Crimean-Congo Hemorrhagic Fever Virus Diversity and Reassortment, Pakistan, 2017-2020.

Sporadic cases and outbreaks of Crimean-Congo hemorrhagic fever (CCHF) have been documented across Pakistan since 1976; however, data regarding the diversity of CCHF virus (CCHFV) in Pakistan is sparse. We whole-genome sequenced 36 CCHFV samples collected from persons infected in Pakistan during 2017-2020. Most CCHF cases were from Rawalpindi (n = 10), followed by Peshawar (n = 7) and Islamabad (n = 4). Phylogenetic analysis revealed the Asia-1 genotype was dominant, but 4 reassorted strains were identified. Strains with reassorted medium gene segments clustered with Asia-2 (n = 2) and Africa-2 (n = 1) genotypes; small segment reassortments clustered with the Asia-2 genotype (n = 2). Reassorted viruses showed close identity with isolates from India, Iran, and Tajikistan, suggesting potential crossborder movement of CCHFV. Improved and continuous human, tick, and animal surveillance is needed to define the diversity of circulating CCHFV strains in Pakistan and prevent transmission.

First report of coinfection and whole-genome sequencing of norovirus and sapovirus in an acute gastroenteritis patient from Pakistan.

Acute gastroenteritis is one of the most common diseases in infants and children in developing countries including Pakistan. In Pakistan, rotavirus (RVA) is known to contribute significantly to pediatric diarrheal illness, but the contribution of other viruses is still unclear. In the current study we have identified a case of mixed infection of norovirus (NoV) and sapovirus (SaV) in a 2-year-old child with acute gastroenteritis. The sample was initially processed for the detection of group A RVA through ELISA followed by NoV using RT-PCR assay. The sample tested positive for NoV RNA and was later subjected to whole-genome sequencing using meta-genome approach on Miseq (Illumina) platform. Sequencing results revealed GII.15 genotype of NoV that clustered with viruses from China and USA from 2017 to 2021. We also retrieved the complete genome of SaV (GI.1 genotype) from the same sample and phylogenetic analysis showed clustering with strains reported from Japan, South Korea, US, and Taiwan during 2012-2016. This is the first report from Pakistan that confirms coinfection of NoV and SaV and elucidates their whole genomes. We recommend initiation of NoV and SaV surveillance program to ascertain disease burden and explore genetic diversity, especially as RVA vaccines have been included in national immunization program.

Whole-genome sequencing of Crimean-Congo hemorrhagic fever virus circulating in Pakistan during 2022.

Pakistan is an endemic country for Crimean-Congo hemorrhagic fever (CCHF) and its Balochistan province is considered a hotspot region for circulation of the virus whereas sporadic cases have been reported from other parts of the country. Our study aims to investigate the genomic diversity of the CCHF virus circulating in Punjab and Khyber Pakhtunkhwa provinces of Pakistan. Between April to September 2022, 46 samples from suspected CCHF patients were tested, with 6 (13%) showing positive RT-PCR results. Among the positive cases, all were male, aged 14-48 years among which 4 were butchers. Three CCHF patients succumbed to the disease. The complete S-M-L-gene fragments of 4 positive samples were sequenced. The S and L segments belonged to the Asia-1 clade and clustered with regional strains from Iran, India, and Afghanistan. One M segment sequence grouped into Africa-2 along with those reported from India during 2016-2019. We report the detection of a reassorted virus (NIH-PAK-CCHF-03|2022) having Asia-1-Africa-2-Asia-1 (S-M-L) segment pattern for the first time from Pakistan. Mutational analysis showed M segments harboring eight mutations (T55A, S80P, T110I, T185A, T189A, A212T, and N239I/T) in the mucin-like domain, five mutations (D250N, T333S, I375V, M401I, A433T), four mutations (N545D, Y657F, K688R, and I824V) in GP38-domain, and three mutations (T1418N, A1431V, and G1449S) in Gc-domain. These findings highlight the significance of whole-genome sequencing of indigenous strains for a better understanding of the CCHFV evolution in Pakistan.

Genomic diversity of SARS-CoV-2 in Pakistan during the fourth wave of pandemic.

