Genetic Analysis of the Neuraminidase (NA) Gene of Equine Influenza Virus (H3N8) from Epizootic of 2008–2009 in India

Abstract

The neuraminidase (NA) gene sequences of four Indian equine influenza viruses (EIVs) isolated from epizootic in 2008 and 2009 were analyzed. The phylogenetic relationship and selection pressure of NA genes were established in comparison to other EIVs circulating worldwide along with the domains and motifs of the encoded protein to find out the significance of mutational changes. Among Indian isolates, two amino acid (aa) changes each in Mysore/12/08 (Asn67Tyr & Asp396Gly), Gopeshwar/1/09 (Ile49Val & Asp396Gly), and Uttarkashi/1/09 (Ile49Val & Asp396Gly) isolates were observed in respect to Jammu-Katra/06/08 isolate. Amino acid (aa) sequence analysis also revealed five consistent aa residue changes viz, Gly/Arg40Glu, Tyr66His, Val191Ile, Val209Ile and Asp235Asn in Asian including Indian isolates, Spain/07 and Spain/09 isolates in comparison to other EIVs circulating worldwide. The topology of the phylogenetic tree revealed that the Indian, Chinese, Mongolian and Kazakhstan isolates together formed a subgroup with Yokohama/10 isolate. Spain/07 & Spain/09 isolates showed closest clustering with Asian isolates. This indicates that non-synonymous mutations in Asian isolates with temporal pattern originating from Spain/07, led to the subgroup of the Asian isolates within Florida clade 2 sublineage. The analysis of the predicted secondary structure has not shown any significant difference in the NA proteins of all Indian isolates. Fixed-effects likelihood (FEL) analysis of the selection pressure revealed three codons (43, 355 & 434) under positive selection pressure. The overall evolutionary changes (ω value) of 3.4 indicates NA gene to be under strong selection pressure. Further, seven putative N-glycosylation sites were observed in the NA protein. The mapping of specific aa changes, their mutational and functional analysis need to be carried out to ascertain their role in pathogenecity of the virus.

Introduction

Equine influenza viruses (EIVs) are the etiologic agents of severe respiratory disease in horses. Antigenically, they are classified as influenza type A viruses of the family Orthomyxoviridae. Two subtypes of EIVs-H3N8 and H7N7 cause infection in horses. In recent times, H3N8 viruses are circulating in horse where as the H7N7 subtype has not been isolated from horses for over 30 years and is presumed to be out of infections cycle at present [14]. Recently, H3N8 virus jumped the species barrier to infect dogs [4, 8, 16] and pigs [25]. Since the first isolation of H3N8 virus (Miami/63), these viruses have diversified into various lineages viz, Predivergent, Eurasian, and American- which on the basis of HA gene analysis has further diversified into Kentucky, South America, and Florida sublineage (clade 1 & 2) [3, 5, 9]. Neuraminidase gene analysis also classified EIVs into different evolutionary lineages which followed similar lineage pattern to the HA gene [2, 19]. In recent times (2007 onwards), Florida clade 1 viruses have been implicated in outbreaks in Australia [www.equineinfluenzainquiry.gov.au] and Japan [34] while clade 2 viruses have been responsible for the outbreaks in Asian countries including Kazakhstan (2007–08), Mongolia (2007), China (2007–08), India (2008–09) [17, 29] and Europe (2006–07) [3]. EI infection re-emerged in India in 2008 after a gap of 20 years since the first outbreak in 1987 [26] and affected equine population in 13 states of the country [28–30].

The genome of EIVs contains 8 single-stranded negative-sense RNA molecules and its segment 6 encodes neuraminidase (NA) protein, the second major surface antigen [33] after haemagglutinin. It is an exosialidase enzyme which cleaves α-ketosidic linkage between the sialic (N-acetylneuraminic) acid and an adjacent sugar residue from respiratory tract mucins [11, 27]. The NA protein is responsible for entry of virus into host cell by fusion of viral and cell membranes [32]. Further, this protein facilitates budding of newly formed viral particles from the surface of the infected cell by cleavage of the terminal sialic acids on the glycosylated haemagglutinin (HA) [32] and amplifies HA haemagglutinating activity by cleavage of the terminal neuraminic acid residues of the oligosaccharides surrounding the receptor-binding site of HA [15]. It has been demonstrated that the NA and HA proteins of the influenza virus act in concert and their evolution proceeds interdependently [7, 12, 32].

