Swine flu viruses are causing a huge amount of death to both human and swine. The World Health Organization (WHO) figures show that worldwide more than 209 countries and overseas territories or communities have reported laboratory confirmed cases of pandemic influenza H1N1 2009, including at least 15174 deaths (WHO, 5 February 2010). Pathogenicity of virulence can be change in viruses while circulating in the poultry population due to high rate of mutation (Stech et al., 1999). The H1N1 subtype is pathogenic swine viral that has been documented to cause an outbreak of respiratory disease in both human and swine. Influenza was first described as a disease of swine in 1918 (Koen, 1919). The first influenza A virus was isolated from swine in 1930 (Brockwell-Staats et al., 2009). A swine influenza virus was isolated from a human in 1974, from 1974 to 2005; there were 43 confirmed cases of transmission of influenza A virus from pigs to humans reported, with six fatalities (Brockwell- Staats et al., 2009). Since there are no unique clinical symptoms to differentiate swine influenza from seasonal influenza in humans, this number is probably having a small fraction of the actual cases. Most of these cases were the result of direct exposure to swine or were human-to-human transmission within a family cluster (Myers et al., 2007).

Swine play an important role in the ecology of influenza A viruses because they are susceptible to viruses of both the avian and mammalian lineages. The cells of the swine respiratory tract contain receptor sialyloligosaccharides possessing both Nacetylneuraminic acid-α2, 3- galactose, which is the preferred receptor for avian influenza viruses and N-acetylneuraminic acid- α2, 6-galactose, which is the preferred receptor for mammalian influenza viruses (Ito et al., 1998; Rogers et al., 1983). This has led to the proposal that swine serve as a “mixing vessel” for influenza viruses of different lineages, providing a place for reassortment and host adaptation to take place (Scholtissek, 1990).

Swine influenza A virus belong to the viral family of Orthomyxoviridae. They are RNA viruses with a segmented genome that is comprised of eight negative-sense, single-stranded RNA segments. These eight segments encode eleven proteins (Brockwell-Staats et al., 2009). The polymerase complex includes the PB2, PB1 and PA proteins as well as the nucleoprotein (NP). There are two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) (Brockwell-Staats et al., 2009).

In view of these outbreaks that occurred due to mutations in influenza A virus as discuss in case of Bird Flu virus subtype H5N1 (Anwar et al., 2006). We initiated our in silico study to analyze the amino acid sequences of neuraminidase and hemagglutinin proteins of swine for the amino acid mutation at different positions compared to the other strains of the same sub-type (H1N1). The great genetic variability in influenza A virus lead to the difficulties in diagnosis, treatment, and prevention of influenza in humans. Therefore, it is significant to analyze these proteins for their mutation and phylogenetic analysis comparing with other strains of influenza virus.

The amino acid sequence of neuraminidase (NA) and hemagglutinin (HA) of swine influenza virus subtype H1N1 of A/Swine/Nebraska/(H1N1) were retrieved from protein sequence database situated at NCBI (http://www.ncbi.nlm.nih.gov/). These protein sequences have GenBank accession number ABR28650 for NA and AAB29091 for HA. Amino acid sequences of NA and HA of swine influenza virus sub-type H1N1 strains were used for screening of 98-99% similar sequences available in nonredundant (nr) database situated at NCBI using BLASTP (http://www.ncbi.nlm.nih.gov/BLAST/).