The emergence of different variants of concern of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in upsurges of coronavirus disease 2019 (COVID-19) cases around the globe. Pakistan faced the fourth wave of COVID-19 from July to August 2021 with 314,786 cases. To understand the genomic diversity of circulating SARS-CoV-2 strains during the fourth wave of the pandemic in Pakistan, this study was conducted. The samples from 140 COVID-19-positive patients were subjected to whole-genome sequencing using the iSeq Sequencer by Illumina. The results showed that 97% (n = 136) of isolates belonged to the delta variant while three isolates belonged to alpha and only one isolate belonged to the beta variant. Among delta variant cases, 20.5% (n = 28) isolates were showing B.1.617.2 while 23.5% (n = 25), 17.59% (n = 19), 14.81% (n = 16), and 13.89% (n = 15) of isolates were showing AY.108, AY.43 AY.127, and AY.125 lineages, respectively. Islamabad was found to be the most affected city with 65% (n = 89) of delta variant cases, followed by Karachi (17%, n = 23), and Rawalpindi (10%, n = 14). Apart from the characteristic spike mutations (T19R, L452R, T478K, P681R, and D950N) of the delta variant, the sublineages exhibited other spike mutations as E156del, G142D, T95I, A222V, G446V, K529N, N532S, Q613H, and V483A. The phylogenetic analysis revealed the introductions from Singapore, the United Kingdom, and Germany. This study highlights the circulation of delta variants (B.1.617.2 and sublineages) during the fourth wave of pandemic in Pakistan.

Genomic surveillance reveals the detection of SARS-CoV-2 delta, beta, and gamma VOCs during the third wave in Pakistan.

SARS-CoV-2 variants of concern (VOCs) have emerged worldwide and gained significant importance due to their high transmissibility and global spread, thus meriting close monitoring. In Pakistan, limited information is available on circulation of these variants as the alpha variant has been reported the main circulating lineage. The current study was designed to detect and explore the genomic diversity of SARS-CoV-2 lineages circulating during the third wave of the pandemic in the indigenous population. From May 01 to June 09, 2021, a total of 16 689 samples were tested using TaqPath™ COVID-19 kit for the presence of SARS-CoV-2. Overall, 2562 samples (15.4%) were COVID-19 positive. Out of these positive samples, 2124 (12.7%) did not show the spike gene amplification (spike gene target failure ([SGTF]), whereas 438 (2.6%) showed spike gene amplification (non-SGTF). A subset (n = 58/438) of non-SGTF samples were randomly selected for whole-genome sequencing. Among VOCs, 45% (n = 26/58) were delta, 46% (n = 27/58) were beta, and one was gamma variant. The delta variant cases were reported mainly from Islamabad (n = 15; 58%) followed by Rawalpindi and Azad Kashmir (n = 1; 4% each). Beta variant cases originated mainly from Karachi (n = 8; 30%) and Islamabad (n = 11; 41%) and the gamma variant case was reported in a traveler from Italy. The delta, beta, and gamma variants possessed lineage-specific spike mutations. Notably, two rare mutations (E484Q and L5F) were found in the delta variant. Furthermore, in the beta variant, two significant rare non-synonymous spike mutations (A879S and K444R) were also reported. High prevalence of beta and delta variants in local population may increase the number of cases in the near future and provides an early warning to national health authorities to take timely decisions and devise suitable interventions to contain a possible fourth wave.

Genomic Characterization of Dengue Virus Outbreak in 2022 from Pakistan.

Pakistan, a dengue-endemic country, has encountered several outbreaks during the past decade. The current study aimed to explore the serotype and genomic diversity of dengue virus responsible for the 2022 outbreak in Pakistan. From August to October 2022, NS-1 positive blood samples (n = 343) were collected from dengue patients, among which, (85%; n = 293) were positive based on RT-PCR. In terms of gender and age, dengue infection was more prevalent in male patients (63%; n = 184), with more adults (21-30 years; n = 94) being infected. The serotyping results revealed DENV-2 to be the most predominant serotype (62%; n = 183), followed by DENV-1 (37%; n = 109) and DENV-3 (0.32%; n = 1). Moreover, a total of 10 samples (DENV-2; n = 8, DENV-1; n = 2) were subjected to whole-genome sequencing. Among these, four were collected in early 2022, and six were collected between August and October 2022. Phylogenetic analysis of DENV-2 sequenced samples (n = 8) revealed a monophyletic clade of cosmopolitan genotype IVA, which is closely related to sequences from China and Singapore 2018, and DENV-1 samples (n = 2) show genotype III, which is closely related to Pakistan isolates from 2019. We also reported the first whole genome sequence of a coinfection case (DENV1-DENV2) in Pakistan detected through a meta-genome approach. Thus, dengue virus dynamics reported in the current study warrant large-scale genomic surveillance to better respond to future outbreaks.

