Immunofluorescence and virus culture are the main methods used to diagnose acute respiratory virus infections. Diagnosing these infections using nucleic acid amplification presents technical challenges, one of which is facilitating the different optimal annealing temperatures needed for each virus. To overcome this problem we developed a diagnostic molecular strip which combined a generic nested touchdown protocol with in-house primer master-mixes that could recognise 12 common respiratory viruses.Over an 18 month period a total of 222 specimens were tested by both immunofluorescence and the molecular strip. The specimens came from 103 males (median age 3.5 y), 80 females (median age 9 y) and 5 quality assurance scheme specimens. Viruses were recovered from a number of specimen types including broncho-alveolar lavage, nasopharyngeal secretions, sputa, post-mortem lung tissue and combined throat and nasal swabs. Viral detection by IF was poor in sputa and respiratory swabs. A total of 99 viruses were detected in the study from 79 patients and 4 quality control specimens: 31 by immunofluorescence and 99 using the molecular strip. The strip consistently out-performed immunofluorescence with no loss of diagnostic specificity.The touchdown protocol with pre-dispensed primer master-mixes was suitable for replacing virus culture for the diagnosis of respiratory viruses which were negative by immunofluorescence. Results by immunofluorescence were available after an average of 4–12 hours while molecular strip results were available within 24 hours, considerably faster than viral culture. The combined strip and touchdown protocol proved to be a convenient and reliable method of testing for multiple viruses in a routine setting. Acute respiratory tract infections are major causes of morbidity and mortality. In 2000, lower respiratory tract infections were globally the number one infectious cause of disability adjusted life-years . The comAs part of service development it was necessary to provide an alternative to virus culture for testing immunofluorescence negative respiratory specimens. Historically and indeed currently immunofluorescence  and viruIncreasingly however, the sensitivity of nucleic acid amplification techniques for diagnosis has become recognised -10. HoweOne such challenge is the different optimal annealing temperatures of the primer sets for each prospective virus target. The ABI PRISM 7000 real-time facility from Applied Biosystems addresses this by using bundled software to design primer/probe combinations that use a common amplification protocol. However this approach is compromised by the inability of software to allow for target heterogeneity. In addition it does not allow users to adopt clinically validated primer sets from the literature.To address these problems we adopted an alternative approach through the development of a generic touchdown amplification protocol. Touchdown protocols involve a pre-designed stepped reduction in the annealing temperature used for primer-to-template binding, which introduces a competitive advantage for specific base-pair priming over non-specific priming . A detai3 copies per ml for human rhinovirus type 1b to 4.2 × 103 copies/ml for RSV-A. Specificity was ascribed through reproducibility, i.e. specimens which were repeatedly positive, following our standard clinical reporting algorithm [Before deciding on the layout of the molecular strip, as described in the methods, we undertook a wide range of preliminary validation steps for each primer set. The complexity of the strip makes it impossible to fully evaluate using the classical approach of applying an individual gold standard to each virus type. Classically this approach works well where a single target is under investigation . Howeverlgorithm , were relgorithm . In addiTo test the feasibility of its routine use we needed to clinically validate its performance in a routine setting on specimens tested in parallel with our standard immunofluorescence protocol for the diagnosis of acute virus respiratory infections. Although the routine immunofluoresence panel lacked capacity for the detection of rhinoviruses, human metapneumovirus and CoV-229E, these were included on the strip for clinical reasons during the period of the study. These findings and their implications are reported.A total of 99 viruses were detected in 84/222 specimens from a total of 79/183 patients and 4/5 National External Quality Assurance Scheme (NEQAS) controls; immunofluorescence did not detect the parainfluenza virus type 2 virus in one of the NEQAS specimens. Viruses were detected in all of the specimen types processed. The molecular strip detected virus in: 16/36 (44.4%) broncho-alveolar lavages, 62/120 (51.6%) nasopharyngeal secretions, 11/35 (31.4%) sputa and 10/31 (32.2%) combined throat and nasal swabs. Immunofluorescence detected virus in: 6/36 (16.6%) broncho-alveolar lavages, 23/120 (19.1%) nasopharyngeal secretions, 1/35 (2.8%) sputa and 1/31 (3.2%) combined throat and nasal swabs.The median age of male and female patients where virus was detected was 3 y (range 2 weeks – 79 years) and 4 y (5 weeks – 81 years) respectively. Sixteen viruses were detected in 14/27 (51.8%) specimens, confirming a respiratory virus in 12 out of 24 (50%) patients investigated in general practice. Seventy-nine viruses were detected in 70/191 (36.6%) specimens, confirming a respiratory virus in 67 out of 159 (42.1%) patients investigated in hospital. Of the 16 viruses detected in specimens from the community, PCR detected all 16 in contrast to a single identification, influenza