The protein sequences of selected strains (Table 1) was then aligned by using multiple sequence alignment tool ClustalW 1.83 (http://www.ebi.ac.uk/tools/clustalw2/index.html) situated at European Molecular Biology Laboratory. ClustalW (Thompson et al., 1994) program calculate the best match for the selected sequences and line them up, so that the identities, similarities and differences can be seen. These alignments were then analyzed for differences in their amino acid at specific positions. A program PROTMUTATION (http://www.biobrainz.com/tools/ protmutation.htm) was developed in Perl CGI programming. It is an interactive, user-friendly program for identifying mutations by comparing protein sequences of two different strains using Needleman-Wunsch algorithm for global sequence alignment to point out mutations in different strains. Hydrophobicity values were obtained from the tool ProtScale (Gasteiger et al., 2005) at ExPASy server (http://www.expasy.org/tools/protscale.html) choosing Kyte & Doolittle hydrophobicity scale (Kyte and Doolittle, 1982). An evolutionary distance matrix was generated from multiple sequence alignment of selected homologous sequences and phylogenetic tree was then drawn using the Neighbour joining method (Saitou and Nei, 1987) by MEGA (Molecular Evolutionary Genetics analysis) version 4.0 (Tamura et al., 2007). Secondary structures of the proteins were predicted using program SOPMA (Geourjon and Deléage, 1995). Domains or motifs among these protein sequences of A/Swine/Nebraska/ (H1N1) were searched using ScanProsite at Expasy Server (http://www.expasy.ch/tools/scanprosite/). Motifs with high probability of occurrence were also included in the search.

Table 1: Selected strains of NA and HA, which are screened using BLASTP containing GenBank accession number.

Sequence analysis

On comparing our sequence of neuraminidase after multiple alignment with the selected sequences of the same subtype having 98-99% similarity, it was found that at position 240 of the sequence the hydrophilic threonine was replaced by hydrophobic alanine and at position 451, hydrophobic glycine was replaced by hydrophilic serine (Table 2). The mutation at position 240 may be important in the sense that here hydrophilic Threonine is being replaced by hydrophobic Alanine, which may help the protein to attain more stable conformation, while at position 451 hydrophobic Glycine is being replaced by hydrophilic Serine, which may help the protein to attain less stable conformation, since glycine is the smallest amino acid, it fits into tight places inside a folded protein and disrupts a-helix formation. In hemagglutinin, mutations were found at position 225, where hydrophilic arginine was substituted by same type of amino acid hydrophilic Lysine and position 283, where more hydrophobic isoleucine was replaced by less hydrophobic valine.

Table 2: Amino acid mutations in neuraminidase and hemagglutinin at specific position and change in properties, secondary structure and hydrophobicity.

The program PROTMUTATION reports all the mutations along wi th their specific posi t ion in the sequence. PROTMUTATION program accepts input sequences by pasting two sequences in raw format in the corresponding text boxes as shown in Figure 1.

Figure 1: Snapshot of PROTMUTATION Tool.

Output of PROTMUTATION produces result instantly in a file containing global alignment of both inputted protein sequences. The results of PROTMUTATION were checked with results produced by manually analyzing mutations after multiple alignments and with known one tool, the results were similar in al l cases, thus, i t was predicted that the program PROTMUTATION produces more than 90% accurate results. This program will be greatly helpful to swine flu researchers who are interested in finding out the rate of mutation in Influenza viruses, as the virus continuously undergoes antigenic shift and antigenic drift.

Secondary structure and hydrophobicity prediction

In order to better understand what sort of changes this mutation possibly might have on the A/Swine/Nebraska/(H1N1) strain, secondary structure and hydrophobicity in this mutation site are examined. The results of secondary structure prediction of neuraminidase and hemagglutinin amino acids are situated at http://www.biobrainz.com/tools/secstr.htm. Secondary structure prediction of NA of A/Swine/Nebraska/(H1N1) strain and all the other strains show that no structure changes were detected at position 240 and 451 and are strand and coil respectively. For HA, no secondary structure change is detected at position 225 and 283 of A/Swine/Nebraska/(H1N1) and all the other strains and are found coil and strand respectively. Hydrophobicity is evaluated using ProtScale software. This software displays the polarity of the initial amino acids and amino acids after the mutation presented in Table 2.

