Isolation and characterization of highly pathogenic avian influenza virus subtype H5N1 from donkeys

Abstract

Background: The highly pathogenic H5N1 is a major avian pathogen that crosses species barriers and seriously affects humans as well as some mammals. It mutates in an intensified manner and is considered a potential candidate for the possible next pandemic with all the catastrophic consequences.

Methods: Nasal swabs were collected from donkeys suffered from respiratory distress. The virus was isolated from the pooled nasal swabs in specific pathogen free embryonated chicken eggs (SPF-ECE). Reverse transcriptase polymerase chain reaction (RT-PCR) and sequencing of both haemagglutingin and neuraminidase were performed. H5 seroconversion was screened using haemagglutination inhibition (HI) assay on 105 donkey serum samples.

Results: We demonstrated that H5N1 jumped from poultry to another mammalian host; donkeys. Phylogenetic analysis showed that the virus clustered within the lineage of H5N1 from Egypt, closely related to 2009 isolates. It harboured few genetic changes compared to the closely related viruses from avian and humans. The neuraminidase lacks oseltamivir resistant mutations. Interestingly, HI screening for antibodies to H5 haemagglutinins in donkeys revealed high exposure rate.

Conclusions: These findings extend the host range of the H5N1 influenza virus, possess implications for influenza virus epidemiology and highlight the need for the systematic surveillance of H5N1 in animals in the vicinity of backyard poultry units especially in endemic areas.

Figure 1

Phylogenetic analyses of HA and NA of an equine H5N1 isolate sequence in comparison to Egyptian human and avian isolates. a, HA gene b, NA gene. Human isolates are shown in blue while avian ones are shown in black whereas equine isolate is shown in red. All sequences were obtained from GenBank. Trees were generated using Neighbour-Joining method. The robustness of individual nodes of the tree was assessed using a bootstrap of 1000 resamplings in per cent (70% and higher) are indicated at key nodes.

Figure 2

Deduced amino acid sequences of the HA protein of equine H5N1 isolate in comparison to closely related Egyptian HPAIV H5N1 isolates. Dots denote identical amino acids, which are given in one-letter code. Consensus sequences for N-glycosylation (NXS or NXT, except where X = P) are underlined. Boxed segments indicate the signal peptide and the polybasic proteolytic cleavage motif, respectively. Shaded letters denote potential sites responsible for receptor binding sites (H3 influenza numbering).

Figure 3

Deduced amino acid sequences of NA protein of equine H5N1 isolate in comparison to two recent closely related and other distant isolates that showed resistant to oseltamivir. Dots indicate residues identical amino acids. Underlined letters are N-glycosylation sites, shaded letters showed site of H274Y and N294S substitutions (H275Y and N295S in N1 influenza numbering).

Figure 4

Beni-Suef map, showed the distribution of H5 seropositive equine samples (donkeys). HI test was performed using local Egyptian antigen (A/chicken/Egypt/F6/2007), red box denotes the locality where the A/Egypt/equine/av1/2009 was isolated.