 5000 (high) hemadsorbing doses (HAD) of the virus via intranasal (IN) or intramuscular (IM) routes, t o investigate the pathogenesis of ASF virus Odintsovo 02/14 isolate. Our results indicated that filtered 10% spleen suspension of ASFV isolate Odintsovo 02/14 induced an acute disease in pigs, resulting in 100% mortality rate. For cultural viral suspension (3rd passage), produced in a PBM cells mortality rate was 85.7%.
We also present an analysis of the complete genome of African swine fever virus (ASF) Odintsovo 02/14 isolate. It is 189 333 nucleotide long and contains more than 160 open reading frames ( ORFs). Complete nucleotide sequence of the genome of Odintsovo 02/14 isolate was obtained using pyrosequencing method and used to determine differences between the nucleotide sequences in the genomes of Odintsovo 02/14 and Georgia 01/2007. The genome of AS F virus Odintsovo 02/14 contains substitutions, insertions and deletions in genes encoding structural, membrane, and regulatory proteins, DNA reparation enzymes, host immune response evasion proteins, and MGF genes.
The intergenic region I73R/I329L of Odi ntsovo 02/14 isolate contains 10 -nucleotide long tandem repeat sequence, missing in Georgia 01/2007.
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Introduction
In 2007 ASFV was detected in the Caucasus region, first in Georgia and subsequently in Armenia, Azerbaijan, and many regions of Russia, including regions that border other countries in Europe and Asia. E pizootiological, pathogenic and immunogenic characteristics of the vir us, as well as lack of an effective vaccine make ASFV a global threat to pig industry.
According to the s equencing of PCR products and the hemadsorption inhibition reaction, this pathogen has been assigned to II genotype and seroimmunotype VIII [3, 33].
Russian isolates of ASFV induce an acute form of the disease, characterized by hemorrhagic syndrome and multisystem damage, high level of viremia and 100% mortality in domestic pigs.
The unique morphology of ASFV causes its high persistence and longevity in environment.
The causative agent of ASF is a large DNA -containing enveloped virus with a virion ~200 nm in diameter. [13]. ASFV virus belongings to the Asfarviridae family. Its particles consist of a nucleoprotein core ~80 nm in diameter. The genome conta ining electron -dense nucleoid enclosed in a lipid membrane and covered by an icosahedral capsid, made of 1892 -2172 capsomeres. The virus acquires an external envelope during the budding process, thus forming extracellular virions about ~200 nm in diameter [2, 19].
Viral dsDNA is 170 -193 kbp long, and it contains 150 -167 ORFs. Generally, changes in the genome length are the result of variation in the number of tandem repeats within a certain gene or intergenic region [1, 27].
ASFV isolate Georgia 2007/1 has a genome of 189 344 bp and contains 166 ORFs. Chapman D. et al. demonstrated the maximum homology of Georgia 2007/1 isolate and Mkuzi 1979 isolate by phylogenetic analysis of the nucleotide sequences of 125 ORFs. In addition, they revealed some differences in the grouping of genes, which may be a result of recombination occurring during the evolution of virus [9].
Page | 27 High variability in groups of genes responsible for the virulence expression and interaction with the host requires constant monitoring of change s in a viral genome [19]. Comparison of the genomes of ASFV isolates of varying virulence can help in determining virulence responsible genes and develop a strategy for making an effective vaccine.
The first complete genome sequence for ASFV was obtained by Yanez et al. in 1995 for Ba71V strain, adapted to Vero cells [31]. Recently, sequences from strains of different genotypes became available for comparative analyses [9, 10, 12].
Currently, next generation sequencing (NGS) is the best method for viral ge nome studying. A single NGS procedure allows determining the entire nucleotide sequence even for large viruses, like Poxviruses, ASF, Herpes, etc. [24]. It is a key tool for detection of variable and marker regions in a genome, which are required for epide miological studies and analysis of the distribution of viral subpopulations [30]. It also allows sequencing of nucleotides at the ends of the viral genome, which cannot be determined using methods based on PCR products sequencing analysis [32].
Comparison of genomic changes and changes in the biological properties of the ASFV will allow us to find out sources of the genome variability; to determine the hallmarks for differentiating isolates by pathogenicity; to track the evolution stages o f the ASFV and to identify possible pathogen introduction sources.
Therefore, the purpose of this study was analysis of the biological properties of Odintsovo 02/14 isolate and its complete genome sequencing.
Conclusion
Cultural properties of Odintsovo 02/14 isolates are significantly different amongst other Russian isolates 2007 -2014. It manifests a decreased amount of RBC’s, attached to an infected cell , which according to L.Dixon is related to attenuation of the virus.
Mortality in infected animals reached 100% for the samples of spleen suspension and 8 5.7% for the 3rd PBM cells passage samples, when we used 50 HAD dose for the challenge, which may indicate the heterogeneity of the viral population of this isolate and presence of viral variants with different pathogenicity.
Page | 36 A comparative analysis of the nucleotide sequences of Odintsovo 02/14 and Georgia 2007/1 ASFV isolates identified differences in genes responsible for virus attachment, cell penetration and host immune system evasion genes. Changes were found in nucleotide sequences of genes I196L, NP419L, ASFV_G_ACD_00070, M448R, MGF_360 -1L, MGF_360 -2L, MGF_110 -1L, MGF_505 -9R and GGAATATATA tandem repeat sequence in the intergenic region I73R/I329L, which is absent in the ASF isolates collected before 2012.
The analysis highlighted group of genes of Odintsovo 02/14 isolate, where changes are undoubted (37.5% of the total variations, where 23.6% are mononucleotide deletions/insertions). These include nucleotide replacement T/C in a NP419L gene encoding the ATP -dependent viral DNA -ligase complex, included in the viral reparation. It is characterized by an extremely high tolera nce for errors in the presence of crosslinking chains by crosslinking nick breaks [15].
Due to variations in a number of tandem repeats in a nucleotide sequence of the ASFV I196L gene it was used for phylogenetic analysis of ASFV isolates [15]. These chang es may have led to an increased inc ubation period in pigs infected with Odintsovo 02/14. The detected changes in the gene sequences require further study.