Domain/motif search

Different domains that were found in the NA and HA proteins are given in the Tables 3 and 4. While in NA, mutations (T240A, G451S) were found at predicted functional sites of Nmyristoylation GScfAI and GVnsST at positions 236 - 241 and 447-452 respectively was shown in Table 3 (Prosite Documentation, http://www.biobrainz.com/tools/pro.htm). The signature for N-myristoylation sites is G - {EDRKHPFYW} - x (2) - [STAGCN] - {P}, at position 5, small-uncharged residues (Ala, Ser, Thr, Cys, Asn and Gly) are allowed and serine is favored (Towler et al., 1988; Grand, 1989). The mutation from T to A at position 240 and G to S at position 451 do not account for any difference in N-myristoylation site. For HA, mutation (I283V) was found at a predicted functional site (GIviSD) of Nmyristoylation at position 281 – 286 was shown in Table 4 (Prosite Documentation, http://www.biobrainz.com/tools/pro.htm). In the signature sequence of N-myristoylation site at position 3 and 4, most, if not all, residues are allowed (Towler et al., 1988; Grand, 1989). Therefore mutation from I to V at position 283 does not account for any difference in N-myristoylation site.

Table 3: Predicted domains/motifs in neuraminidase representing the functional site name, its position on the sequence and the sequence of the site using prosite tool situated at expasy server.

Table 4: Predicted domains/motifs in hemagglutinin representing the functional site name, its position on the sequence and the sequence of the site using Prosite tool situated at expasy server.

Phylogenetic analysis

Phylogenetic trees of NA and HA sequences with selected strains (Table 1) from swine were shown in Figure 2 and Figure 3. It is evident from phylogenetic analysis of protein sequences that isolates were having close relationship.

Figure 2: Neighbour-joining tree of test NA sequence with selected strains obtained from BLASTP search using MEGA (Molecular Evolutionary Genetics Analysis) version 4.0 software. It shows relationship between test NA sequence with closely related representative strains.

Figure 3: Neighbour-joining tree of test HA sequence with selected strains obtained from BLASTP search using MEGA (Molecular Evolutionary Genetics Analysis) version 4.0 software. It shows relationship between test HA sequence with closely related representative strains.

Analysis of the sequence comparison shows that mutations were observed in HA (2sites) and NA (2 sites). Thus, we can say that HA and NA of swine influenza virus sub-type H1N1 are sensitive towards mutations. Then we find that these mutations involve in change polarity or hydrophobicity. Furthermore, not only the polarity or hydrophobicity is significantly altered by most mutations but also the propensity of each amino acid residue to stabilize the secondary structure. In this work, no structural conformation changes were observed in HA (at position 225 and 283) and in NA (at position 240 and 451). All the important domains of NA and HA protein sequences were tracked (Table 3 and Table 4). In NA and HA mutations were found at predicted functional sites of N-myristoylation. These mutation were not affecting N-myristoylation according to our in silico study but in future if mutation occurs at this position the virus may become more lethal to humans.

Although we have not yet been predicted any mutation that may lead to an outbreak of swine flu rather we can in principle monitor the mutations along the time course, and predict the trend of mutations. Thus, further mutational analysis would have to be carried out to map a specific amino acid change in a protein causing the change in pathogenicity of the virus.

Our study revealed that HA and NA are prone to mutations, thus we conclude that HA and NA might be an important proteins involved in the pathogenesis of swine influenza virus. We also found that these mutations involve change in polarity or hydrophobicity. Furthermore, it is not only the polarity or hydrophobicity significantly altered by most mutations but also the propensity of each amino acid residue to stabilize the secondary structure. Mutations at N-myristoylation site in NA (T240A, G451S) and HA (I283V) do not make any difference but if another mutation occurs at this point then it might be fatal. Secondary structure prediction and prosite documentation files of these proteins are available at http://www.biobrainz.com/tools/secstr.htm and http://www.biobrainz.com/tools/pro.htm. The program PROTMUTATION available at http://www.biobrainz.com/tools/protmutation.htm can be used to predict mutations in new strains.

The authors are grateful to Prof. Ashok Kumar (Dean, I.B.M.E.R, Mangalayatan University Aligarh, U.P, India) for providing necessary facilites and encouragement. The authors are also thankful to all faculty members of the Institute of Biomedical Education and Research, Mangalayatan University Aligarh, U.P, India for their generous help and suggestions during the course of experimental work and manuscript preparation.