Current challenges: from the path of "original antigenic sin" towards the development of universal flu vaccines.

Annual flu led by influenza viruses is contemplated to be one of the foremost global health challenges due to its rapid spread leading to the life-threatening epidemic or pandemic. An enormous number of people die due to flu and its associated intricacies every year. Annual vaccination is considered to be the gold standard strategy to protect the individual from acquiring infection and further decimation, although recent estimates suggest that overall flu vaccine effectiveness was within 19% to 53% in last five years. A significant weakness of current vaccination is its inability to protect an individual from different or mutant flu strain. Host immune system performs a vital role during natural infection or after vaccination leading to influenza-specific immunities. Previous imprints of common flu or vaccination may alter the outcomes of the current vaccination. Current flu vaccine regime does not consider the host immune status before vaccination. Irrespective of the previous influenza exposure history or prior flu vaccination, individual get flu vaccination based on WHO recommendation with selected strains which may be the reason why induction of broad immunities does not transpire with their testimonial. Over the last few decades, scientific research had identified the role of preexisting immunities on vaccination or natural infection outcome. In this review, we are proposing the concept of personalized flu vaccines depending on individual immune status. We will also discuss why individual was unable to induce broader immunities to protect itself from diverse influenza viruses and how we can accomplish that goal with the current findings.

Isolation and Characterization of Novel Broad Host Range Bacteriophages of Vibrio cholerae O1 from Bengal.

OBJECTIVES: We have isolated a total of five newer cholera phages which are novel broad host range to incorporate with the existing phage typing schemes for an extended typing scheme. MATERIALS AND METHODS: These newly isolated phages were well characterized including the electron micrograph. A total of 300 Vibrio cholerae strains were isolated from the different endemic region in India were included in phage typing study. RESULTS: These phages were found different from the existing phages. Electron microscopic results showed that the phages belonged to myophage and podophage group. Characterization of the phages based on pH, temperature, and organic solvent sensitivity showed differences among the phages used in this study. All the strains of Vibrio O1 were typeable (100%) with the five set of cholera phages. Of these, 40% strains were clustered under Type-1. CONCLUSION: The newer Vibrio phages are novel and broad host range and will be useful to incorporate with the existing phage typing system for more precisely discriminate the strains of Vibrio cholerae.

Apoptotic and Early Innate Immune Responses to PB1-F2 Protein of Influenza A Viruses Belonging to Different Subtypes in Human Lung Epithelial A549 Cells.

PB1-F2 is a multifunctional protein and contributes to the pathogenicity of influenza A viruses. PB1-F2 is known to have strain and cell specific functions. In this study we have investigated the apoptotic and inflammatory responses of PB1-F2 protein from influenza viruses of diverse pathogenicities in A549 lung epithelial cells. Overexpression of PB1-F2 resulted in apoptosis and heightened inflammatory response in A549 cells. Comparison revealed that the response varied with each subtype. PB1-F2 protein from highly pathogenic H5N1 virus induced least apoptosis but maximum inflammatory response. Results indicated that apoptosis was mediated through death receptor ligands TNFα and TRAIL via Caspase 8 activation. Significant induction of cytokines/chemokines CXCL10, CCL5, CCL2, IFNα, and IL-6 was noted in A549 cells transfected with PB1-F2 gene construct of 2008 West Bengal H5N1 virus (H5N1-WB). On the contrary, PB1-F2 construct from 2007 highly pathogenic H5N1 isolate (H5N1-M) with truncated N-terminal region did not evoke as exuberant inflammatory response as the other H5N1-WB with full length PB1-F2, signifying the importance of N-terminal region of PB1-F2. Sequence analysis revealed that PB1-F2 proteins derived from different influenza viruses varied at multiple amino acid positions. The secondary structure prediction showed each of the PB1-F2 proteins had distinct helix-loop-helix structure. Thus, our data substantiate the notion that the contribution of PB1-F2 to influenza pathogenicity is greatly strain specific and involves multiple host factors. This data demonstrates that PB1-F2 protein of influenza A virus, when expressed independently is minimally apoptotic and strongly influences the early host response in A549 cells.

Gene expression profile of high-fat diet-fed C57BL/6J mice: in search of potential role of azelaic acid.

