An outbreak of equine influenza (EI) was reported in Algeria between May and July, 2011. The outbreak started in Tiaret, in west province of Algeria, and spread to the other parts of the country affecting almost 900 horses in many provinces. The population studied was composed of 325 horses from different groups of age. Clinical sign expression was age dependent. Indeed, a morbidity rate of 14.9% was observed in horses under 15 months old and a rate of 4.95% in horses over 8 years old. Interestingly, the morbidity rate raised sharply to reach 100% in horses aged between 18 months and 7 years. The virus (H3N8) was detected in nasopharyngeal swabs (n = 11) from non-vaccinated horses using a qRT-PCR targeting a portion of the gene encoding the matrix protein (M). The virus isolates were identified as H3N8 by sequencing the haemagglutinin (HA) and neuraminidase (NA) genes and were named from A/equine/Tiaret/1/2011 to A/equine/Tiaret/10/2011. Alignment of HA1 amino acid sequence confirmed that viruses belong to Clade 2 of the Florida sublineage in the American lineage. Moreover, they are closely related to A/equine/Yokohama/aq13/2010, A/equine/Eyragues/1/2010, A/equine/Bokel/2011 and A/equine/Lichtenfeld/2012. Our data indicate that this strain was also circulating in the European horse population in 2010, 2011 and 2012.

SummaryIn 2009, a major outbreak of equine infectious anaemia (EIA) was reported in the south-east of France. This outbreak affected three premises located in the Var region where the index case, a 10-year-old mare that exhibited clinical signs consistent with EIA, occurred at a riding school. Overall, more than 250 horses were tested for EIAV (equine infectious anaemia virus) antibodies, using agar gel immunodiffusion test, and 16 horses were positive in three different holdings. Epidemiological survey confirmed that the three premises were related through the purchase/sale of horses and the use of shared or nearby pastures. Molecular characterization of viruses was performed by sequencing the full gag gene sequence (1,400 bp) of the proviral DNAs retrieved from the spleen of infected animals collected post-mortem. Phylogenetic analysis confirmed epidemiological data from the field, as viruses isolated from the three premises were clustering together suggesting a common origin whereas some premises were 50 km apart. Moreover, viruses characterized during this outbreak are different from European strains described so far, underlying the high genetic diversity of EIAV in Europe. K E Y W O R D Sepidemiology, equine infectious anaemia virus, France, horse, phylogeny

BackgroundEquine arteritis virus (EAV) is responsible for infections in equids. It can spread easily within the horse population and has a major impact on the horse breeding industry. No EAV outbreak has ever been reported in Serbia. To determine whether EAV is nonetheless circulating there, especially in the Vojvodina region, 340 horse serum samples were subjected to serology testing to detect EAV antibodies. In parallel, semen samples from three seropositive stallions were collected to evaluate their EAV status, using RT-qPCR and virus isolation on cell culture.ResultsHorse sera with EAV antibodies represented 15.88% (54/340) of the tested samples, 83.23% (283/340) being negative, and just three samples (0.89%) being uninterpretable due to cytotoxicity. Only 7.2% (10/138) of horses kept by private owners on their own property were seropositive for EAV, whereas 21.8% (44/202) of horses kept on stud farms had EAV antibodies. Phylogenetic analysis showed that the Serbian EAV isolate was most closely related to isolates from the neighbouring Hungary.ConclusionsEAV is circulating in the Serbian horse population, especially among the breeding population certainly due to the use of EAV shedder stallions since there is no surveillance programme in Serbia and only limited checks on racehorses. Moreover, phylogenetic analysis indicates that the EAV isolated from a Lipizzaner stallion in Serbia is closely related to isolates from Hungary, and together form a new cluster.

