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The application of molecular diagnostic techniques along with nucleotide sequence determination to permit contemporary phylogenetic analysis of European field isolates of equine infectious anemia virus (EIAV) has not been widely reported. As a result, of extensive testing instigated following the 2006 outbreak of equine infectious anemia in Italy, 24 farms with a history of exposure to this disease were included in this study. New PCR-based methods were developed, which, especially in the case of DNA preparations from peripheral blood cells, showed excellent correlation with OIE-approved agar gel immunodiffusion (AGID) tests for identifying EIAV-infected animals. In contrast, the OIE-recommended oligonucleotide primers for EIAV failed to react with any of the Italian isolates. Similar results were also obtained with samples from four Romanian farms. In addition, for the first time complete characterization of gag genes from five Italian isolates and one Romanian isolate has been achieved, along with acquisition of extensive sequence information (86% of the total gag gene) from four additional EIAV isolates (one Italian and three Romanian). Furthermore, in another 23 cases we accomplished partial characterization of gag gene sequences in the region encoding the viral matrix protein. Analysis of this information suggested that most Italian isolates were geographically restricted, somewhat reminiscent of the "clades" described for human immunodeficiency virus type 1 (HIV-1). Collectively this represents the most comprehensive genetic study of European EIAV isolates conducted to date.

Although equine infectious anemia (EIA) was described more than 150 years ago, complete genomic sequences have only been obtained from two field strains of EIA virus (EIAV), EIAV Wyoming and EIAV Liaoning . In 2011, EIA was detected within the distinctive feral Misaki horse population that inhabits the Toi-Cape area of southern Japan. Complete proviral sequences comprising a novel field strain were amplified directly from peripheral blood of one of these EIAVinfected horses and characterized by nucleotide sequencing. The complete provirus of Miyazaki2011-A strain is 8208 bp in length with an overall genomic organization typical of EIAV. However, this field isolate possesses just 77.2 and 78.7 % nucleotide sequence identity with the EIAV Wyoming and EIAV Liaoning strains, respectively, while similarity plot analysis suggested all three strains arose independently. Furthermore, phylogenetic studies using sequences obtained from all EIAV-infected Misaki horses against known viral strains strongly suggests these Japanese isolates comprise a separate monophyletic group.

Equine infectious anemia virus (EIAV), the causative agent of equine infectious anaemia (EIA), possesses the least-complex genomic organization of any known extant lentivirus. Despite this relative genetic simplicity, all of the complete genomic sequences published to date are derived from just two viruses, namely the North American EIAV WYOMING (EIAV WY) and Chinese EIAV LIAONING (EIAV LIA) strains. In 2006, an outbreak of EIA occurred in Ireland, apparently as a result of the importation of contaminated horse plasma from Italy and subsequent iatrogenic transmission to foals. This EIA outbreak was characterized by cases of severe, sometimes fatal, disease. To begin to understand the molecular mechanisms underlying this pathogenic phenotype, complete proviral genomic sequences in the form of 12 overlapping PCR-generated fragments were obtained from four of the EIAV-infected animals, including two of the index cases. Sequence analysis of multiple molecular clones produced from each fragment demonstrated the extent of diversity within individual viral genes and permitted construction of consensus wholegenome sequences for each of the four viral isolates. In addition, complete env gene sequences were obtained from 11 animals with differing clinical profiles, despite exposure to a common EIAV source. Although the overall genomic organization of the Irish EIAV isolates was typical of that seen in all other strains, the European viruses possessed ¡80 % nucleotide sequence identity with either EIAV WY or EIAV LIA. Furthermore, phylogenetic analysis suggested that the Irish EIAV isolates developed independently of the North American and Chinese viruses and that they constitute a separate monophyletic group. The GenBank/EMBL/DDBJ accession numbers for the sequences of EIAV IRE described in this paper are JX480631-JX480841. Six supplementary figures and a table are available with the online version of this paper.

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