The segmented nature of influenza genome and lack of proof reading capability of RNA polymerase lead to constant antigenic shift and drift, respectively [33]. The antigenic drift in EIVs is more common and point mutations especially in HA and NA genes lead to change in antigenicity of the surface glycoproteins [33]. This leads to reduced vaccine efficiency, difficulty in diagnosis, and thus compromising the management and control of influenza. Therefore, continuous characterization of various genes of all influenza virus isolates especially those expressing the surface glycoproteins is of strategic importance, as it gives an insight into the accumulation of point mutations which can be of significance and have bearing on the virus getting selective advantage to evade immune system. Further, the phylogenetic analysis of the matrix gene of EIVs (H3N8) from epizootic in India in 2008–09 clustered the isolates in a separate Asian Clade along with other isolates from Asia (Mongolia and China) during this period, although HA gene analysis had placed them along with the EIVs of Florida clade 2 [29]. Thus the concern is to analyze and compare the nucleotide and protein sequences of other genes from Indian isolates for their mutational changes and evolutionary relationship with other EIVs isolated across the globe. In view of the important role played by the neuraminidase gene in disease pathogenesis, herein we analyzed NA gene and protein sequences of four Indian EIVs isolated from epizootic in 2008 and 2009 and attempt to establish the phylogenetic relationship and selection pressure in comparison to other EIVs circulating worldwide along with details of the domains and motifs to find out the significance of mutational changes observed in the structure of NA protein.

Materials and Methods

Viral Isolation

The clinical materials in the form of nasal swabs and blood samples were collected from the infected horses during epizootic of equine influenza in different states viz, Katra, Jammu and Kashmir; Mysore, Karnataka and Gopeshwar and Uttarkashi, Uttarakhand. Nasal swabs collected from the animals exhibiting respiratory signs were processed for virus isolation in embryonated chicken eggs of 9–11 days of incubation. For this, an aliquot of 0.1 ml of processed sample was inoculated into embryonated eggs. The eggs were incubated at 35 °C for 72 h after which they were chilled overnight at 4 °C. The allantoic fluid was harvested from the eggs and subjected to HA test with chicken erythrocytes (0.5 % in PBS) in microtitre plates. The isolated virus were identified and characterized using HI test employing standard antiserum against various strains of EIV H3N8 (A/eq/Newmarket/1/93, A/eq/Newmarket/2/93, A/eq/Kentucky/1/81, A/eq/Miami/63) and EIV H7N7 (A/eq/Prague/56), obtained from NIBSC, UK and by sequence analysis of HA gene. The isolates from the above places were propagated in embryonated hen’s egg and used for the present study.

Amplification, Cloning and Sequencing

Briefly, viral RNA was extracted from the allantoic fluid (200 μl) of infected embryos using AuPrep Viral RNA Extraction Kit [Life Technologies (India) Pvt. Ltd., India] as per the manufacturer’s protocol. The cDNA was synthesized using random hexamer primer and AMV reverse transcriptase (MBI Fermentas, Burlington, Canada) in a 20 μl reaction volume. For the amplification of full-length NA gene, cDNA was subjected to PCR using the gene-specific primer (forward primer: 5′-AAAGCAGGAGTTTAAAATGAATC3′and reverse primer: 5′-TTTTTCGTAAATTACATCTTATC-3′) designed based on sequence of GenBank accession no AY855343. The reaction mix contained 5 μl cDNA as template, 20 pmol of each primer, 200 mM dNTPs and 0.5 μl (3 U) of Maxima Hot Start Taq DNA polymerase (MBI Fermentas, Burlington, Canada) in a 50 μl reaction volume. The thermal cycling programme included initial denaturation at 95 °C for 4 min followed by 34 cycles of 95 °C for 1 min, 50 °C for 1 min, 72 °C for 2 min with final extension at 72 °C for 10 min. An aliquot of 5 μl of PCR amplified product was analyzed in a 1 % (w/v) agarose gel to visualize the amplicon, after staining with ethidium bromide (0.5 mg/ml).