A (H3), by immunofluorescence.PCR identified one or more viruses in specimens from 84 of the 183 patients and the 4 NEQAS positive specimens, detecting a total of 99 viruses as shown in Table Dual infections were detected in 11/79 (13.9%) patients. The dual infections were: influenza A (H3) and adenovirus (4); influenza A (H3) virus and rhinovirus (2); influenza A (H1) and adenovirus (1); adenovirus and rhinovirus (3); respiratory syncytial virus type B and rhinovirus (1).Nine patients had more than one specimen taken on the same day in which a virus was detected in at least one specimen by PCR. For 5 of the patients the same virus was detected in each of the 2 specimens. The viruses identified were rhinovirus (3), adenovirus (1) and parainfluenza type 3 (1); the latter was also immunofluorescence positive. In 2 cases a rhinovirus was detected in only one of the specimens. As part of a separate rhinovirus validation protocol one of these specimens was subjected to retesting coupled with limited sequencing of the 5' non-coding region amplicon which confirmed the presence of a rhinovirus sequence. Additionally, premature twin boys admitted to intensive care on the same day with severe bronchiolitis, both had evidence of acute rhinovirus infection by PCR. Limited sequencing of the 5' non-coding region of these viruses as part of the rhinovirus validation protocol confirmed the presence of an identical sequence of rhinovirus in both specimens.Immunofluorescence identified a virus in specimens from 28 of the 183 patients and 3/4 NEQAS positive specimens (16.4%), detecting a total of 31 viruses as shown in Table Although touchdown PCR has been used successfully to help overcome some of the uncertainties associated with the thermal amplification of microbial nucleic acid targets -22, its In this study the large number of targets is the main problem encountered. Using an empirical approach a series of amplification steps linked to a stepped reduction in annealing temperature from 55°C to 46°C was constructed. This proved extremely resilient when used with a wide range of primer sets and included the apparent anomaly of putting adenovirus through an initial reverse transcription step to stream line all of the targets on to a single strip; we have previously reported this approach for testing group F adenovirus alongside norovirus, astrovirus and rotavirus . The touWhere multiple viral targets are sought in clinical practice, we believe that it is only feasible to correlate the performance of the new assay in a head-to-head comparison with that already in routine use. Unfortunately for many clinical laboratories there is an elusion of testing for a wider range of viruses than is the case, by the inoculation of cell lines with a theoretical ability to grow the respective viruses. The annual reports of most clinical laboratories of one of the commonest human respiratory viruses, human rhinovirus, is an example of this; using the touchdown protocol we now report approximately 450 HRV infections per annum. The under reporting of adenovirus by standard methods  and the 3 and 2 × 103 copies per ml) with a generic matrix set (copy number sensitivity of 1 × 103 copies per ml).The ability to simultaneously validate the performance of multiple molecular primer sets in a routine clinical setting is a major accomplishment of the current methodological approach. The results demonstrated that a range of primers from both the medical literature and from in-house development could be amplified with a single generic touchdown protocol. It therefore confirmed the feasibility of directly incorporating primer sets into a standard operating procedure without the necessity for the individual optimisation of cycling parameters. As such the study results should facilitate primer selection and formal critical evaluation as here described. As an example of this enhanced flexibility we have recently replaced the primer sets for influenza A H1 and H3  cross-contamination during manual extraction and (b) contamination of second round adjacent wells with product from a first round positive amplification. The use of the QIAGEN BioRobot for the extraction of all specimens reduced the former while the nature of the strip prevents the latter, since all the wells have separate mixes Table . With boThe results obtained were very encouraging. Although the strip was constructed to detect a wider range of viruses than immunofluorescence, over the period of validation it almost doubled (59 versus 31) the number of viruses that could have been detected by immunofluorescence, including a positive NEQAS specimen which was missed by immunofluorescence. Of this group of viruses the detection of adenovirus showed the most dramatic increase, an observation we have also previously made in a separate study  and whicAs mentioned in the introduction a factor which often impacts negatively on a laboratory's decision to use molecular diagnostics is one of cost. It is worth considering that no matter which assay is chosen for use, it will attract the same overheads needed to provide the infrastructure of a laboratory set-up i.e. building, utilities, staff and equipment. In this regard there are no cheap tests and to use reagent costs as the sole factor in determining which assay to use is somewhat perverse. While the reagent costs of the strip are higher that commercial immunofluorescence reagents by a factor of 3, including extraction, this would undoubtedly narrow if immunofluorescence were capable of closing the pathogen gaps that currently exist e.g. HRV, hMPV. Currently using this approach, we