Comparable Overall Survival in Patients with Hepatocellular Carcinoma Diagnosed within and outside a Surveillance Programme: The Potential Impact of Liver Cirrhosis.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death, and its incidence is rising. Mortality from HCC is predicted to increase by 140% by 2035. Surveillance of high-risk patients with cirrhosis or chronic liver disease may be one means of reducing HCC mortality, but the level of supporting evidence for international guidelines is low/moderate. This study explores the real-world experience of HCC surveillance at a tertiary referral centre. Electronic patient records for all new HCCs diagnosed between August 2012 and December 2021 were retrospectively reviewed. Patient and tumour characteristics were evaluated, including the co-existence of chronic liver disease, cancer treatment and survival, and categorised according to HCC diagnosis within or outside a surveillance programme. Patients with HCC who presented through surveillance had smaller tumours diagnosed at an earlier stage, but this did not translate into improved overall survival. All patients in surveillance had chronic liver disease, including 91% (n = 101) with cirrhosis, compared to 45% (n = 29) in the non-surveillance cohort. We propose that the immune dysfunction associated with cirrhosis predisposes patients to a more aggressive tumour biology than the largely non-cirrhotic population in the non-surveillance group.

Identification and Functional Characterization of Mutation in FYCO1 in Families with Congenital Cataract.

Congenital cataract (CC) causes a third of the cases of treatable childhood blindness worldwide. CC is a disorder of the crystalline lens which is established as clinically divergent and has complex heterogeneity. This study aimed to determine the genetic basis of CC. Whole blood was obtained from four consanguineous families with CC. Genomic DNA was extracted from the blood, and the combination of targeted and Sanger sequencing was used to identify the causative gene. The mutations detected were analyzed in silico for structural and protein-protein interactions to predict their impact on protein activities. The sequencing found a known FYCO1 mutation (c.2206C>T; p.Gln736Term) in autosomal recessive mode in families with CC. Co-segregation analysis showed affected individuals as homozygous and carriers as heterozygous for the mutation and the unaffected as wild-type. Bioinformatics tools uncovered the loss of the Znf domain and structural compactness of the mutant protein. In conclusion, a previously reported nonsense mutation was identified in four consanguineous families with CC. Structural analysis predicted the protein as disordered and coordinated with other structural proteins. The autophagy process was found to be significant for the development of the lens and maintenance of its transparency. The identification of these markers expands the scientific knowledge of CC; the future goal should be to understand the mechanism of disease severity. Ascertaining the genetic etiology of CC in a family member facilitates establishing a molecular diagnosis, unlocks the prospect of prenatal diagnosis in pregnancies, and guides the successive generations by genetic counseling.

Vaccinomics Approach for Multi-Epitope Vaccine Design against Group A Rotavirus Using VP4 and VP7 Proteins.

Rotavirus A is the most common cause of Acute Gastroenteritis globally among children <5 years of age. Due to a segmented genome, there is a high frequency of genetic reassortment and interspecies transmission which has resulted in the emergence of novel genotypes. There are concerns that monovalent (Rotarix: GlaxoSmithKline Biologicals, Rixensart, Belgium) and pentavalent (RotaTeq: MERCK & Co., Inc., Kenilworth, NJ, USA) vaccines may be less effective against non-vaccine strains, which clearly shows the demand for the design of a vaccine that is equally effective against all circulating genotypes. In the present study, a multivalent vaccine was designed from VP4 and VP7 proteins of RVA. Epitopes were screened for antigenicity, allergenicity, homology with humans and anti-inflammatory properties. The vaccine contains four B-cell, three CTL and three HTL epitopes joined via linkers and an N-terminal RGD motif adjuvant. The 3D structure was predicted and refined preceding its docking with integrin. Immune simulation displayed promising results both in Asia and worldwide. In the MD simulation, the RMSD value varied from 0.2 to 1.6 nm while the minimum integrin amino acid fluctuation (0.05-0.1 nm) was observed with its respective ligand. Codon optimization was performed with an adenovirus vector in a mammalian expression system. The population coverage analysis showed 99.0% and 98.47% in South Asia and worldwide, respectively. These computational findings show potential against all RVA genotypes; however, in-vitro/in-vivo screening is essential to devise a meticulous conclusion.