The amplified products were purified using PCR Purification Kit (Stratagene Inc., La Jolla, CA, USA) according to manufacturer’s protocol and cloned into pTZ57R/T vector (MBI Fermentas, Burlington, Canada) as per the standard protocol. Recombinant clones were confirmed by colony PCR and restriction enzyme analysis with EcoRI and NotI. All clones were sequenced commercially employing automated DNA sequencer (ABI 3130 Genetic Analyzer) by Chromas Biotech Asia Pvt. Ltd., Bangalore, India. Two clones for each gene were selected for sequencing and a consensus sequence was obtained for analysis.

Sequence and Phylogenetic Analysis

The open reading frame (ORF) of nucleotide sequence of NA gene was subjected to homology search using NCBI BLAST server [1]. The NA gene sequences of all the members of the EIV were retrieved from the NCBI database for comparative analysis. The nt and deduced aa sequences were aligned using Clustal W method of Molecular Evolutionary Genetic Analysis (MEGA) version 5 program [24] and analyzed for differences at specific positions. Hydrophathicity values of the each amino acid were calculated using the ProtScale tool at ExPASy server (http://www.expasy.org/tools/protscale.html) choosing the hydrophobicity scale of Kyte and Doolittle with window size 19.

Phylogenetic trees were constructed based on nucleotide sequences of the ORF of genes using maximum likelihood method in MEGA5 [24] and the tree topologies were evaluated by bootstrap using 1,000 replicates of the data set. The bootstrap consensus tree is taken to represent the evolutionary history of the taxa analyzed [6].

Secondary Structure, Domains and Motifs Prediction

Secondary structure of amino acid sequences of NA protein was predicted employing PSIPRED program (http://bioinf.cs.ucl.ac.uk/psipred/) and the surface accessibility of the aa residues were predicted using NetSurfP program (http://www.cbs.dtu.dk/services/NetSurfP/). Domains or motifs of the protein sequences were searched using the tool ScanProsite at Expasy Server (http://ca.expasy.org/tools/scanprosite/). Motifs with high probability of occurrence were also included in the search. The transmembrane protein helices were predicted using TMHMM server v.2.0 (http://www.cbs.dtu.dk/services/TMHMM/).

Estimation of Selection Pressure

All the available complete ORF of NA gene(s) were retrieved from the FLU database (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html) for estimation of selection pressure. The accessible 103 coding sequences of NA genes of H3N8 EIVs were used to calculate the relative rate of site-by-site substitutions of non-synonymous (dN) and synonymous (dS) nucleotides of NA gene using “Datamonkey” (www.datamonkey.org) web server [21, 22]. Fixed-effects likelihood (FEL) and single likelihood ancestor counting (SLAC) methods were adopted for the evaluation of the selection pressure. The magnitude of the selection pressure on each codon is indicated by the ratio of “dN/dS”. The selection pressure of a site was inferred as positive or negative on the basis of values of dN/dS as >1 or <1, respectively. Evolutionary finger printing analysis was carried out to estimate relative rate of dN and dS substitutions over the entire alignment which was defined as omega (ω).