have been able to replace both immunofluorescence and viral culture and this ability makes molecular diagnostics a more cost effective method for diagnosing viral infections. Taking into account the superior range, sensitivity, ability to quantify and speed of molecular techniques it is incredible how little they are used in routine laboratories. With the advent of SARS and the threat of avian influenza, this deficit is now beginning to disturb health care planners at the highest level.Because specimen sampling was not contiguous seasonal peaks were not detected, accounting for the small numbers of respiratory syncytial virus detected and the lack of detection of human metapneumoviruses, parainfluenza virus type 1 and coronavirus 229E; subsequent (unpublished) data from the routine use of the molecular strip support an important role for human metapneumovirus in acute respiratory infections and the sporadic nature of infections caused by parainfluenza type 1 and coronavirus 229E.Several interesting observations need highlighting. First, for immunofluorescence to perform reliably it was essential that a good nasopharyngeal specimen was available. The use of throat and/or nasal swabs with immunofluorescence alone is inappropriate. Second, immunofluorescence was very poor at detecting viruses from patients in the community, again almost certainly because of the universal use of swabs in that setting. Third, the rapid results of immunofluorescence were complemented by the touchdown protocol which can report definitive results within 24 hours, considerably faster than culture. Fourth, the molecular strip was better at detecting multiple infections. Even allowing for the inability of immunofluorescence to detect rhinoviruses, it should have detected the mixed adenovirus and influenza virus infections. Although immunofluorescence is capable of diagnosing dual infections, its routine use along with culture probably grossly underestimates their prevalence. The most plausible explanation is that the molecular technique detects infections where one of the viruses is below the detection threshold of immunofluorescence. These low level viruses are either just starting or more likely reaching the end of an infectious episode (latency is less likely) and this raises the previously unaddressed question of their role in viral respiratory pathogenesis. Fifth, the extent of rhinovirus infections was very significant. Their clinical significance ranged from acting as a definitive respiratory pathogen to a less certain role when acting as the most frequently detected co-pathogen in mixed infections.In conclusion the use of the touchdown protocol with pre-dispensed and quality checked primer master-mixes was suitable for replacing virus culture for the diagnosis of respiratory viruses for immunofluorescence negative specimens. Immunofluorescence results were available after an average of 4–12 hours while molecular strip results were available within 24 hours, considerably faster than viral culture. The combined strip and touchdown protocol is a convenient and reliable method of testing for multiple viruses in a routine setting. Its generic nature makes it especially useful for introducing test repertoire modifications e.g. incorporating primers for the newly identified coronaviruses SARS-CoV and HCoV-NL63.A total of 222 specimens were included in the validation between January 2002 and June 2003, including 14 from an influenza surveillance scheme. The specimens were collected from 183 patients including: 103 male, median age 3.5 y (7 m – 84 y); 80 female patients, median age 9 y (7 m – 84 y); both male and female ages were skewed towards the lower age ranges, and 5 national external quality assurance scheme (NEQAS) specimens . One hundred and fifty-nine patients were in hospital and 24 were in the community at the time of sampling. Specimens tested consisted of a wide range of specimens including: broncho-alveolar lavage (36), nasopharyngeal secretions (120), sputum (35) and combined throat and nasal swabs (31).Nasopharyngeal secretions, broncho-alveolar lavage and sputum specimens were received in dry sterile containers at ambient temperature. Upon receipt they were re-suspended in 2 ml of virus transport medium (VTM) consisting of phosphate buffered saline pH 7.1, bovine serum albumin 7.5 μg/ml, penicillin G sodium 1000 units/ml, streptomycin sulphate 1000 μg/ml and amphotericin B 2.5 μg/ml. Throat and nasal swabs were received in 2 ml of VTM and vortexed on arrival to release cells attached to the fibres of the swab. An aliquot of 410 μl was taken off for extraction after which the specimens were centrifuged at 2600 g for 5 min and the resulting cell deposits air-dried on glass multi-well slides and fixed in acetone prior to testing. Immunofluorescence was set up on the respiratory specimens using commercial reagents according to the manufacturer's instructions, and was able to detect: influenza A, influenza B, respiratory syncytial virus, adenovirus, parainfluenza type 1, parainfluenza type 2 and parainfluenza type 3 .A volume of 200 μl of the respiratory specimen suspension was extracted on a QIAGEN BioRobot 9604 using the Blood and Body Fluid Vacuum Protocol of the QIAamp DNA Blood Kit . This protocol allows the co-extraction of both RNA and DNA simultaneously.Simultaneous amplification of all targets was facilitated by using a standard 8 well multi-well PCR strip to which all mixes were pre-dispensed and stored frozen; this format is referred to in the paper as the "respiratory strip" because of the respiratory nature of the targets. The respiratory