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.

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.

Gene expression profiles and retinal potential of stem/progenitor cells derived from human iris and ciliary pigment epithelium.

The aim of our study was to isolate and characterize the properties of neurospheres and differentiated cellular progeny derived from iris and ciliary pigment epithelial (IPE and CPE) cells of human cadaveric eyes. In this study we investigated the gene expression profiles of the stem/progenitor cells and the differentiated progeny derived from IPE and CPE cells, as the changes underlying differentiation of the stem/progenitor derived from the IPE and CPE cells from human cadaveric eye are essentially unknown. The IPE and CPE cells from human cadaver eyes were cultured in the presence of mitogens to generate neurospheres (NS) and the growth characteristics were evaluated. The Neurospheres (NS) were plated under conditions inducing differentiation and their potential was analyzed by RT-PCR, immunocytochemistry, calcium imaging studies and microarray studies to measure the changes involved in the process of differentiation. The IPE and CPE cells can generate NS containing progenitor cells in the presence of mitogens and were capable of producing different retinal cell types as demonstrated by RT-PCR and immunocytochemistry. The cluster analyses of the differentially expressed genes show the dynamic changes that occur during the course of IPE and CPE neurospheres differentiating into retinal progeny. The study gives clues towards the changes that occur during differentiation from NS into retinal progeny. In the present study we have demonstrated the expansion and maintenance of SCs from IPE and CPE of cadaveric eyes. These cells maintain their self-renewal properties and the ability to differentiate along retinal cell lineages and hence could be a practical source of donor cells for ex-vivo stem cell therapy during retinal degeneration.

Fluorescence amplified fragment length polymorphism for subtyping of genotypes of Acanthamoeba isolated from patients with keratitis.

BACKGROUND & OBJECTIVES: Acanthamoeba keratitis (AK) is a painful and vision-threatening ocular infection. The differentiation of Acanthamoeba at the species and subspecies level is complicated. Nearly all the AK isolates have been shown to belong to T4 genotype when analysed by ribosomal RNA gene sequences and there is no universally acceptable method for differentiation of different subtypes of T4. The purpose of this study was to attempt further discrimination of T4 genotypes. METHODS: In the present investigation, 15 Acanthamoeba isolates obtained from cornea of keratitis patients were subjected to fluorescence amplified fragment length polymorphism (FAFLP) genotyping to differentiate T4 subtypes. RESULTS: FAFLP profiles showed five distinct clusters (I to V) within T4 clonal complex which clearly depicted genetic differences among the isolates of T4 sequence type of Acanthamoeba. INTERPRETATION & CONCLUSIONS: Our study demonstrated the usefulness of FAFLP for reliable differentiation of T4 clonal complex of Acanthamoeba.

Expression of high mobility group A2 protein in retinoblastoma and its association with clinicopathologic features.

Retinoblastoma (RB) is the commonest primary intraocular tumor in children. Overexpression of the high mobility group (HMG) A2 protein has been observed in a variety of malignant tumors and often correlates with poor prognosis. We studied the expression of HMGA2 in primary tumor samples and correlated with clinicopathologic features such as invasion, differentiation, and laterality of the tumors. Among 64 tumors, there were 29 tumors with invasion of the optic nerve, choroid, and/or orbit and 35 tumors without invasion. HMGA2 immunoreactivity was evaluated on archival paraffin sections and the results confirmed by Western blotting on 12 fresh tumor samples. Among 29 tumors with invasion, HMGA2 was strongly positive (++) in 10 tumors, moderately positive (+) in 11 tumors. Among 35 tumors without invasion, HMGA2 was strongly positive (++) in 6 tumors, moderately positive (+) in 6 tumors. Tumors with invasion showed significantly higher expression of HMGA2 compared with tumors without invasion (P<0.01). Non-neoplastic retina was negative for HMGA2. There was no correlation between HMGA2 expression and differentiation/laterality. Western blotting revealed that 7 tumors were strongly positive, 2 were moderately positive, and 1 was faintly positive for HMGA2. Our study has demonstrated the HMGA2 expression in a large cohort of primary retinoblastoma tumors and its correlation with invasiveness.