fThis study shows that an unbiased amplification method applied to equine arteritis virus RNA significantly improves the sensitivity of the real-time reverse transcription-quantitative PCR (RT-qPCR) recommended by the World Organization for Animal Health. Twelve viral RNAs amplified using this method were hybridized on a high-density resequencing microarray for effective viral characterization. Equine arteritis virus (EAV), the causative agent of equine viral arteritis (EVA), is a member of the Arteriviridae family. Its genome is made up of a single positive-strand RNA molecule (1, 2). EAV infects Equidae and may be transmitted through the respiratory and venereal routes. EAV can persist in the reproductive tract of stallions only (3). Following the primary infection, up to 70% of stallions can be persistently infected and shed the virus in their semen, sometimes for life. These "shedder" stallions spread the virus in the horse population during breeding or when their semen is used for artificial insemination (4). The financial consequences are potentially huge, since shedder stallions lose their value. Moreover, infection may cause abortion in pregnant mares and the death of young foals (5). The World Organization for Animal Health (OIE) prescribes viral isolation (VI) on cell culture to detect EAV for international trade (6). However, a recent study showed that under certain conditions, the real-time reverse transcription-quantitative PCR (RT-qPCR) assay developed by Balasuriya et al. in 2002 (7) is as sensitive as VI for detecting EAV in semen (8). Therefore, RT-qPCR targeting open reading frame 7 (ORF7) of the EAV genome, encoding the viral nucleoprotein, is increasingly used for diagnosis.The main challenge to EAV surveillance is detecting EAV in the semen of shedder stallions and the organs of aborted fetuses to prevent costly outbreaks. In particular, when the viral load is very low and thus the amount of viral nucleotides targeted is limited (9). The aim of our study was to increase the sensitivity of the OIE-recommended RT-qPCR method (6) by combining it with an unbiased amplification method using the Phi29 polymerase coupled to a high-density resequencing microarray (RMA) to genotype the viruses detected.Sixty different samples were used in this study. Of the 48 EAVpositive samples, 31 were from semen (Table 1), 12 were from virus isolation cell culture supernatants, and 5 were tissue samples from the lungs, spleen, or liver of 1 aborted foetus, 3 young foals, and 1 adult. Viral RNA was extracted from semen, cell culture supernatant, or 1 g of tissue sample (previously homogenized in 5 ml of cell medium) with the QIAmp viral RNA minikit (Qiagen, Valencia, CA) following the manufacturer's instructions.In order to evaluate the gain in sensitivity of the newly developed method, extracted and purified viral RNA was amplified in triplicate using three different protocols (Fig. 1). First, RNA samples were amplified using the one-step RT-qPCR (assay 1) method recommended by the OIE (7). In parallel, the extra...

Background Equine infectious anemia (EIA) is a viral disease, caused by the Equine Infectious Anemia virus (EIAV) belonging to the Retroviridae family, genus Lentivirus. Horses (or equids) infected with EIAV are lifelong carriers and they remain contagious for other horses even in the absence of clinical signs. So far, EIAV infection has been reported among horses in North and South America, France, Germany, Italy, Hungary and Romania, with no publication regarding the presence of EIAV in horses in Serbia. To determine the circulation of EIAV among, approximately, the 5000 horses of the Vojvodina region, northern part of Serbia, 316 serum undergone serological testing for EIA. Then, identification and full genome sequencing using next generation sequencing was performed from one EIA positive horse. Results the 316 sera were tested with 3 different commercial agar gel immunodiffusion (AGID) tests and two different commercial enzyme-linked immunosorbent assay (ELISA). With the three AGID kits, 311 (98.4%) among the 316 tested sera were negative and only five (1.6%) sera were positive for EIA. Some discrepancies were seen for the two ELISA kits tested since one exhibited the same results as AGID test and the second gave 295 sera with negative results, five with a positive result and 16 with doubtful outcome. Phylogenetic analysis performed using the full genome sequence showed that EIAV characterized from a horse in Serbia is different from those identify so fare around the world and form a distinct and separate group together with another EIAV strain. Conclusions This study demonstrate for the first time that EIAV is circulating at a low level in the horse population from the Northern part of Serbia. Interestingly, phylogenetic data indicates that this EIAV from the western Balkan region of Europe belongs to a new cluster.