strip targeted the following 12 common respiratory viruses: influenza A H3), influenza A (H1), influenza B, respiratory syncytial virus type A, respiratory syncytial virus type B, adenovirus, coronavirus 229E, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, human rhinovirus and human metapneumovirus. The final configuration of the single and multiplex primer mixes in the 8 well strip are shown in Table , influenEach primer master-mix was made-up and titrated against a known positive control before being aliquoted and dispensed into its respective well of the 8-well microtube strip. The strips were stored frozen at -20°C until used. A positive control was also aliquoted and stored separately at -20°C until used. For the duration of the study the positive control was the cloned target of parainfluenza virus type 1; a negative control was not deemed necessary.4, 1 unit AMV reverse transcriptase, 1 unit Tfl DNA polymerase, 0.2 mM each deoxynucleoside triphosphate  and 1 μM outer primers.First round volumes were made-up in Access RT-PCR buffer  and in the final 10 μl volume contained the following reagent amounts: 1.5 mM MgSO2, 50 mM KCl, 0.1% Triton X-100, 0.2 mM of each deoxynucleoside triphosphate , 0.25 units of Taq DNA polymerase (Promega) and 0.2 μM inner primers.Second round volumes were made-up in Taq Buffer B (Promega) and in the final 10 μl volume contained the following final amounts: 10 mM Tris-HCl (pH 9.0), 3.5 mM MgClFirst round amplification was performed on 2 μl of extract added to 8 μl of first round primer master-mix per well. Second round amplification was performed on 0.2 μl of the first round reaction added to 9.8 μl of second round primer master-mix per well; a multi-channel pipette facilitated the transfer of the 8 volumes in one step. The positive control was run on the eighth well of each strip. The second round products were run on ethidium bromide stained 2% agarose gels and photographed. Specimens were reported positive when respectively the correct size bands and the positive control bands were present.Amplification was carried out on a range of thermal cyclers including the Applied Biosystems GeneAmp 2400 and 9700 series and a DNA Engine Tetrad PTC 225 . The first and second round amplification protocols consisted of 36 identical cycles with the exception that (a) a reverse transcription step of 48°C-10 min preceded the first round and (b) a hot-start preceded the second round by transferring the strip directly from ice to a thermal cycler held at 94°C. The touchdown protocol consisted of 6 steps as follows: (a) initial denaturation (94°C-2 min); (b) 3 cycles of denaturation (94°C-30 s), annealing (55°C-30 s) and extension (72°C-30 s); (c) 3 cycles of denaturation (94°C-30 s), annealing (52°C-30 s) and extension (72°C-30 s); (d) 20 cycles of denaturation (94°C-30 s), annealing (49°C-30 s) and extension (72°C-30 s); (e) 10 cycles of denaturation (94°C-30 s), annealing (46°C-30 s) and extension (72°C-30 s); (f) 72°C for 5 mins.PVC: Touchdown and molecular strip design and manuscript preparation.GMO: Early application of touchdown cycling to respiratory samples.HJO'N: Study protocol and manuscript preparation.CMcC: Study protocol and manuscript preparation.DDEO: Routine application of touchdown protocol during study.FM: Routine application of touchdown protocol during study.SJM: Routine application of touchdown protocol during study.SAF: Primer-mastermix manufacture and quality control.DEW: Early application of touchdown cycling to respiratory samples and manuscript preparation.MF: Routine application of immunofluorescence protocol during study.JS: Design and validation of primers for human metapneumonavirus detection.


E. coli have yet to be refolded from inclusion bodies, and although leadered MOMP can be expressed in E. coli cells, it often misfolds and aggregates. We aimed to improve the surface expression of correctly folded MOMP to investigate the membrane topology of the protein, and provide a system to display native and modified MOMP epitopes.Chlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin  with important structural and, in many species, immunity-related roles. MOMP forms extensive disulphide bonds with other chlamydial proteins, and is difficult to purify. Leaderless, recombinant MOMPs expressed in C. trachomatis MOMP was expressed on the surface of E. coli cells (including "porin knockout" cells) after optimizing leader sequence, temperature and medium composition, and the protein was functionally reconstituted at the single-channel level to confirm it was folded correctly. Recombinant MOMP formed oligomers even in the absence of its 9 cysteine residues, and the unmodified protein also formed inter- and intra-subunit disulphide bonds. Its topology was modeled as a (16-stranded) β-barrel, and specific structural predictions were tested by removing each of the four putative surface-exposed loops corresponding to highly immunogenic variable sequence (VS) domains, and one or two of the putative transmembrane strands. The deletion of predicted external loops did not prevent folding and incorporation of MOMP into the E. coli outer membrane, in contrast to the removal of predicted transmembrane strands.C. trachomatis MOMP was functionally expressed on the surface of E. coli cells under newly optimized conditions. Tests of its predicted membrane topology were consistent with β-barrel oligomers in which major immunogenic regions are displayed on surface-exposed loops. Functional surface expression, coupled with improved understanding of MOMP's topology, could provide modified antigens for