High-fat diet (HFD) elevates circulatory fatty acids and influences glucose and fat metabolism. Azelaic acid (AzA), a naturally occurring α,ω-dicarboxylic acid in wheat, rye, barley, oat seeds and sorghum, has been reported to exert antidiabetic effects in HFD-induced type 2 diabetes mellitus (T2DM) C57BL/6J mice. The present study was undertaken to identify the genes that are differentially modulated by treatment with AzA in HFD-fed mice. Mice were fed HFD for 10 weeks and subjected to intragastric administration of 80 mg/kg body weight (BW) of AzA daily along with HFD from 11 to 15 weeks. Lipid profile, adipokines and cytokines were examined in the plasma/liver of mice. Whole genome profiling was performed in the liver of mice using microarray and validated by qRT-PCR, Western blot and immunohistochemical analyses. HFD intake resulted in significantly elevated lipids (except high-density lipoproteins), resistin, tumour necrosis factor alpha and interleukin-6 with marked reduction in adiponectin. Administration of AzA to HFD-fed mice significantly restored the lipids, adipokines and cytokines to near normal. Transcript profiling revealed that HFD intake activated the genes involved in stress response, cell cycle regulation and apoptosis. Treatment with AzA caused increased expression of genes involved in reactive oxygen species (ROS) scavenging, receptor-mediated signalling, transcription, protein modification and insulin signal transduction. AzA activates insulin signal molecules leading to insulin sensitivity. The ability of AzA to modulate the expression of these genes supports the notion that AzA is a promising drug candidate for the treatment of insulin resistance associated with T2DM.

Gene expression profile of high-fat diet-fed C57BL/6J mice: in search of potential role of azelaic acid

High-fat diet (HFD) elevates circulatory fatty acids and influences glucose and fat metabolism.Azelaic acid (AzA), a naturally occurring alpha,ω-dicarboxylic acid in wheat, rye, barley, oat seeds and sorghum, has been reported to exert antidiabetic effects in HFD-induced type 2 diabetes mellitus (T2DM) C57BL/6J mice. The present study was undertaken to identify the genes that are differentially modulated by treatment with AzA in HFD-fed mice. Mice were fed HFD for 10 weeks and subjected to intragastric administration of 80 mg/kg body weight (BW) of AzA daily along with HFD from 11 to 15 weeks. Lipid profile, adipokines and cytokines were examined in the plasma/liver of mice. Whole genome profiling was performed in the liver of mice using microarray and validated by qRT-PCR, Western blot and immunohistochemical analyses. HFD intake resulted in significantly elevated lipids (except high-density lipoproteins), resistin, tumour necrosis factor alpha and interleukin-6 with marked reduction in adiponectin. Administration of AzA to HFD-fed mice significantly restored the lipids, adipokines and cytokines to near normal. Transcript profiling revealed that HFD intake activated the genes involved in stress response, cell cycle regulation and apoptosis. Treatment with AzA caused increased expression of genes involved in reactive oxygen species (ROS) scavenging, receptor-mediated signalling, transcription, protein modification and insulin signal transduction. AzA activates insulin signal molecules leading to insulin sensitivity. The ability of AzA to modulate the expression of these genes supports the notion that AzA is a promising drug candidate for the treatment of insulin resistance associated with T2DM

Infection with influenza A viruses causes changes in promoter DNA methylation of inflammatory genes.

BACKGROUND: Replication of influenza virus in the host cells results in production of immune mediators like cytokines. Excessive secretion of cytokines (hypercytokinemia) has been observed during highly pathogenic avian influenza virus (HPAI-H5N1) infections resulting in high fatality rates. OBJECTIVE: The exact mechanism of hypercytokinemia during influenza virus infection is still not known completely. As promoter DNA methylation changes are linked with expression changes in genes, we intend to identify whether changes in promoter DNA methylation have any role in expression of cytokines during influenza A virus infection. METHODS: A panel of 24 cytokine genes and genes known to be involved in inflammatory response were analyzed for their promoter DNA methylation changes during influenza A virus infections. Four different strains of influenza A viruses, viz. H5N1, H1N1, pandemic (2009) H1N1, and a vaccine strain of H5N1, were used for the study. RESULTS: We found seven of the total 24 inflammatory genes studied, showing significant changes in their promoter methylation levels in response to virus infection. These genes included proinflammatory cytokines CXCL14, CCL25, CXCL6, and interleukines IL13, IL17C, IL4R. The changes in DNA methylation levels varied across different strains of influenza viruses depending upon their virulence. Significant promoter hypomethylation in IL17C and IL13 genes was observed in cells infected with HPAI-H5N1 virus compared with other influenza viruses. This decrease in methylation was found to be positively correlating with the increased expression of these genes. Analysis of IL17C promoter region using bisulfite sequencing resulted in identification of a CpG site within Retinoid X receptor-alpha (RXR-α) transcription factor binding site undergoing demethylation specifically in H5N1-infected cells but not in other influenza-infected cells. CONCLUSION: Thus, the study could demonstrate that changes in promoter methylation in certain specific cytokine genes actually have a possible role in their expression changes during influenza A virus infection.

