A proposed update of African swine fever virus (genotype II) subgenotyping based on the central variable region (CVR) of Russian isolates.

African swine fever virus (ASFV) isolates are grouped and tracked through analysis of their central variable region (CVR) sequences. In this study, sequences of 70 ASFV isolates collected from different regions of Russia between 2018 and 2022 were analyzed. The analysis based on the CVR sequences indicated that the isolates belonged to three distinct groups. Group 1 shared 100% sequence identity to the isolate Georgia 2007/1. Group 5 had a C > A single-nucleotide polymorphism (SNP) at position 601, while group 13 is new and unique to the Far East of Russia, with five isolates from the Amur, Khabarovsk, and Primorsky regions. These findings demonstrate a new approach to phylogenomics and cladistics of ASFV isolates within genotype II on the basis of the CVR.

Novel Protocol for the Preparation of Porcine Bone Marrow Primary Cell Culture for African Swine Fever Virus Isolation.

Isolation of African swine fever virus (ASFV) is a critical step towards the identification, titration, characterization, and even modification of the virus. Therefore, it is important to identify a suitable cell line that supports the efficient replication of ASFV for these purposes. This should be achieved even when starting with a low virus load, as in the case of isolating the virus from field samples. This article presents a detailed protocol on the preparation of porcine bone marrow primary (PBMP) cell culture, which has a high sensitivity towards ASFV, resulting in high viral yields with a minimal risk of bacterial contamination.

A Guide to Molecular Characterization of Genotype II African Swine Fever Virus: Essential and Alternative Genome Markers.

African swine fever is a contagious viral disease that has been spreading through Europe and Asia since its initial report from Georgia in 2007. Due to the large genome size of the causative agent, the African swine fever virus (ASFV), the molecular epidemiology, and virus evolution are analyzed by employing different markers. Most of these markers originate from single nucleotide polymorphisms or disparities in the copy number of tandem repeat sequences observed during the comparisons of full genome sequences produced from ASFVs isolated during different outbreaks. Therefore, consistent complete genome sequencing and comparative analysis of the sequence data are important to add innovative genomic markers that contribute to the delineation of ASFV phylogeny and molecular epidemiology during active circulation in the field. In this study, the molecular markers currently employed to assess the genotype II ASFVs circulating in Europe and Asia have been outlined. The application of each of these markers to differentiate between ASFVs from related outbreaks is described to implement a guideline to their suitability for analyzing new outbreaks. These markers do not signify the complete repertoire of genomic differences between ASFVs, but will be beneficial when analyzing the first outbreaks in a new region or a large number of samples. Furthermore, new markers must be determined via complete genome sequence analyses for enabling in-depth insights into the molecular epidemiology of ASFV.

Genetic Characterization of the Central Variable Region in African Swine Fever Virus Isolates in the Russian Federation from 2013 to 2017.

African swine fever virus (ASFV), classified as genotype II, was introduced into Georgia in 2007, and from there, it spread quickly and extensively across the Caucasus to Russia, Europe and Asia. The molecular epidemiology and evolution of these isolates are predominantly investigated by means of phylogenetic analysis based on complete genome sequences. Since this is a costly and time-consuming endeavor, short genomic regions containing informative polymorphisms are pursued and utilized instead. In this study, sequences of the central variable region (CVR) located within the B602L gene were determined for 55 ASFV isolates submitted from 526 active African swine fever (ASF) outbreaks occurring in 23 different regions across the Russian Federation (RF) between 2013 and 2017. The new sequences were compared to previously published data available from Genbank, representing isolates from Europe and Asia. The sequences clustered into six distinct groups. Isolates from Estonia clustered into groups 3 and 4, whilst sequences from the RF were divided into the remaining four groups. Two of these groups (5 and 6) exclusively contained isolates from the RF, while group 2 included isolates from Russia as well as Chechnya, Georgia, Armenia, Azerbaijan and Ukraine. In contrast, group 1 was the largest, containing sequences from the RF, Europe and Asia, and was represented by the sequence from the first isolate in Georgia in 2007. Based on these results, it is recommended that the CVR sequences contain significant informative polymorphisms to be used as a marker for investigating the epidemiology and spread of genotype II ASFVs circulating in the RF, Europe and Asia.