immunological studies and vaccination, including live subunit vaccines, and might be useful to co-express MOMP with other chlamydial membrane proteins. Chlamydiales is an obligate intracellular pathogen  undeC. trachomatis MOMP is highly immunogenic. Antibodies to the protein neutralised EB infectivity . Howectivity , with THecretion . C. tracCh. psittaci (formerly known as C. psittaci) OmcA is a 9 kDa lipid-anchored protein with 14 cysteine residues )pAb Fig. .The absence of a signal from control cells and cells expressing MOMP in its non-leadered, mature form confirmed the incubation and blotting conditions did not cause cell lysis and expose unincorporated protein. Both OmpT- and native-leadered MOMP were detected on the cell surface Fig.  showed ME. coli, cross the inner membrane, and be processed in the periplasm. Furthermore, under modified incubation and induction conditions , MOMP could be folded and incorporated into the outer membrane.We concluded that MOMP constructs encoding appropriate leaders could be expressed in C. trachomatis MOMP was inserted into the OM of E. coli cells, we set out to investigate how the protein was organized in the membrane. While noting that predictive algorithms must always be deployed with care, and with reference to established findings for a given protein, we first analyzed MOMP's primary sequence for membrane crossings using a neural network trained with OM proteins of known structure [C. trachomatis MOMP generally corresponded to regions of the protein predicted to be extracellular.Having confirmed that tructure . The anaWe then reanalyzed the sequence using two β-strand prediction programs Fig. . The comC. trachomatis MOMP constructs  in which substantial regions of VS domains 1, 2, 3 or 4  buffer , or the addition of 2 mM EDTA, failed to "unmask" any immunoreactivity  was -31 ± 1.5 mV . This corresponded to a relative cation vs anion selectivity of 3.8 under these specific ionic conditions. Control preparations (detailed under Methods), including membrane proteins from control BL21omp8 cells subjected to the same experimental conditions, where OM proteins were solubilised and subjected to GE chromatography in exactly the same way, did not give rise to similar channel activity (6 experiments).Fully processed and correctly folded MOMP should function as a porin-like ion channel . We test or OmpC . We thenC. trachomatis MOMP at the single-channel (single molecule) level from cells lacking many endogenous porins provides very strong evidence that MOMP adopted its native fold when expressed in E. coli under suitable conditions. Although a leadered version of recombinant chlamydial MOMP was expressed and functionally analysed previously [E. coli porins for liposome-swelling studies. Although MOMP may have contributed additional porin-like activity, functional modification of endogenous porins could not be ruled out.Functional reconstitution of recombinant eviously , membranE. coli cells depends on the precise leader sequence, as well as on the specific protein. PorB is less "toxic" with its native leader, in contrast to MOMP, which is less "toxic" with the E. coli OmpT leader, and native-leadered C. muridarum MOMP is less deleterious to E. coli than Ch. abortus MOMP. Although a full investigation of the role of leader sequences could not be undertaken here, it is known that successful OM insertion, as well as prior transport across the inner membrane and processing, is also signal sequence-dependent. For example, a large proportion of E. coli LamB porins with signal sequence mutations remained "tethered" to the inner membrane , even though the protein was also closely associated with the OM [C. trachomatis MOMP, use of the Omp-T leader and induction at 16°C , in either "wild-type" cells or "porin knockout" cells in a supportive medium (SOC), provides improved processing and OM insertion, and there is also significant insertion at 25°C in "wild-type" E. coli.Interestingly, the successful expression and processing of recombinant chlamydial porins in h the OM . For C. C. trachomatis MOMP are consistent with previous data on bacterial [The single-channel properties of acterial  and putaacterial  porins. acterial , and pooC. trachomatis MOMP, like putative porins in the intracellular pathogens Burkholderia thailandensis and B. pseudomallei [Despite the lack of sequence similarity to known bacterial porins, a combination of different predictive approaches (none of which was entirely satisfactory in isolation), set in the context of elegant and pioneering work from many laboratories on the properties of VS domains, predicted that domallei , could bdomallei ,40. The C. trachomatis MOMP differs in some significant respects from the recent prediction for C. muridarum MOMP [C. muridarum prediction. This would preclude the significant interactions with OmcB and OmcA, described in the Background. We also assigned L2, 4, 6 and 7 as C. trachomatis VS domains, not L2, 3, 5 and 6, the homologous regions in C. muridarum MOMP. Experimental tests of the predicted membrane topology of C. trachomatis MOMP are consistent with our model, because individual VS domains can be substantially truncated without preventing incorporation of the protein into the bacterial OM. If MOMP is a β-barrel porin, as suggested, and VS domains are confined to specific extracellular loops, it is conceivable that MOMP can continue to fold into a β-barrel in the absence of one of these domains. On the other hand, the removal of β-strands would disrupt folding. Removal of a single strand, bringing periplasmic residues into direct contact with