these include isolates from disease outbreaks in africa , europe , the caribbean , and , more recently , from the russian / trans - caucasian epizootic and attenuated variants . in brief ,
swine red bone marrow cell culture was used for asfv isolate amplification , and swine anti - asfv serum and erythrocytes were subsequently added to the culture .
asfv isolates for which the hemadsorption phenomenon was inhibited by serum belonging to the same group within serogroups 18 were clustered into a homologous serogroup .
only asfv hemadsorbing strains could be analyzed by this method , and some hemadsorbing asfv isolates could not be placed into existing serogroups because hai was not observed with available reference serum ( 8,9 ) .
asfv isolates from the depository at vniivvim were also classified by using a standard asfv genotyping protocol previously published by bastos et al .
, the variable part of the p72 ( b646l ) gene was amplified by conventional pcr , and the amplicons were directly sequenced by using a 3130xl genetic analyzer ( applied biosystems , foster city , ca , usa ) according to manufacturer s recommendations .
sequences determined at vniivvim were aligned with other publically available asfv sequences and analyzed by using minimum evolution ; a rooted tree was constructed with mega 5.0 software ( 14 ) and edited with figtree v1.4 ( http://tree.bio.ed.ac.uk/ ) ( figure 1 ) .
phylogenetic tree of african swine fever virus ( asfv ) isolates maintained in a collection at the national research institute for veterinary virology and microbiology in okrov , russia ; the variable part of b646l gene relative to the 22 known p72 genotypes ( labeled i - xxii ) was used for analysis .
the tree was reconstructed by using the minimum evolution method with 1,000 replicates .   *
the isolates were selected from the african swine fever virus ( asfv ) collection at the national research institute for veterinary virology and microbiology ( vniivvim ) in okrov , russia .
asfv genotypes are assigned according to p72 ( b646l ) nucleotide sequencing and phylogenetic tree reconstruction .
serogroups are defined on the basis of results of hemadsorption inhibition assay ( hai ) with reference serum ( serogroups 18 ) .
democratic republic of congo ;  , not applicable ; nk , not known ; nd , not defined . 
if a country name changed since the virus was isolated , the current name of the country is shown in parentheses . 
 isolates bartlett , magadi , and davis could not be placed into an existing serogroup because no hai was observed with available reference serum , so exact serogroups for these isolates are not defined . 
isolate spenser does not belong to any of the 22 known genotypes and remains untyped . newly identified asfv genotypes and known serogroups were mapped together so their geographic distribution in africa and europe ( including the european part of the russian federation ) could be visualized .
the results ( figure 2 ) show that genotypic and serogroup diversity are greatest in a relatively limited area , mainly in southeastern africa .
in contrast , non - asfv  endemic countries , where asf outbreaks were caused by asfv of a single genotype , exhibited low or no serogroup diversity .
world distribution of african swine fever virus ( asfv ) isolates maintained in a collection at the national research institute for veterinary virology and microbiology in okrov , russia .
results of p72 genotyping and hemadsorption inhibition assay of asfv isolates are summarized on the map .
genotype ii of asfv isolates from lithuania , latvia , estonia , poland , and belarus was identified by cisa - inia ( animal health research center ; european union reference laboratory for african swine fever ) .
asfv isolate o-77 , which was isolated in 1977 from odessa , ukraine ( at the time , part of the union of soviet socialist republics ) , was used in this study . on the basis of cisa - inia results , currently circulating isolates in ukraine belong to genotype ii .
the oval with a 1 inside indicates switzerland ; the oval with a 2 inside indicates so tome and principe .
country names are presented as 2-letter country codes as designated by the international organization for standardization country codes ( iso 3166 , http://www.iso.org/iso/country_codes.htm ) .
single genotype clades of asfv were observed to contain viruses of multiple serogroups ( table ) .
for example , asfv isolates belonging to serogroups 1 , 2 , and 4 were specifically clustered within genotype i , and did not group with other genotypes .
we also found several serogroups of asf viruses within genotype x. the asfv isolates tsp80 ( serogroup 5 ) and ts-7 ( serogroup 6 ) were subsequently isolated from 1 field sample derived from a naturally infected pig in tanzania .
of note , 1 serogroup 2 isolate ( spenser ) demonstrated a novel genotype within the p72 phylogenetic tree and relative to other serogroup 2 viruses ( figure 1 ) , indicating that the p72 genotype , in addition to lacking serotype resolution , has potential to be incongruous relative to serogroup .
together , these data indicate that the antigenic heterogeneity of asfv strains is not fully captured by using the standard genotyping approach .
the virus elements responsible for protective and cross - protective immune responses are not well known .
given the structural and genetic complexity of asfv , it is likely that genes encoding different antigens will be more suited for virus typing .
substantial genetic variability can exist between strains and predominate in specific genomic regions , and it is these regions that may provide improved targets for genotyping .
our findings support that of a previous study that showed that geographic areas with asfv of high genotypic and serotypic diversity are located in countries where multiple mechanisms of asf transmission ( mixed sylvatic and domestic cycle ) are established ( 11 ) .
hai serology provides a measure of asfv typing that , compared with p72 genotyping , better discriminates biologically pertinent phenotypes .
viruses belonging to one p72 genotype may be serotypically heterogeneous : strains that are closely related genetically , even from a single isolate , may have different phenotypes and form homologous serogroups .
our assessment of asfv genotyping relating to hai serotyping shows the serologic diversity within a p72 genotype .
our results highlight the potential for using serogroup classification to understand issues of homologous cross - protection among asfv isolates and virus determinants that influence disease emergence .
the key finding from our study is that p72 asfv phylogenetic analysis fails to accurately define asfv hai serogroups .
thus , conventional asfv genotyping can not discriminate between viruses of different virulence or predict the efficacy of a specific asfv vaccine .
this finding also suggests that for vaccination - based control of asf , it may be more important to determine serogroups rather than genotypes of asfv isolates .
 african swine fever virus ( asfv ) causes highly lethal hemorrhagic disease among pigs , and asfv s extreme antigenic diversity hinders vaccine development . 
 we show that p72 asfv phylogenetic analysis does not accurately define asfv hemadsorption inhibition assay serogroups . 
 thus , conventional asfv genotyping can not discriminate between viruses of different virulence or predict efficacy of a specific asfv vaccine . 
The Study
Conclusions