Detection of human papillomavirus DNA in retinoblastoma samples: a preliminary study.

Recent studies have shown the presence of human papillomavirus (HPV) genome in retinoblastoma (RB) tumor samples. There is no information on the HPV status in the RB tumors of Indian patients. We studied the presence of HPV genome in RB tumor samples from patients with unilateral tumor. Forty-four fresh RB tumor samples and 30 non-neoplastic donor retinas were analyzed for the presence of HPV 16 and 18 genome by nested and seminested polymerase chain reaction. Tumor tissue sections were also used to assess the expression of the retinoblastoma (Rb) protein. All 30 control tissues were negative for HPV genome. Among the 44 tumor samples, there were 23 tumors with invasion of optic nerve/choroid and 21 tumors with no invasion. HPV DNA was present in 21/44 (47%) RB tumors. Among 21 unilateral RB tumors that were positive for HPV DNA, HPV 16 was detected in 12/21 (57%) tumors. However, HPV 18 was negative in all the tumors. Rb protein was absent in 16 (71%) of 21 tumors that had HPV DNA. However, Rb was also absent in 20 (86%) of 23 tumors that were HPV negative. Children younger than 18 months old were significantly associated with the presence of HPV DNA compared with children above 24 months old (P<0.014). Our study shows the presence of HPV and HPV 16 in a subset of RB tumor samples. However, further studies are in progress to know the role played by HPV in RB.

Effect of human tears on acanthamoeba-induced cytopathic effect.

OBJECTIVE: To determine whether tears of healthy individuals provide protection against Acanthamoeba-induced cytopathic effect (CPE) in vitro. METHODS: Acanthamoebae were added to confluent cultures of corneal epithelium in 24-well plates, and co-cultures were incubated overnight in a serum-free medium containing varying amounts of tears or immunoglobulin A (IgA)-depleted tears. At the end of the incubation period, the cells were stained with Giemsa, and the extent of target cell damage (ie, CPE) was quantified. RESULTS: Acanthamoebae produced extensive CPE. The presence of even a low concentration of tears (10 microL of undiluted tears per milliliter of media) almost completely inhibited Acanthamoeba-induced CPE. The CPE was inhibited by pretreatment of the parasites with tears. In contrast, the pretreatment of host cells with tears was not protective. This finding suggests that the target of the inhibitory factor is the parasite. IgA-depleted tears also inhibited Acanthamoeba-induced CPE, albeit with a lower potency than total tears. CONCLUSION: In addition to known IgA-dependent protective factors, human tears contain factors that inhibit Acanthamoeba-induced CPE independently of IgA. Clinical Relevance Identification and characterization of factors that protect against Acanthamoeba-induced CPE should help in the development of novel, rationally designed strategies to manage and protect against keratitis.

Acanthamoeba keratitis in non-contact lens wearers in India: DNA typing-based validation and a simple detection assay.

OBJECTIVES: To establish that the protozoan Acanthamoeba is one of the causative organisms associated with non-contact lens-related keratitis in the Indian population and to develop a simple and sensitive diagnostic assay for clinical testing. DESIGN: DNA sequencing of nuclear 18S and 26S ribosomal DNA motifs was performed and compared with the reference Acanthamoeba strains, to establish the genetic identity of the putative amoeba isolates obtained from the corneal scrapings of non-contact lens-wearing patients with keratitis. Ribosomal DNA typing of clinical corneal scrapings from the patients with keratitis was performed by means of a simple agarose gel-based multiplex polymerase chain reaction assay, to detect the cases of Acanthamoeba keratitis. RESULTS: The ribosomal DNA analysis of 15 putative amoeba isolates obtained from the corneal scrapings of 14 patients with keratitis and 1 from the patients' environment established the isolates to be pathogenic formsof Acanthamoeba belonging to type T4 ribosomal DNA genotype. Multiplex polymerase chain reaction assay was specific and sensitive enough to detect as low as 5 pg of Acanthamoeba DNA. Its utility as a reliable diagnostic assay was demonstrated directly with the use of 34 additional corneal scrapings. CONCLUSIONS: Acanthamoeba is one of the causative organisms of keratitis in Indian patients with no history of contact lens usage. Moreover, the Acanthamoeba infection can be easily detected in the clinical samples by means of the simple multiplex polymerase chain reaction assay based on ribosomal DNA typing. Clinical Relevance This study suggests the need and means to determine the incidence and prevalance of Acanthamoeba keratitis in India and elsewhere. Moreover, the polymerase chain reaction assay would help in early and definitive diagnosis, leading to better prognosis of Acanthamoeba keratitis condition.