external residues, is predicted to be particularly destructive to the global fold. Removal of more than one strand might be better tolerated, provided the β-barrel can form with a significantly reduced diameter. In practice, it appears that C. trachomatis MOMP cannot accommodate either type of strand modification.Our working model for the membrane topology of rum MOMP  . HoweveCh. abortus MOMP forms SDS-resistant oligomers of ~100 K [E. coli OMs. We speculate that this may be because native MOMP oligomers are stabilised by interactions with other chlamydial components , and poond Fig. .C. trachomatis MOMP, an immunodominant, cysteine-rich, chlamydial surface protein of crucial importance in the immune response to infection, is a major subunit vaccine target. However, unlike many other bacterial porins, it has been difficult to refold from inclusion bodies or to achieve and demonstrate functional surface expression. This study is the first to report unambiguous functional analysis, by single-channel recording, of recombinant chlamydial MOMP recovered from bacterial outer membranes. The modified expression system described in the present study provided a means to test specific hypotheses provided by a working model for the C. trachomatis protein. However, although our results are consistent with a working model of MOMP as a 16-stranded β-barrel, more mutations or other approaches are needed before a specific model can be accepted. The protein also formed oligomers, even in the complete absence of cysteine residues. The surface display of modified, functional MOMP in E. coli cells , together with a working topological model, could guide the removal of unwanted or harmful epitopes from engineered proteins, and it might also be possible to display external loops containing specific MOMP epitopes on other porin "scaffolds" in living cells. However, it is important to note that such approaches will be limited if essential disulphide bonds in the native chlamydial envelope, including bonds involving non-MOMP cysteines, stabilise the conformation of key immunogenic VS domains.C. trachomatis ompA  and Ch. abortus ompA were cloned without their leader sequences into the Nde-I/Nco-I sites of pET22b(+) (Novagen) after destroying an internal Nde-I site in C. trachomatis ompA by Quik-Change PCR mutagenesis (Stratagene). This did not alter the encoded protein. C. muridarum ompA and porB were amplified with and without their leaders from genomic DNA and cloned into the Nde-I/Bam-HI sites and Nde-I/Nco-I sites, respectively, of the same vector . The E. coli OmpT protease leader sequence or the native C. trachomatis MOMP leader sequence was added to the 5' end of the leaderless C. trachomatis and Ch. abortus inserts by sequential gene extension PCR using three overlapping primers. A 5' Nde-I site was again used to provide the starting methionine codon in the final full-length construct. Quik-Change PCR was also used to create pairs of unique internal restriction sites in native-leadered C. trachomatis ompA to permit the deletion of specific domains by plasmid restriction and religation [Age-I; for VS2, Bcl-I; and for VS3, VS4 and the predicted β-strands, Aat-II. Successful deletions were confirmed by hemi-nested single-colony PCR (using Taq polymerase) to identify clones that could be amplified by gene-spanning primers but not by primers complementary to regions that had been removed. We also generated C. trachomatis MOMP expression constructs containing inserts in which all 9 cysteine residues  were replaced by alanine using Quik-Change PCR. Most of the modifications were carried out in a pSTBlue-I/NovaBlue system, and the fidelity of each insert was confirmed by automated DNA sequencing (MWG Biotech).ligation . These sE. coli BL21(DE3) or BL21(DE3)omp8 [DE3)omp8  cells wevs the logarithm of the molecular mass of Precision Plus unstained protein markers (BioRad). For Western blotting, proteins were electrophoretically transferred to PVDF membranes under conditions compatible with the transfer of high-MW proteins including native MOMP oligomers [C. trachomatis MOMP antibody  for 1 hour at room temperature. Following 2 × 30 sec and 3 × 5 min washes in PBS-T, membranes were incubated in 1:10,000 HRP-conjugated anti-goat/sheep antibody (Sigma) for 1 hr at room temperature. After washing, immunoreactive proteins were detected by ECL.Unless otherwise indicated, SDS-PAGE was carried out under reducing conditions using 10–12% (w/v) gels. Molecular masses were estimated from plots of relative mobility ligomers . The memC. trachomatis MOMP polyclonal antibody as described above. Immunofluorescence was carried out as described previously [10 ml of LB medium was seeded 1:100 with cultures grown to saturation overnight, and incubated until the OD reached 0.6. The cells were pelleted by centrifugation  and resuspended in fresh medium. Following incubation at the selected temperature for 10 mins, 0.1–1 mM IPTG was added and incubation was continued for another 2–16 hrs. Intact cells were harvested by gentle centrifugation  and washed in 1 ml PBS. The pellets were resuspended in 200 μl PBS, and 10 μl was applied to a nitrocellulose membrane and allowed to dry. The membrane was blocked and probed with anti-eviously , with fiVt 320 ml) was calibrated in the presence of detergent using standard proteins. V0 (the void volume) was 115 ml, and Kav was calculated as (Ve - V0)/(Vt - V0), where Ve is the elution volume.Solublised OM proteins were separated by GE chromatography using a high resolution 26/60 HiLoad Superdex 200 prep grade column (Amersham Pharmacia Biotech) freshly equilibrated in 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 50 mM NaCl and 5 mM DTT (omitting the latter for the cysteine-less MOMP mutant). The buffer also contained either 0.05% (w/v) LDAO or 0.05% (w/v) Zwittergent 3–14. 