An insight into the PB1F2 protein and its multifunctional role in enhancing the pathogenicity of the influenza A viruses.

PB1F2 is the 11th protein of the influenza A virus. The protein has variable sizes with truncations either at the C- or N-terminal ends. The most recent example being the 2009 pandemic H1N1 virus which codes for only 11 amino-acids of the C-terminus. A review of the reports since the discovery of PB1F2 in 2001 suggests a multifunctional role for this protein that includes a proapoptotic function in immune cells and an ability to cause increased pathogenesis in animal models by dysregulating cytokines and inducing inflammation. It has also been suggested that PB1F2 regulates polymerase activity via co-localization with PB1 and causes enhanced secondary bacterial pneumonia. This review primarily focuses on understanding the proapoptotic ability of PB1F2, its sub-cellular localization and the mechanism through which it brings about apoptosis. We believe there is much more to learn about PB1F2, as many of its proposed functions are strain, host or cell-line specific.

Comprehensive global amino acid sequence analysis of PB1F2 protein of influenza A H5N1 viruses and the influenza A virus subtypes responsible for the 20th-century pandemics.

BACKGROUND: PB1F2 is the 11th protein of influenza A virus translated from +1 alternate reading frame of PB1 gene. Since the discovery, varying sizes and functions of the PB1F2 protein of influenza A viruses have been reported. Selection of PB1 gene segment in the pandemics, variable size and pleiotropic effect of PB1F2 intrigued us to analyze amino acid sequences of this protein in various influenza A viruses. METHODS: Amino acid sequences for PB1F2 protein of influenza A H5N1, H1N1, H2N2, and H3N2 subtypes were obtained from Influenza Research Database. Multiple sequence alignments of the PB1F2 protein sequences of the aforementioned subtypes were used to determine the size, variable and conserved domains and to perform mutational analysis. RESULTS: Analysis showed that 96·4% of the H5N1 influenza viruses harbored full-length PB1F2 protein. Except for the 2009 pandemic H1N1 virus, all the subtypes of the 20th-century pandemic influenza viruses contained full-length PB1F2 protein. Through the years, PB1F2 protein of the H1N1 and H3N2 viruses has undergone much variation. PB1F2 protein sequences of H5N1 viruses showed both human- and avian host-specific conserved domains. Global database of PB1F2 protein revealed that N66S mutation was present only in 3·8% of the H5N1 strains. We found a novel mutation, N84S in the PB1F2 protein of 9·35% of the highly pathogenic avian influenza H5N1 influenza viruses. CONCLUSIONS: Varying sizes and mutations of the PB1F2 protein in different influenza A virus subtypes with pandemic potential were obtained. There was genetic divergence of the protein in various hosts which highlighted the host-specific evolution of the virus. However, studies are required to correlate this sequence variability with the virulence and pathogenicity.

Non structural protein of avian influenza A (H11N1) virus is a weaker suppressor of immune responses but capable of inducing apoptosis in host cells.

