Γρίπη ιπποειδών: απομόνωση, μοριακός χαρακτηρισμός και φυλογενετική ανάλυση στελεχών του ιού από τις επιζωοτίες 2003 και 2007 στην Ελλάδα

Abstract

Equine Influenza (EI) 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 Orthomyxoviridae. 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 Subtype 1 has not been isolated since 1977, and is presumed that has been replaced by the subtype 2, which is the causative agent of many recent outbreaks.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 regions that rarely report equine influenza outbreaks. The aim of the present study is isolation, molecular and phylogenetic analysis of the Greek strains, from outbreaks in 2003 and 2007. 30 nasal swabs from horses with fever and respiratory signs were used. Swabs were initially tested by a screening rapid test for Influenza A (Directigen) and then by diagnostic RT-PCR for the M gene, and the presence of influenza A virus was demonstrated. Isolation was succeeding in MDCK cell line, which was confirmed by both haemmaglutination with turkey RBCs and RT-PCR. RT-PCR with specific sets of primers for HAH3 and NAN8 was used for the characterization of the strains, as well. Five primer pairs were designed during this study in order to detect NP, PA, PB1, PB2 and NS genes of H3N8 viruses. Molecular analysis confirmed that the Greek isolates were members of H3N8 equine influenza viruses. HA sequences appeared to be more closely related to viruses isolated in Europe in early 1990s, but sequences of the other genes appeared to be more closely related to recent isolates of the last decade in Europe and Asia, suggesting that reassortment had occurred. Although, most studies up to date were limited to the HA characterization, our results Γξίπε Ηππνεηδψλ 10 suggest that sequencing of other genes, in addition to that encoding HA, is now being increasingly interesting for investigating the origin or pathogenicity of equine influenza viruses. These findings reinforce the ―frozen evolution‖ theory, which describes strains that show reduced amount of antigenic drift compared to the majority of circulating EIVs, and continue to circulate among equines. Thus, reassortment of co-circulation of genetically distinct strains may be threat to equine population. Further evidence that the horse may not be a dead-end host has arisen from the characterisation of two viruses isolated from pigs in 2005 and 2006 and dogs during 2003-2008. For first time we report isolation of H3N8 equine influenza virus in Greece. In addition, isolation, molecular and phylogenetic analysis of the reassorted strain as presented in this study is a useful tool to investigate evolution pathways of H3N8 equine influenza virus in order to control the outbreaks.