Results

Sequence and Phylogenetic Analysis

The nt sequence of the full-length NA gene of all four Indian EIVs comprised of 1,413 nucleotides encoding predicted 470aa protein. The nucleotide sequences were deposited in NCBI GenBank and assigned accession numbers: JN674065, JN674066, JN674067, and JN674068. The NA protein of the Indian isolates shared ~99 % homology at both nt and aa levels with clade 2 isolates of Asian origin. Comparative aa substitutions are depicted in the Table 1. There were two aa changes each in Mysore/12/08 (Asn67Tyr & Asp396Gly), Gopeshwar/1/09 (Ile49Val & Asp396Gly), and Uttarkashi/1/09 (Ile49Val & Asp396Gly) isolates in respect to Jammu-Katra/06/08 isolate. Among these aa changes, two specific mutations (Val59Ile & Thr434Arg) were observed only in Indian isolates compared to all other EIVs. Amino acid sequence analysis also revealed that there were five consistent aa residue changes viz. Gly/Arg40Glu, Tyr66His, Val191Ile, Val209Ile, and Asp235Asn in Indian isolates, other Asian (Chinese, Mongolian and Kazakhstan), Japan (Yokohama/10) and Spain/07 isolates in comparison to other EIVs (Table 1). However, amino acid valine is present at position 209 in Yokohama/10 and Spain/07 isolates along with all other compared sequences. Earlier Indian EIV isolate (Ludhiana/87) showed several aa changes similar to pre-divergent isolates (Miami/63, Fontainebleau/79, Kentucky/81, and Santiago/85). Further, two specific aa changes (Ala9Thr and Phe12Ser) were observed only in European isolates.

Table 1.

Comparative amino acid substitutions in the predicted neuraminidase (NA) protein compared to A/equine/Jammu-Katra/6/08 (H3N8) isolate with other EI virus isolates

Strain	8	9	11	12	17	18	20	35	40	42	43	46	49	51	59	61	62	63	66	67	69	70
KAT/6/08	I	T	G	S	I	L	I	V	E	G	L	K	I	R	I	K	I	T	H	N	S	A
MYS/12/08	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	Y	.	.
GOP/1/09	.	.	.	.	.	.	.	.	.	.	.	.	V	.	.	.	.	.	.	.	.	.
UTR/1/09	.	.	.	.	.	.	.	.	.	.	.	.	V	.	.	.	.	.	.	.	.	.
GAN/7/08	.	.	.	.	.	.	.	.	.	.	.	.	.	.	V	.	.	.	.	.	.	.
HUB/6/08	.	.	.	.	.	.	.	.	.	.	.	.	.	.	V	.	.	.	.	.	.	.
LIA/9/08	.	.	.	.	.	.	.	.	.	.	.	.	.	.	V	.	.	.	.	.	.	.
XIN/1/07	.	.	.	.	.	.	.	.	.	.	.	.	.	.	V	.	.	.	.	.	.	.
MON/8/08	.	.	.	.	.	.	.	.	.	.	.	.	.	.	V	.	.	.	.	.	.	.
YOK/aq13/10	.	.	.	.	.	.	.	.	.	.	.	.	.	.	V	.	.	.	.	.	.	.
SPAIN/1/07	.	.	.	.	.	.	.	.	.	.	.	.	.	.	V	.	.	.	.	.	.	.
NEW/T5/03	.	A	.	F	.	.	.	.	G	.	.	.	.	.	V	.	.	.	Y	.	.	.
CAL/8560/02	.	A	.	F	.	.	.	.	G	.	.	.	.	.	V	.	.	.	Y	.	.	.
KEN/5/02	.	A	.	F	.	.	.	.	G	.	.	.	.	.	V	.	.	.	Y	.	.	.
OHI/1/03	.	A	.	F	.	.	.	.	R	D	.	.	.	.	V	.	.	.	Y	.	.	T
WIS/1/03	.	A	.	F	.	.	.	.	R	D	.	.	.	.	V	.	.	.	Y	.	.	T
NEW/07	M	A	.	F	.	.	.	A	R	D	.	.	.	K	V	.	.	.	Y	.	.	T
KAN/1/07	M	A	.	F	.	.	.	A	R	D	.	.	.	K	V	.	.	.	Y	.	.	T
IBA/1/07	.	.	.	.	.	.	.	.	R	D	.	.	.	K	V	.	.	.	Y	.	.	T
NEW/1/93	.	.	.	.	.	.	.	.	G	.	.	.	.	.	V	R	.	.	Y	.	.	T
NEW/2/93	.	.	.	.	V	.	L	.	G	.	.	N	.	.	V	R	.	.	Y	.	.	T
SAN/85	.	.	.	.	.	.	L	.	G	.	.	N	.	.	V	R	V	I	Y	.	N	T
LUD/87	.	.	.	.	.	.	L	.	G	.	.	N	.	.	V	R	.	.	Y	.	N	T
KEN/1/81	.	.	.	.	.	.	L	.	G	.	.	N	.	.	V	R	.	.	Y	.	N	T
MIA/1/63	.	.	.	.	L	V	L	.	G	.	P	N	.	.	V	R	.	.	Y	.	N	T
FON/1/79	.	.	.	.	.	.	L	.	G	.	.	N	.	.	V	R	.	.	Y	.	N	T