BACKGROUND: The Non-Structural (NS1) protein of Influenza A viruses is an extensively studied multifunctional protein which is commonly considered as key viral component to fight against host immune responses. Even though there has been a lot of studies on the involvement of NS1 protein in host immune responses there are still ambiguities regarding its role in apoptosis in infected cells. Interactions of NS1 protein with host factors, role of NS1 protein in regulating cellular responses and apoptosis are quite complicated and further studies are still needed to understand it completely. RESULTS: NS1 genes of influenza A/Chicken/India/WBNIV2653/2008 (H5N1) and A/Aquatic bird/India/NIV-17095/2007(H11N1) were cloned and expressed in human embryonic kidney (293T) cells. Microarray based approach to study the host cellular responses to NS1 protein of the two influenza A viruses of different pathogenicity showed significant differences in the host gene expression profile. NS1 protein of H5N1 resulted in suppression of IFN-ß mediated innate immune responses, leading to down-regulation of the components of JAK-STAT pathway like STAT1 which further suppressed the expression of pro-inflammatory cytokines like CXCL10 and CCL5. The degree of suppression of host immune genes was found considerable with NS1 protein of H11N1 but was not as prominent as with H5N1-NS1. TUNEL assay analyses were found to be positive in both the NS1 transfected cells indicating both H5N1 as well as H11N1 NS1 proteins were able to induce apoptosis in transfected cells. CONCLUSIONS: We propose that NS1 protein of both H5N1 and H11N1 subtypes of influenza viruses are capable of influencing host immune responses and possess necessary functionality to support apoptosis in host cells. H11N1, a low pathogenic virus without any proven evidence to infect mammals, contains a highly potential NS1 gene which might contribute to greater virus virulence in different gene combinations.

Pandemic (H1N1) 2009 influenza virus induces weaker host immune responses in vitro: a possible mechanism of high transmissibility.

BACKGROUND: The world has recently overcome the first influenza pandemic of the 21st century caused by a novel H1N1 virus (pH1N1) which is a triple reassortant comprising genes derived from avian, human, and swine influenza viruses and antigenically quite different from seasonal H1N1 strains. Although the case fatality rates have decreased in many developed countries, the situation is still alarming in many developing countries including India where considerable numbers of new cases are appearing everyday. There is still a high morbidity and mortality of susceptible adult as well as young population without having underlying health issues due to the influenza infection. RESULTS: To achieve a better understanding of the risk posed by the pH1N1 and to understand its pathogenicity, we studied the host gene expression response to Indian isolate of pH1N1 infection and compared it with seasonal H1N1 infection. The response was studied at four different time points (4, 8, 16 and 24 h) post infection (hpi) in A549 cells using microarray platform. We found that pH1N1 induces immune response earlier than seasonal H1N1 viruses, but at the later stages of infection there is a suppression of host immune responses. The infection with pH1N1 resulted in considerable decrease in the expression of cytokine and other immune genes namely IL8, STAT1, B2 M and IL4 compared to seasonal H1N1. CONCLUSION: We propose that the inability to induce strong innate immune response could be a reason for the high transmissibility, pathogenicity and mortality caused by pH1N1 virus.

Host gene expression profiling in influenza A virus-infected lung epithelial (A549) cells: a comparative analysis between highly pathogenic and modified H5N1 viruses.

BACKGROUND: To understand the molecular mechanism of host responses to highly pathogenic avian influenza virus infection and to get an insight into the means through which virus overcomes host defense mechanism, we studied global gene expression response of human lung carcinoma cells (A549) at early and late stages of infection with highly pathogenic avian Influenza A (H5N1) virus and compared it with a reverse genetics modified recombinant A (H5N1) vaccine virus using microarray platform. RESULTS: The response was studied at time points 4, 8, 16 and 24 hours post infection (hpi). Gene ontology analysis revealed that the genes affected by both the viruses were qualitatively similar but quantitatively different. Significant differences were observed in the expression of genes involved in apoptosis and immune responses, specifically at 16 hpi. CONCLUSION: We conclude that subtle differences in the ability to induce specific host responses like apoptotic mechanism and immune responses make the highly pathogenic viruses more virulent.

Characterization of the influenza A H5N1 viruses of the 2008-09 outbreaks in India reveals a third introduction and possible endemicity.