2 ml aliquots of solubilised OM proteins  were loaded, and the column was eluted with the same buffer for 800 min. at a flow rate of 0.5 ml/min. 5 ml fractions were collected and 10 μl of each protein-containing fraction was deposited onto a pre-prepared PVDF membrane and probed for MOMP as described earlier under Western blotting. The column  were added to the cis chamber, followed by aliquots of 5 M KCl to raise the salt concentration to 500 mM. Channel incorporation usually occurred within 30 min, accelerated by switching the holding potential between +/- 60 mV. Experimental protocols were programmed and the digitised data were low-pass filtered  and recorded using pClamp8 software (Axon Instruments), and analysed offline. The bilayer potential was slowly and repeatedly ramped between -100 mV and +100 mV (each sweep taking 32 s) in the presence of an asymmetric  gradient of KCl, or with equimolar 500 mM or 1 M KCl. At least 3 voltage ramps were recorded and analysed for each experiment, and equilibrium recordings were obtained at defined holding potentials. Holding potentials refer to the cis chamber, and upgoing deflections represent net movement of cations from cis to trans or of anions from trans to cis. Relative ionic permeabilities were determined from the equilibrium solution of the Nernst-Planck flux equations [Planar bilayers were cast from diphytanoyl phosphatidylcholine (Avanti) between two 0.5 ml chambers containing 50 mM KCl, 20 mM Tris-HCl (pH 8.0) and 1 mM DTT, designated nd trans . The cisquations . When thEr is the equilibrium  potential in asymmetric KCl, and R, T and F have their usual significance.  (the permeability ratio of K+ to Cl-) was calculated using appropriate activity coefficients (a) from standard tables. Control experiments were carried out using equivalent amounts of detergent and with equivalent amounts of solubilised OM proteins purified from non-transformed bacteria, selecting identical GE column fractions.C. trachomatis MOMP and the known characteristics of antiparallel, amphipathic β-barrel strands in porins.The number of membrane crossings was predicted using a neural network based-outer membrane protein topology prediction program trained with known porins . We discHEF and HM carried out most of the experiments. HEF analysed and organised the data, and drafted the first version of the manuscript. RHA conceived the overall project, provided experimental guidance, carried out some of the experiments, and redrafted the manuscript. All the authors read and approved the final manuscript.Detergent extraction of recombinant MOMP. Immunoblot with ECL detection following SDS-PAGE of OM proteins (10 μg per lane) from BL21 cells expressing OmpT-leadered C. trachomatis MOMP, induced for 2 hrs at 37°C. OM proteins (see Methods) were solubilised in 1% (w/v or v/v) octylglucoside, Triton X-100, Zwittergent 3–14 or LDAO, as indicated. NB & B are non-boiled and boiled samples, respectively. NE = non-expressing (control) cells.Click here for fileC. trachomatis MOMP expression and processing in BL21omp8 cells. Optimisation of Growth curves of BL21omp8 cells expressing C. trachomatis MOMP with its native leader, in LB or SOC medium .Click here for fileMOMP epitopes are not unmasked by Tris buffer or EDTA in whole cell immunoblots. Control BL21 cells and cells expressing "strand-deleted" constructs were suspended in 100 mM NaCl containing 50 mM Tris-HCl (pH 7.4) with or without 2 mM EDTA, applied to nitrocellulose membranes, and probed with anti-C. trachomatis MOMP polyclonal antibody.Click here for fileRecombinant MOMP does not form SDS-resistant oligomers. SDS-PAGE and immunoblot analysis of C. trachomatis MOMP expressed with its native leader in BL21omp8 cells at 16°C (induced for 12 hrs in the presence of 0.1 mM IPTG). Lanes 1 & 2 contain 10 μg non-boiled and boiled OM proteins, respectively, solubilised in 1% (w/v) OG. Note successful transfer of high-MW proteins.Click here for file


Aluminum has recently been recognized as a causative agent in dialysis encephalopathy, osteodystrophy, and microcytic anemia occurring in patients with chronic renal failure who undergo long-term hemodialysis. Only a small amount of Al(III) in dialysis solutions may give rise to these disorders.3O4). Then, metal complexing ligand alizarin yellow was covalently attached onto mPHEMA beads. Alizarin yellow loading was 208 μmol/g. These beads were used for the removal of Al(III) ions from tap and dialysis water in a magnetically stabilized fluidized bed.Magnetic poly(2-hydroxyethyl methacrylate) (mPHEMA) beads in the size range of 80–120 μm were produced by free radical co-polymerization of HEMA and ethylene dimethacrylate (EDMA) in the presence of magnetite particles (FeAl(III) adsorption capacity of the beads decreased with an increase in the flow-rate. The maximum Al(III) adsorption was observed at pH 5.0. Comparison of batch and magnetically stabilized fluidized bed (MSFB) maximum capacities determined using Langmuir isotherms showed that dynamic capacity (17.5 mg/g) was somewhat higher than the batch capacity (11.8 mg/g). The dissociation constants for Al(III) were determined using the Langmuir isotherm equation to be 27.3 mM (MSFB) and 6.7 mM (batch system), indicating medium affinity, which was typical for pseudospecific affinity ligands. Al(III) ions could be repeatedly adsorbed and desorbed with