Strain	72	76	78	84	109	125	134	147	150	160	161	174	180	191	198	201	205	209	213	218	219	220	235	250	252
KAT/6/08	K	R	P	N	R	S	Q	V	R	K	I	S	T	I	N	I	N	I	I	A	G	D	N	K	R
MYS/12/08	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
GOP/1/09	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
UTR/1/09	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
GAN/7/08	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
HUB/6/08	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	E	.	.	.	.	.
LIA/9/08	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	E	.	.	.	.	.
XIN/1/07	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	E	.	.	.	.	.
MON/8/08	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	E	.	.	.	.	.
YOK/aq13/10	.	.	.	.	K	.	.	.	.	.	.	.	.	.	.	.	.	V	.	.	.	.	.	.	.
SPAIN/1/07	.	.	.	.	.	.	.	.	Q	.	.	.	A	.	.	.	.	V	.	.	.	.	.	E	G
NEW/T5/03	.	.	.	.	.	.	.	.	.	.	.	.	.	V	.	.	.	V	.	.	.	.	.	.	.
CAL/8560/02	.	.	.	.	.	.	.	.	.	.	.	.	.	V	.	.	.	V	.	.	.	.	D	.	.
KEN/5/02	.	.	.	.	.	.	.	.	.	.	.	.	.	V	.	.	.	V	.	.	.	.	D	.	.
OHI/1/03	.	.	S	.	.	.	.	.	.	.	.	.	.	V	.	.	.	V	.	.	.	.	D	.	.
WIS/1/03	.	.	S	.	.	.	.	.	.	.	.	.	.	V	.	.	.	V	.	.	.	.	D	.	.
NEW/07	.	.	S	.	.	.	.	.	.	.	.	.	.	V	.	.	.	V	.	.	.	.	D	.	.
KAN/1/07	.	.	S	.	.	.	.	I	.	.	.	.	.	V	.	.	.	V	.	.	.	N	D	.	.
IBA/1/07	.	.	S	.	.	.	.	I	.	.	.	.	.	V	.	.	.	V	.	.	.	N	D	.	.
NEW/1/93	E	.	S	.	.	.	.	.	.	.	.	.	.	V	.	.	.	V	.	.	.	.	D	.	.
NEW/2/93	E	.	S	.	.	.	.	.	.	.	.	.	.	V	.	.	.	V	.	.	W	.	D	.	.
SAN/85	E	.	S	.	.	.	.	.	.	.	.	.	.	V	.	V	.	V	.	.	.	.	D	.	.
LUD/87	E	.	S	.	.	.	.	.	.	.	.	.	.	V	.	V	.	V	.	.	.	.	D	.	.
KEN/1/81	E	.	S	.	.	L	.	.	.	.	V	.	.	V	.	V	.	V	.	.	.	.	D	.	.
MIA/1/63	E	E	S	S	.	L	.	.	.	E	V	A	.	V	A	V	H	V	V	.	.	.	D	Q	.
FON/1/79	E	.	S	.	.	L	H	.	.	.	V	A	.	V	T	V	H	V	.	.	.	.	D	.	.

