Phylogenetic and molecular characterization of equine H3N8 influenza viruses from Greece (2003 and 2007): evidence for reassortment between evolutionary lineages

Abstract

Background: For 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.

Methods: Equine 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.

Results: HA 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.

Conclusion: Our 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.

Figure 1

CPE of equine influenza virus: MDCK cells incubated in the absence [control (a)] and presence of 100 μl swab extract for 24 h (b), 48 h(c) and 72 h (d). (Invertoscope, 10× magnification).

Figure 2

Viability of MDCK cell cultures 72 h post infection at each passage expressed as a percentage of live cells in comparison to the control cell culture.

Figure 3

Evolutionary relationships of H3 protein: The evolutionary history was inferred using the NJ method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method and are in the units of the number of base substitutions per site. Phylogenetic analyses were conducted in MEGA4.

Figure 4

Evolutionary relationships of N8 protein: The evolutionary history was inferred using the NJ method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method and are in the units of the number of base substitutions per site. Phylogenetic analyses were conducted in MEGA4.