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 Deleage, 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.

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.