Strain	261	268	271	289	301	306	337	341	342	355	381	390	394	396	397	415	426	434	451	452	462	470	471
KAT/6/08	V	S	G	I	I	S	S	N	K	T	T	R	I	D	P	K	M	R	K	I	I	M	*
MYS/12/08	.	.	.	.	.	.	.	.	.	.	.	.	.	G	.	.	.	.	.	.	.	.	*
GOP/1/09	.	.	.	.	.	.	.	.	.	.	.	.	.	G	.	.	.	.	.	.	.	.	*
UTR/1/09	.	.		.	.	.	.	.	.	.	.	.	.	G	.	.	.	.	.	.	.	.	*
GAN/7/08	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	T	.	.	.	.	*
HUB/6/08	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	T	.	.	.	.	*
LIA/9/08	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	T	.	.	.	.	*
XIN/1/07	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	T	.	.	.	.	*
MON/8/08	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	T	.	.	.	.	*
YOK/aq13/10	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	R	.	S	.	.	.	.	*
SPAIN/1/07	.	.	.	.	.	.	.	.	R	.	.	.	.	.	.	.	.	T	.	.	.	.	*
NEW/T5/03	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	T	.	.	.	.	*
CAL/8560/02	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	T	.	.	.	.	*
KEN/5/02	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	T	.	.	.	.	*
OHI/1/03	.	.	E	.	.	.	.	.	.	.	.	.	.	.	.	.	.	T	.	.	.	.	*
WIS/1/03	.	.	.	.	.	.	R	.	.	.	.	.	.	.	.	.	.	T	.	.	.	.	*
NEW/07	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	T	.	.	.	.	*
KAN/1/07	.	N	.	.	.	.	N	.	.	.	.	.	.	.	.	.	.	T	.	.	.	.	*
IBA/1/07	.	N		.	.	.	N	.	.	.	.	.	.		.	.	.	T	.	.	-	-	*
NEW/1/93	I	.	.	.	.	.	.	.	.	.	.	.	.	N	.	.	.	T	.	.	.	.	*
NEW/2/93	I	.	.	.	.	.	.	.	.	.	.	.	.	N	.	.	.	T	.	.	.	.	*
SAN/85	I	.	.	V		P	.	.	.	.	.	.	.	N	.	.	.	T	.	.	.	.	*
LUD/87	I	.	.	V		P	.	.	.	N	.	K	.	N	L	.	S	T	.	.	.	I	*
KEN/1/81	I	.	.	V		P	.	.	.	N	.	K	.	N	L	.	.	T	.	.	.	.	*
MIA/1/63	I	N	.	V	V	P	.	S	Q	N	.	K	V	N	L	.	.	T	.	V	.	.	*
FON/1/79	I	N	.	V		p	.	.	Q	N	I	K	V	N	L	.	.	T	R	.	V	.	*

Amino acid residues are numbered from the N-terminal methionine. Residue identity to A/eq/Katra-Jammu/06/08 is shown with a dot, where as single alphabet represents difference in the amino acid sequence. The stop codons are represented with an asterisk. Other reference strains are A/eq/Mysore/12/08, A/eq/Gopeshwar/1/09, , A/eq/Uttarkashi/1/09, A/eq/Gansu/7/08, A/ eq/Hubei/6/08, A/eq/Lianjiang/1/07, A/eq/Inner Mongolia/8/08, A/eq/Yokohama/aq13/10, A/eq/Spain/1/07, A/eq/Newmarket/5/03, A/eq/California/8560/02, A/eq/Kentucku/5/02, A/eq/Ohio/1/03, A/eq/Wisconsin/1/03, A/eq/Kanazawa/1/07, A/eq/Ibaraki/1/07, A/eq/Newmarket/1/93, A/eq/ Newmarket/2/93, A/eq/Santiago/85, A/eq/Ludhiana/87, A/eq/Kentucku/1/81, A/eq/Miami/1/63 and A/eq/Fontainebleau/1/79

We have also analyzed whether these mutations involve any change in polarity or hydrophobicity. In most of the cases, overall hydropathicity did not change. Amino acid changes (Asn67Tyr and Asp396Gly in Mysore, Asp396Gly in Gopeshwar and Uttarkashi isolates) cause change of properties from hydrophilicity to hydrophobicity. However, in Asian isolates consensus aa changes (Gly/Arg40Glu and Tyr66His) favoured the polarity shifting from hydrophobicity to hydrophilicity. Analysis of the N8 proteins also revealed 17 highly conserved cysteine residues; one is in the stalk region and the others in the head section.

The topology of the phylogenetic tree clustered the EIVs into five lineages as pre-divergence, Eurasian, American, Florida clade 1 and 2 along with grouping of the Asian isolates (Indian, Chinese, Mongolian and Yokohama/10) together in a subgroup with bootstrap value 100 (Fig. 1). However, Spain/07 isolate also clustered with in Asian subgroup.