Multiplex polymerase chain reaction for the detection of herpes simplex virus, varicella-zoster virus and cytomegalovirus in ocular specimens.

PURPOSE: A majority of ocular viral diseases are caused by herpes group of viruses. Such infections, especially atypical herpetic keratitis, iridocyclitis and intra-ocular inflammations, can often present with overlapping clinical manifestations misleading the diagnosis. Molecular techniques are most useful in such instances for an accurate and rapid diagnosis since conventional methods are time consuming and less sensitive. A multiplex PCR was developed and used for the detection of herpes simplex virus (HSV), varicella zoster virus (VZV), and cytomegalovirus (CMV) in ocular samples. METHODS: One hundred and forty six ocular samples (corneal scrapings - 52, aqueous fluid - 36, vitreous fluid - 31, tissues - 26, skin vesicle scraping - 1) were included in the study. The sensitivity of the assay was determined using serial dilutions of standard strains of HSV, VZV, and CMV vis-à-vis plaque forming assay. RESULTS: The sensitivity of the assay was 4, 4 and 12 PFU/ml or 20, 20 and 60 genome copy numbers of HSV, VZV and CMV respectively. Using DNA from various sources (fungal, bacterial, human leukocytes, tissues) along with standard positive controls, the assay was found to be highly specific. HSV DNA was detected in majority of the clinical samples (33.6%), most frequent being corneal samples. Comparatively, VZV and CMV infections were detected in small number of samples (VZV-3, CMV-2). CONCLUSIONS: We found the assay very useful in our set-up whenever a differential diagnosis of herpetic infections was suggested by the ophthalmologist. The multiplex PCR we have described here can be of greater value in clinics with larger number of patients suspected of having HSV, VZV or CMV infections.

Use of 18S rRNA gene-based PCR assay for diagnosis of acanthamoeba keratitis in non-contact lens wearers in India.

Identification of Acanthamoeba cysts and trophozoites in ocular tissues requires considerable expertise and is often time-consuming. An 18S rRNA gene-based PCR test, highly specific for the genus Acanthamoeba, has recently been reported in the molecular diagnosis of Acanthamoeba keratitis. This PCR assay was compared with conventional microbiological tests for the diagnosis of Acanthamoeba keratitis. In a pilot study, the PCR conditions with modifications were first tested on corneal scrapings from patients with culture-proven non-contact lens-related Acanthamoeba, bacterial, and fungal keratitis. This was followed by testing of corneal scrapings from 53 consecutive cases of microbial keratitis to determine sensitivity, specificity, and predictive values of the assay. All corneal scrapings from patients with proven Acanthamoeba keratitis showed a 463-bp amplicon, while no amplicon was obtained from patients with bacterial or fungal keratitis. Some of these amplified products were sequenced and compared with EMBL database reference sequences to validate these to be of Acanthamoeba origin. Out of 53 consecutive cases of microbial keratitis included for evaluating the PCR, 10 (18.9%) cases were diagnosed as Acanthamoeba keratitis on the basis of combined results of culture, smear, and PCR of corneal scrapings. Based on culture results as the "gold standard," the sensitivity of PCR was the same as that of the smear (87.5%); however, the specificity and the positive and negative predictive values of PCR were marginally higher than the smear examination (97.8 versus 95.6%, 87.5 versus 77.8%, and 97.8 versus 97.7%) although the difference was not significant. This study confirms the efficacy of the PCR assay and is the first study to evaluate a PCR-based assay against conventional methods of diagnosis in a clinical setting.