Molecular docking and pharmacophore models to probe binding hypothesis of inhibitors of hypoxia inducible factor-1.

Hypoxia inducible factor-1 is a heterodimeric transcription factor that regulates cellular responses to hypoxia and is involved in tumor progression and resistance to chemotherapy. Dimerization between HIF-1α and ß subunits has been recognized crucial for DNA binding and transcriptional activity of HIF-1. Therefore, inhibitors of α and ß dimerization subunits of HIF-1 may potentially evade HIF-1-mediated chemotherapy resistance. In the current study, ligand-based pharmacophore model was developed to determine 3 D binding features of HIF-1 inhibitors. The selected pharmacophore model comprises of one hydrogen bond donor, one hydrogen bond acceptor and one hydrophobic feature. The selected model was used for virtual screening of publically available data base by ChemBridge Corporation. Overall, six potential hits against HIF-1α and ß dimerization have been identified. These include, Hit 1 (4-(4-chlorophenyl)-2,6-dimethyl-3,5-pyridinedicarboxylic acid), 3 (2-[2-(2-hydroxybenzoyl)carbonohydrazonoyl]benzoic acid) and 5 (3-(4-methoxyphenyl)-2,4-quinolinedicarboxylic acid) nicotonic acid derivatives, Hit 2 (3-[(1-adamantylamino)sulfonyl]benzoic acid), 4 (5-{[(2-fluorophenyl)amino]sulfonyl}-2-methylbenzoic acid), and 6 (4-({[2-(trifluoromethyl)phenyl]sulfonyl}amino)benzoic acid) sulfonamide derivatives. Additionally, adamantyl moiety of compound 2 shows interactions with the experimentally known hydrophobic amino acid residues (V336, C334, E245) of HIF-1α and ß dimerization site. The identified hits showed lower to higher µM biological activity (IC50) values and thus, after further structure optimization may serve as potential inhibitor of HIF-1 dimerization in cancer chemotherapy.Communicated by Ramaswamy H. Sarma.

Breakthrough cases of Omicron and Delta variants of SARS-CoV-2 during the fifth wave in Pakistan.

COVID-19 pandemic has severely affected Pakistan with 1,557,134 cases as of August 4, 2022. However, the data regarding breakthrough infections in Pakistan is scant. Hence, the objective was to analyze SARS-CoV-2 breakthrough infections with respect to vaccines and variants during the fifth wave in Pakistan. Therefore, the Department of Virology (NIH, Pakistan) genotyped 2,467 randomly selected individuals between November 2021 and February 2022 using the SNPsig® SARS-CoV-2 (EscapePLEX) kit (PrimerDesign, UK). P681R and K417N mutations were used to distinguish delta and omicron. Data on the patient's age, gender, date of collection, variant, and vaccination status were analyzed using Statistical Package for Social Sciences (SPSS) software. Among 2,467 genotyped samples, Omicron was detected in 58.6% (n = 1445), Delta in 40.4% (n = 998) and undetermined/wildtype variant in 24 samples. The vaccination status of omicron-positive patients showed (49.7%; n = 718/1445) and Delta-positive patients (39.67%; n = 396/998) to be fully vaccinated. Of note, a high percentage 85% of breakthrough cases (n = 947) were identified among fully vaccinated individuals (n = 1114). Among them, 85.9% (n = 617/718) belonged to omicron and 83.3% (n = 330/396) to delta. Moreover, 76.7% (n = 855) of vaccinated individuals (n = 1114) received Sinopharm (n = 432) and Sinovac (n = 423) vaccines. The majority of breakthrough subjects who contracted Omicron were vaccinated with Sinopharm (93.0%, n = 256) and delta with Cansino (100%, n = 44). Individuals vaccinated with Sinovac showed the most frequent breakthrough cases for both Omicron and Delta variant between the 4th and 6th months (n = 278) after primary vaccination as compared to the 7th to 9th months (n = 24) category. While in case of Sinopharm, maximum breakthrough cases occurred between 7th to 9th months (n = 234) as compared to the 4th to 6th months (n = 120) after primary vaccination. Omicron and Delta breakthrough cases in men (n = 364 and 193) are more frequently seen than women (n = 253 and 138) respectively and breakthrough majority cases (n = 392) occurred in individuals aged 18-33 years. Breakthrough cases limiting monitoring in Pakistan impose a substantial constraint on policymakers' ability to take timely effective decisions. Since the current study consists of only a 2,467-genotyped sample, comprehensive data should be analyzed.