Fig. 1.

Phylogenetic tree based on the nucleotide sequences of neuraminidase gene of EIV (H3N8) isolates. Maximum likelihood tree was constructed using MEGA5. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. The representative sequences reported in this manuscript are marked by black boxes. Eq stands for equine and Can stands for Canine

Secondary Structure Prediction

The predicted helical structure of the protein consisted of three regions: cytoplasmic (1–12); transmembrane helix (13–35) and extracellular region (36–470). The analysis of the predicted secondary structure has not shown any significant difference in the predicted regions of NA proteins of all Indian isolates. However, two helical regions varied among Indian isolates: 6–12 and 48–52 in Katra/08; 6–12 and 49–51 in Mysore/08; Gopeshwar/09 and Uttarkashi/09 isolates and 6–11 and 49–51 in other Asian isolates. The aa changes (Ile→Val) at position 49 has led to change of secondary structure from strand to coil in Gopeshwar/09 and Uttarkashi/09 isolates. No much changes have been observed in the relative surface accessibility due to aa changes except Mysore isolate where aa changes at 59 (Val→Ile) and 67 (Asn→Tyr) caused positional changes from buried to exposed and exposed to buried, respectively.

Domain/Motif Search

Different domains viz, N-glycosylation (7 sites), N-myristoylation (3 sites), Protein kinase C phosphorylation (7 sites), Casein kinase II phosphorylation (5 sites), and Amidation (1 site) in the NA proteins of different isolates have been predicted. The mutation at position 67 (Asn→Tyr) in Mysore/08 isolate abolished N-glycosylation site (NTSA at position 67–70). The change of aa (Gly40Glu) in Asian isolates compared to all other EIV lineages caused change of N-glycosylation domain (NETG→NGTG) and abolition of N-myristoylation domain (GTglNC). The role of these changes in N-glycosylation and N-myristoylation pattern are to be assessed in light of infectivity of the viruses. The numbering of the active sites of the NA protein was done after comparing with the N2 numbering pattern as followed in structural and functional analysis.

Analysis of Selection Pressure (ω)

All coding sequences of NA gene(s) across different geographical regions throughout the world over a larger time span were included in estimation of the selection pressure for the better inference of the evaluation. FEL analysis of the selection pressure revealed that three codons (43, 355 and 434) are under positive selection pressure and 163 sites under negative selection pressure at significance level of 0.1. The mean dN/dS value was 0.195 as estimated by SLAC analysis. The overall evolutionary changes (ω value) of 3.4 indicates NA gene to be under strong selection pressure.

Discussion

Considering the important role of the NA protein at different stages of infection in conjunction with HA protein, we have analysed the sequences and phylogeny of this gene of four EIVs isolated from recent epizootic (2008–09) in India to elucidate mutational changes as well as to find out the evolutionary relationship with other circulating EIVs. The polypeptide chain of NA comprised of 470 aa residues similar to that of the all other NA subtypes of influenza A viruses [32]. Indian isolates revealed maximum homology (~99 %) at both nt and aa levels with Chinese, Mongolian, Kazakhstan and Japanese (Yokohama/2010) isolates indicating EIVs circulating in Asian regions during 2007 to 2010 have almost similar sequences. In the previous studies, the HA gene sequence analysis of EIVs from current outbreak revealed highest amino acid identity amongst Asian isolates [29]. The point mutations observed amongst Indian isolates (Mysore/08, Gopeshwar/09 & Uttarkashi/09) in comparison to Jammu-Katra/06/08 isolate. No literature pertaining to complete mapping of antigenic sites of NA8 protein of influenza virus is available as of now which impede the comparative analysis of antigenic sites. Comparative sequence analysis also revealed that mutations (Gly/Arg40Glu, Tyr66His and Val191Ile) originated from Spain isolate (2007) and point mutation (Asp235Asn) originated from New Market isolate in 2003. These mutations are also detected in viruses isolated from Kazakhstan (2007–08), Mongolia (2007–08), China (2007–08), India (2008–09) and Japan (2010). However, another aa change (Val209Ile) was observed in all Asian isolates except Yokohama/2010 isolate. Amino acid change (Gly/Arg40Glu) led to change in N-glycosylation domain (NGTG to NETG) and abolition of N-Myristoylation site (GTglNC). The significance of these post-translational modifications needs to be carried out to ascertain their role in pathogenesis. Two changes (Val59Ile & Thr434Arg) in amino acids were observed only in Indian EIV isolates; however, these changes do not have any effect on hydropathicity as well as secondary structure. Further, two non-synonymous point mutations at position 9 and 12 in European isolates did not sustain in Asian isolates where the sequence reverted back (Ala9Thr and Phe12Ser). Similar reverse mutation was also observed at position 218 (Glu to Ala) in Mongolian and Chinese isolates. These reverse mutations might have occurred due to less selective advantage to the virus.

