BackgroundFor first time in Greece equine influenza virus infection was confirmed, by isolation and molecular analysis, as the cause of clinical respiratory disease among unvaccinated horses during 2003 and 2007 outbreaks.MethodsEquine influenza virus (EIV) H3N8 was isolated in MDCK cells from 30 nasal swabs from horses with acute respiratory disease, which were tested positive by Directigen Flu A. Isolation was confirmed by haemagglutination assay and RT-PCR assay of the M, HA and NA gene.ResultsHA sequences of the Greek isolates appeared to be more closely related to viruses isolated in early 1990s in Europe. These results suggested that viruses with fewer changes than those on the main evolutionary lineage may continue to circulate. On the other hand, analysis of deduced NA amino acid sequences were more closely related to viruses isolated in outbreaks in Europe and Asia during 2003-2007. Phylogenetic analysis characterized the Greek isolates as a member of the Eurasian lineage by the haemagglutinin (HA) protein alignment, but appeared to be a member of the Florida sublineage clade 2 by the neuraminidase (NA) protein sequence suggesting that reassortment might be a possible explanation.ConclusionOur findings suggest that the Greek strains represent an example of "frozen evolution" and probably reassortment between genetically distinct co-circulated strains. Therefore expanding current equine influenza surveillance efforts is a necessity.

To amplify the NS, NP, PB1, PB2 and PA internal genes of two equine H3N8 influenza A viruses isolated in Greece in 2003 and 2007 five primer pairs were designed. The derived sequences were analysed from a phylogenetic point of view and compared with the evolutionary patters of the HA and NA proteins. Comparison of nucleotide sequences of the five internal genes of the Greek strains showed high similarity (99.3% - 99.7%) to strains isolated from outbreaks in Europe and Asia during 2002-2008. A total of 11 amino acid substitutions of the surface protein NA and the RNP complex proteins were identified in the Greek strains compared to those of progenitor viruses circulating up to 2003. These substitutions were repeated in Chinese and Mongolian isolates from outbreaks in 2007-2008. Notably NS1 protein did not acquired amino acid substitutions and moreover, a stop codon introduced at position 220 was stably maintained in the Greek strains. Phylogenetic trees of the five internal genes did not show the same separation in clades. Greek strains classified them into the American sublineage (as for the PA) Florida clade II (as for the NP, NS1 and PB1) and among Chinese strains of 2007-2008 outbreaks (as for the PB2). Additionally, evolutionary profiles of these internal proteins, except PB2, indicated a parallel evolution fashion to the HA protein, suggesting the possible occurrence of genetic reassortment between H3N8 viruses of district evolutionary lineages. In conclusion, phylogenetic analysis of the internal genes reported in this study could establish a candidate framework for future scientific communications on the phylogenetic diversity and evolution of the equine influenza viruses

Equine Influenza (ΕΙ) is an acute, highly contagious, respiratory disease of equine. The causative agent of EI infections is a type A influenza virus, classified into the family Orthomyxovirìdae. Up to today two subtypes of EI are known, subtype 1 (H7N7) and subtype 2 (H3N8). Subtype 1 has not been isolated since 1977 and is presumed that has been replaced by the subtype 2, which is the causative agent of many recent outbreaks. Antigenic drift of H3N8 viruses resulted in the divergence of strains into two distinct evolutionary lineages, which co-circulate. The high morbidity of equine influenza disease was demonstrated in all resent widespread outbreaks all over the world. On the other hand, the mortality rate of influenza disease in equids is generally low, unless secondary bacterial infections occurred. Devastating economic loss of the disease in breeding and race animals reinforced the importance of vaccination. Despite the extensive use of vaccines, outbreaks of equine influenza continue to occur. In 2003 there were widespread outbreaks of equine influenza among un vaccinates and regularly vaccinated horses in Europe and later all over the world, even in regions that rarely report equine influenza outbreaks. However, studies have shown that vaccination does not prevent transmission and on the other hand multiple booster doses could result to paralysis of the immune system. Furthermore, all these developments including transmission to swine and dogs, shows the unpredictable evolutionary pathways the equine influenza virus follows. In conclusion, influenza surveillance and research should go on and provide useful tools to better evaluate when vaccine strains should be updated.