Proliferation of SARS-CoV-2 B.1.1.7 Variant in Pakistan-A Short Surveillance Account.

The emergence of a more transmissible variant of SARS-CoV-2 (B1. 1.7) in the United Kingdom (UK) during late 2020 has raised major public health concerns. Several mutations have been reported in the genome of the B.1.1.7 variant including the N501Y and 69-70deletion in the Spike region that has implications on virus transmissibility and diagnostics. Although the B.1.1.7 variant has been reported by several countries, only three cases have been reported in Pakistan through whole-genome sequencing. Therefore, the objective of the study was to investigate the circulation of B.1.1.7 variant of concern (VOC) in Pakistani population. We used a two-step strategy for the detection of B.1.1.7 with initial screening through TaqPathTM COVID-19 CE-IVD RT-PCR kit (ThermoFisher Scientific, Waltham, US) followed by partial spike (S) gene sequencing of a subset of samples having the spike gene target failure (SGTF). From January 01, 2021, to February 21, 2021, a total of 2,650 samples were tested for SARS-CoV-2 and 70.4% (n = 1,867) showed amplification of all the 3 genes (ORF, N, and S). Notably, 29.6% (n=783) samples have been SGTF that represented numbers from all the four provinces and suggest a rather low frequency during the first 3 weeks of January (n = 10, n = 13, and n = 1, respectively). However, the numbers have started to increase in the last week of January, 2021. During February, 726 (93%) cases of SGTF were reported with a peak (n = 345) found during the 3rd week. Based on the partial sequencing of SGTF samples 93.5% (n = 29/31) showed the characteristic N501Y, A570D, P681H, and T716I mutations found in the B.1.1.7 variant. In conclusion, our findings showed an upsurge of B.1.1.7 cases in Pakistan during February, 2021 affecting 15 districts and warranting large scale genomic surveillance, strengthening of laboratory network and implementation of appropriate control measures in the country.

Co-expression of HIF-1α, MDR1 and LAPTM4B in peripheral blood of solid tumors.

The hypoxic tumor microenvironment is the major contributor of chemotherapy resistance in solid tumors. One of the key regulators of hypoxic responses within the cell is the hypoxia inducible factor-1α (HIF-1α) that is involved in transcription of genes promoting cell survival and chemotherapy resistance. Multidrug resistance gene-1 (MDR1) and Lysosome-associated protein transmembrane 4B-35 (LAPTM4B-35) are among those notable players which augment their responses to cellular hypoxia. MDR1 is the hypoxia responsive gene involved in multidrug resistance phenotype while LAPTM4B-35 is involved in chemotherapy resistance by stabilizing HIF-1α and overexpressing MDR1. Overexpression of HIF-1α, MDR1 and LAPTM4B has been associated with poor disease outcome in many cancers when studied individually at tissue level. However, accessibility of the tissues following the course of chemotherapy for ascertaining chemotherapy resistance is difficult and sometimes not clinically feasible. Therefore, indication of hypoxic biomarkers in patient's blood can significantly alter the clinical outcome. Hence there is a need to identify a blood based marker to understand the disease progression. In the current study the expression of hypoxia associated chemotherapy resistance genes were studied in the peripheral blood lymphocytes of solid tumor patients and any potential correlation with disease progression were explored. The expression of HIF-1α, MDR1 and LAPTM4B was studied in blood of 72 breast, 42 ovarian, 32 colon and 21 prostate cancer patients through real time PCR analysis using delta cycle threshold method. The statistical scrutiny was executed through Fisher's Exact test and the Spearman correlation method. There was 12-13 fold increased in expression of HIF-1α, two fold increased in MDR1 and 13-14 fold increased in LAPTM4B mRNA level in peripheral blood of breast, ovarian, prostate and colon cancer patients. In the current study there was an association of HIF-1α, MDR1 and LAPTM4B expression with advanced tumor stage, metastasis and chemotherapy treated group in breast, ovarian, prostate and colon cancer patients. The Spearman analysis also revealed a positive linear association among HIF-1α, MDR1 and LAPTM4B in all the studied cancer patients. The elevated expression of HIF-1α, MDR1 and LAPTM4B in peripheral blood of solid tumor patients can be a predictor of metastasis, disease progression and treatment response in these cancers. However, larger studies are needed to further strengthen their role as a potential biomarker for cancer prognosis.