The three-dimensional structure of NA consists of four main domains: the cytoplasmic (1–12 aa), transmembrane (13–35 aa), stem (36–86 aa) and head domain (90–470 aa) [13, 23]. The catalytic site of the enzyme is found in the head region [23]. The first two domains (cytoplasmic and transmembrane) were conserved among isolates from Asia and Spain when compared to other isolates across various lineages. However, five mutational changes were observed in the stem region. The aa residues (Arg116, Asp149, Arg150, Arg223, Glu275, Arg291, Arg368 and Tyr402) which account for the catalytic function of the enzyme as depicted by Shtyrya et al. [23], were intact in all the Indian as well as other EIVs. Numbering of aa sequences constituting active site was done following numbering pattern of N2 protein [23]. However, Saito et al. [20] identified two antibody recognition sites through screening with a panel of monoclonal antibodies. One epitope located at the head region which consists of peptide loop involving amino acid residues at positions 150, 199, 344 to 346, 367, 399, and 400 and another epitope consists of the peptide loop containing residues 344–346 as per N2 protein numbering. We have not observed any changes among these critical amino acid residues in the Indian and Spain/07 isolates.

Domain and motif search revealed 7 putative N-glycosylation sites in the NA protein of Indian isolates. Such post-translational modification plays important role in pathogenesis as has been demonstrated in mutational studies in H1N1 (A/WSN/33) [10] and H3N8 (A/duck/Ukraine/1/63) viruses [18]. Asn144 glycosylation—which is essential for regulatory function and has been demonstrated in determining the influenza virus neuro-virulence in H1N1 virus (A/WSN/33) [10] and substrate specificity profile N8 of duck isolate (A/duck/Ukraine/1/63) [18]—was highly conserved in all NAs including Indian EIVs. However, further studies are needed to elucidate the functional role of the change of Asian isolates (Gly/Arg40Glu) in relation to their virulence.

The evolutionary study revealed that the mean dN/dS value estimated to be 0.195 which indicates localized adaptive evolution in NA gene. The phylogenetic analysis of NA gene showed the clustering of Indian isolates with those of Kazakhstan, Mongolian, Chinese, and Japanese isolates. These broadly grouped in Florida clade 2 and followed lineage pattern of EIVs as that of HA genes of our earlier studies [29]. However, in the present study, we could also find that a new subgroup of Asian isolates showing robustness in phylogeny as supported by high bootstrap value (100) emerged from Kazakhstan/07 isolate and all the viruses isolated subsequently from Asia in chronology belonged to this group. Previously authors have also demonstrated the grouping of Asian isolates in a separate cluster for Matrix gene as well [31]. The mutational changes and phylogeny revealed that the equine influenza outbreaks in Asian country routed through Spain/07 outbreak and subsequent to Kazakhstan (2007), Mongolia (2007–08), China (2008) and India (2008–09). However, sequence information for NA gene from other clade 2 viruses incriminated for outbreaks across the globe during this time is lacking which confound the further analysis. The NA protein along with HA protein come in direct contact with host immune defence system as these are major surface glycoproteins. This leads to the frequent changes in them owing to point mutations. Hence, both HA and NA genes are of significance for evolutionary studies.