Widespread infection of highly pathogenic avian influenza A H5N1 was reported from backyard and commercial poultry in West Bengal (WB), an eastern state of India in early 2008. Infection gradually spread to Tripura, Assam and Sikkim, the northeastern states, with 70 outbreaks reported between January 2008 and May 2009. Whole genome sequence analysis of three isolates from WB, one isolate from Tripura along with the analysis of hemagglutinin (HA) and neuraminidase (NA) genes of 17 other isolates was performed during this study. In the HA gene phylogenetic tree, all the 2008-09 Indian isolates belonged to EMA3 sublineage of clade 2.2. The closest phylogenetic relationship was found to be with the 2007-09 isolates from Bangladesh and not with the earlier 2006 and 2007 Indian isolates implying a third introduction into the country. The receptor-binding pocket of HA1 of two isolates from WB showed S221P mutation, one of the markers predicted to be associated with human receptor specificity. Two substitutions E119A (2 isolates of WB) and N294S (2 other isolates of WB) known to confer resistance to NA inhibitors were observed in the active site of neuraminidase. Several additional mutations were observed within the 2008-09 Indian isolates indicating genetic diversification. Overall, the study is indicative of a possible endemicity in the eastern and northeastern parts of the country, demanding active surveillance specifically in view of the critical mutations that have been observed in the influenza A H5N1 viruses.

A unique influenza A (H5N1) virus causing a focal poultry outbreak in 2007 in Manipur, India.

BACKGROUND: A focal H5N1 outbreak in poultry was reported from Manipur, a north-eastern state, of India, in 2007. The aim of this study was to genetically characterize the Manipur isolate to understand the relationship with other H5N1 isolates and to trace the possible source of introduction of the virus into the country. RESULTS: Characterization of the complete genome revealed that the virus belonged to clade 2.2. It was distinctly different from viruses of the three EMA sublineages of clade 2.2 but related to isolates from wild migratory waterfowl from Russia, China and Mongolia. The HA gene, had the cleavage site GERRRRKR, earlier reported in whooper swan isolates from Mongolia in 2005. A stop codon at position 29 in the PB1-F2 protein could have implications on the replication efficiency. The acquisition of polymorphisms as seen in recent isolates of 2005-07 from distinct geographical regions suggests the possibility of transportation of H5N1 viruses through migratory birds. CONCLUSION: Considering that all eight genes of the earlier Indian isolates belonged to the EMA3 sublineage and similar strains have not been reported from neighbouring countries of the subcontinent, it appears that the virus may have been introduced independently.

Role of transmembrane domain and cytoplasmic tail amino acid sequences of influenza a virus neuraminidase in raft association and virus budding.

Influenza virus neuraminidase (NA), a type II transmembrane glycoprotein, possesses receptor-destroying activity and thereby facilitates virus release from the cell surface. Among the influenza A viruses, both the cytoplasmic tail (CT) and transmembrane domain (TMD) amino acid sequences of NA are highly conserved, yet their function(s) in virus biology remains unknown. To investigate the role of amino acid sequences of the CT and TMD on the virus life cycle, we systematically mutagenized the entire CT and TMD of NA by converting two to five contiguous amino acids to alanine. In addition, we also made two chimeric NA by replacing the CT proximal one-third amino acids of the NA TMD [NA(1T2N)NA] and the entire NA TMD (NATRNA) with that of human transferrin receptor (TR) (a type II transmembrane glycoprotein). We rescued transfectant mutant viruses by reverse genetics and examined their phenotypes. Our results show that all mutated and chimeric NAs could be rescued into transfectant viruses. Different mutants showed pleiotropic effects on virus growth and replication. Some mutants (NA2A5, NA3A7, and NA4A10) had little effect on virus growth while others (NA3A2, NA5A27, and NA5A31) produced about 50- to 100-fold-less infectious virus and still some others (NA5A14, NA4A19, and NA4A23) exhibited an intermediate phenotype. In general, mutations towards the ectodomain-proximal sequences of TMD progressively caused reduction in NA enzyme activity, affected lipid raft association, and attenuated virus growth. Electron microscopic analysis showed that these mutant viruses remained aggregated and bound to infected cell surfaces and could be released from the infected cells by bacterial NA treatment. Moreover, viruses containing mutations in the extreme N terminus of the CT (NA3A2) as well as chimeric NA containing the TMD replaced partially [NA(1T2N)NA] or fully (NATRNA) with TR TMD caused reduction in virus growth and exhibited the morphological phenotype of elongated particles. These results show that although the sequences of NA CT and TMD per se are not absolutely essential for the virus life cycle, specific amino acid sequences play a critical role in providing structural stability, enzyme activity, and lipid raft association of NA. In addition, aberrant morphogenesis including elongated particle formation of some mutant viruses indicates the involvement of NA in virus morphogenesis and budding.