The Route to 'Chemobrain' - Computational probing of neuronal LTP pathway.

Chemotherapy causes deleterious side effects during the course of cancer management. The toxic effects may be extended to CNS chronically resulting in altered cognitive function like learning and memory. The present study follows a computational assessment of 64 chemotherapeutic drugs for their off-target interactions against the major proteins involved in neuronal long term potentiation pathway. The cancer chemo-drugs were subjected to induced fit docking followed by scoring alignment and drug-targets interaction analysis. The results were further probed by electrostatic potential computation and ligand binding affinity prediction of the top complexes. The study identified novel off-target interactions by Dactinomycin, Temsirolimus, and Everolimus against NMDA, AMPA, PKA and ERK2, while Irinotecan, Bromocriptine and Dasatinib were top interacting drugs for CaMKII. This study presents with basic foundational knowledge regarding potential chemotherapeutic interference in LTP pathway which may modulate neurotransmission and synaptic plasticity in patient receiving these chemotherapies.

Structural insights and characterization of human Npas4 protein.

Npas4 is an activity dependent transcription factor which is responsible for gearing the expression of target genes involved in neuro-transmission. Despite the importance of Npas4 in many neuronal diseases, the tertiary structure of Npas4 protein along with its physico-chemical properties is limited. In the current study, first we perfomed the phylogenetic analysis of Npas4 and determined the content of hydrophobic, flexible and order-disorder promoting amino acids. The protein binding regions, post-translational modifications and crystallization propensity of Npas4 were predicted through different in-silico methods. The three dimensional model of Npas4 was predicted through LOMET, SPARSKS-X, I-Tasser, RaptorX, MUSTER and Pyhre and the best model was selected on the basis of Ramachandran plot, PROSA, and Qmean scores. The best model was then subjected to further refinement though MODREFINER. Finally the interacting partners of Npas4 were identified through STRING database. The phylogenetic analysis showed the human Npas4 gene to be closely related to other primates such as chimpanzees, monkey, gibbon. The physiochemical properties of Npas4 showed that it is an intrinsically disordered protein with N-terminal ordered region. The post-translational modification analyses indicated absence of acetylation and mannosylation sites. Three potential phosphorylation sites (S108, T130 and T136) were found in PAS A domain whilst a single phosphorylation site (S273) was present in PAS B domain. The predicted tertiary structure of Npas4 showed that bHLH domain and PAS domain possess tertiary structures while the rest of the protein exhibited disorder property. Protein-protein interaction analysis revealed NPas4 interaction with various proteins which are mainly involved in nuclear trafficking of proteins to cytoplasm, activity regulated gene transcription and neurodevelopmental disorders. Moreover the analysis also highlighted the direct relation to proteins involved in promoting neuronal survival, plasticity and cAMP responsive element binding protein proteins. The current study helps in understanding the physicochemical properties and reveals the neuro-modulatory role of Npas4 in crucial pathways involved in neuronal survival and neural signalling hemostasis.

Characterization of evolutionary changes in hemagglutinin of influenza H1N1 virus: a computational analysis.

Influenza virus continues to evolve due to changes in the genome and the new strain of virus is more pathogenic then the previous strain. These changes may also help the virus to cross specie barrier and may also affect the binding pattern of virus.The main theme of the current study is the identification of changes in the hemagglutinin sequence of H1N1 virus from 1960 to 2011 and also how these changes affect the binding properties of virus. From 1960 to 2000 following important changes were observed: Ala198Asp and Gly225Glu in 1980; and Gly225Asp in 1999. From 1999 to 2011 many changes were observed, most of the changes were transient, but two of the changes, Gly225Asp and Ala227Glu, were consistent in the period of 1999-2010. These residues make the binding stronger. The important conserved residues are Asp190, Tyr98, His183 and Gln226. The current study will provide an understanding how virus evolve with the passage of time. The current study also helps to understand the changes in the binding pattern of virus. It will also help for the identification of new therapeutic targets.