Burashev Y.D., Sultankulova K.T., Strochkov V.M., Sansyzbay A.R., Sandybayev N.T., Orynbayev M.B. Phylogenetic analysis of surface HA gene, of equine influenza A/equine/LKZ/09/2012 (H3N8) virus strains

Research Institute for Biological Safety Problems» Zhambulskaya oblast, Korday region, Gvardeyskyi

Аннотация

This paper presents the results of epizootological monitoring of equine influenza in the Republic of
Kazakhstan, which were organized the expeditions to South Kazakhstan, Zhambyl, Almaty, East Kazakhstan, Kostanai, Aktobe and West Kazakhstan regions. For laboratory investigations in different regions of
the Republic of Kazakhstan, 2404 samples were collected and delivered to RIBSP. The outbreak of equine
influenza virus (EIV) in Kazakhstan was confirmed by isolating the virus in chick embryos, sequencing the
surface hemagglutinin HA gene, and then analyzing the resulting nucleotide sequence at the BLAST base.
All investigated samples were taken from un-vaccinated horses with expressed clinical signs of respiratory
disease in 2007 and 2012 in southern and northern Kazakhstan. EIV H3N8 was isolated in chicken eggs
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.
HA sequences of the Kazakhstan isolates appeared to be more closely related to viruses isolated in early
2000 in Asia. 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 Kazakhstan EIV 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. The
topology of the maximum-likehood tree for both HA and NA sequences showed four clades corresponding
to the Pre-diverge, American, Eurasian and Florida lineage viruses. The Kazakhstan isolates were well supported within the Eurasian clade, as seen for the HA protein with bootstrap value 90% and 99% within the
Florida sublineage clade II for the HA protein. Our findings suggest that the Kazakhstan strains represent
an example of “evolution” and probably reassortment between genetically distinct co-circulated strains.
Key words: virus, equine influenza, PCR, sequencing, phylogenetic analysis.