Whole-genome sequencing of African swine fever virus from wild boars in the Kaliningrad region reveals unique and distinguishing genomic mutations.

Introduction: Since the first report of outbreaks of African swine fever (ASF) in Georgia in 2007, the disease has expanded into Europe, Russia, and Asia, spreading rapidly via contact with infected animals including domestic pigs and wild boars. The vast expansion of this Genotype II African swine fever virus (ASFV) across wide-ranging territories and hosts inevitably led to the acquisition of novel mutations. These mutations could be used to track the molecular epidemiology of ASFV, provided that they are unique to strains restricted within a certain area. Whilst whole-genome sequencing remains the gold standard for examining evolutionary changes, sequencing of a single locus with significant variation and resolution power could be used as a rapid and cost-effective alternative to characterize multiple isolates from a single or related outbreak. Material and methods: ASFVs obtained during active ASF outbreaks in the Russian region of Kaliningrad between 2017 and 2019 were examined. Since all of the viruses belonged to Genotype II and no clear differentiation based on central variable region (CVR) sequencing was observed, the whole-genome sequences of nine ASFV isolates from this region were determined. To obtain insights into the molecular evolution of these isolates, their sequences were compared to isolates from Europe, Asia, and Africa. Results: Phylogenetic analysis based on the whole-genome sequences clustered the new isolates as a sister lineage to isolates from Poland and Germany. This suggests a possible shared origin followed by the addition of novel mutations restricted to isolates from this region. This status as a sister lineage was mirrored when analyzing polymorphisms in MGF-505-5R and MGF-110-7L, whilst a polymorphism unique to sequences from Kaliningrad was identified at locus K145R. This newly identified mutation was able to distinguish the isolates obtained from Kaliningrad with sequences of Genotype II ASFVs available on GenBank. Discussion: The findings of this study suggest that ASFVs circulating in Kaliningrad have recently obtained this mutation providing an additional marker to the mutations previously described.

Comparative Analysis of Full Genome Sequences of African Swine Fever Virus Isolates Taken from Wild Boars in Russia in 2019.

In this study, we report on the full genome phylogenetic analysis of four ASFV isolates obtained from wild boars in Russia. These samples originated from two eastern and two western regions of Russia in 2019. Phylogenetic analysis indicated that the isolates were assigned to genotype II and grouped according to their geographical origins. The two eastern isolates shared 99.99% sequence identity with isolates from China, Poland, Belgium, and Moldova, whereas the western isolates had 99.98% sequence identity with isolates from Lithuania and the original Georgia 2007 isolate. Based on the full genome phylogenies, we identified three single locus targets, MGF-360-10L, MGF-505-9R, and I267L, that yielded the same resolving power as the full genomes. The ease of alignment and a high level of variation make these targets a suitable selection as additional molecular markers in future ASFV phylogenetic practices.

A Long-Term Study of the Biological Properties of ASF Virus Isolates Originating from Various Regions of the Russian Federation in 2013-2018.

Biological properties of the African swine fever (ASF) virus isolates originating from various regions of the Russian Federation (2013-2018) were studied in a series of experimental infections. Comparative analysis allowed us to establish the differences in the key characteristics of the infection, such us the duration of the incubation periods, disease, and the onset of death. The incubation period averaged 4.1 days, varying from 1 to 13 days. An average duration of the disease was 6.3 days and varied from 0 to 18 days. Overall case fatality was 94.5%, and antibodies were detected only in 19.3% of the animals. The biological properties of isolates Odintsovo 02/14 and Lipetsk 12/16 were significantly different from others. For this two, the presence of antibodies to the virus was detected in 71.4% and 75% of animals respectively and mortality levels were of 87.5% and 50%.