these beads without noticeable loss in their Al(III) adsorption capacity.Adsorption of Al(III) demonstrate the affinity of magnetic dye-affinity beads. The MSFB experiments allowed us to conclude that this inexpensive sorbent system may be an important alternative to the existing adsorbents in the removal of aluminium. About 8% of the Earth's crust is comprised of aluminium. This element is the most abundant metal naturally present in air, soil and water. Consequently, environmental exposure to aluminium is potentially possible. Its ingestion is unavoidable since aluminium compounds are added not only to most water supplies but also to many processed foods and medicines. Aluminium is a known neurotoxicant. It enters the brain, where it contributes to some neuro-degenerative diseases including dialysis encephalopathy, osteomalacia, osteodystrophy, in particular those related to dialysis treatment of uremic subjects . Only a Positively charged aqua and hydroxy-monomeric forms have been found to be the most toxic species of aluminium to living organisms in the terrestrial and aquatic environments . GeneralThe selective removal of aluminium ions have been extensively investigated by applying several techniques -9. AmongThere have been several separation approaches performed under magnetic field . The mosIn the present study, we attempted to use alizarin yellow-attached magnetic poly(2-hydroxyethyl methacrylate) (mPHEMA) beads as specific adsorbent for aluminium removal from aqueous solutions in a magnetically stabilized fluidized bed. Al(III) adsorption on the alizarin yellow-affinity beads from aqueous solutions containing different amounts of Al(III) ions and at different pHs is reported here. Finally, reuse of the dye-affinity beads is also discussed.3O4, diameter < 1 μm) were obtained from Aldrich (USA). Alizarin yellow  was purchased from BDH . All other chemicals were obtained from Merck as analytical grade. All water used in the adsorption experiments was purified using a Barnstead  ROpure LP® reverse osmosis unit with a high flow cellulose acetate membrane (Barnstead D2731) followed by a Barnstead D3804 NANOpure® organic/colloid removal and ion exchange packed bed system.2-hydroxyethyl methacrylate (HEMA), was purchased from Sigma , and was purified by vacuum distillation under a nitrogen atmosphere. The comonomer, ethylene dimethacrylate  was used as the crosslinking agent. Magnetite particles  (10 ml). This reaction medium was boiled in rotary evaporator for 6 h. Then, 2.5 g alizarin yellow was dissolved in absolute ethanol (30 ml). Alizarin yellow-attachment process was performed in ethanol solution for 24 h. At the end of this reaction period, the alizarin yellow-attached beads were removed by filtration and washed with ethanol, water and tetrahydrofuran several times until all the unbound dye molecules were removed. The dye attached beads were stored at 4°C with 0.02% sodium azide to prevent microbial growth.Preparation and characterization of the alizarin yellow-attached mPHEMA beads were reported in our previous paper in detail . In ordeThe leakage of the alizarin yellow from the dye-attached beads was investigated within the media at the selected pH in the range of 2.0–7.0. These media were the same which were used in the Al(III) adsorption experiments. The medium with the dye attached magnetic beads was stirred for 24 h at room temperature. Then, magnetic beads were separated from the medium, and the alizarin yellow concentration was measured in the liquid phase by spectrophotometry at 500 nm.Al(III) adsorption studies were carried out in a magnetically stabilized fluidized bed. Beads suspended in pure water were degassed under reduced pressure (by using water suction pump) and magnetically stabilized into a column (10 cm × 0.9 cm inside diameter) equipped with a water jacket for temperature control. The vertically oriented magnetic field was produced by passing DC current through two modified Helmholtz coils (1.5 cm diameter × 2.5 cm thick) spaced 4 cm apart. At a current of 1.6 A (50 W), each coil produced a magnetic field of 40 Gauss. Equilibration of the column was performed by passing four column volumes of phosphate buffer (pH: 7.4) before injection of the Al(III) solution. In a typical adsorption system, 50 ml of the aqueous Al(III) solution was passed through the column containing magnetic beads, by a peristaltic pump for 2 h. After loading, the column was washed with deionized water to wash out Al(III) impurities. The concentrations of the Al(III) ions in the aqueous phases after the desired treatment periods were measured by using a graphite furnace atomic absorption spectrophotometer . Deuterium background correction was used. Pyrolitic graphite coated tubes were used for AAS measurements. The instrument response was periodically checked with known Al(III) solution standards. The experiments were performed in replicates of three and the samples were analyzed in replicates of three as well. For each set of data present, standard statistical methods were used to determine the mean values and standard deviations. Confidence intervals of 95% were calculated for each set of samples in order to determine the margin of error.In the first group of experiments, the flow rate of the aqueous solution  content of 50 mg/L) was changed between 0.5–3.0 mL/min. In the second group of experiments, Al(III) adsorption from aqueous solution was studied at different pH's (2.0–7.0). Adsorption isotherm was also obtained in the magnetically stabilized fluidized bed. Aqueous solutions containing different amount of Al(III) were used in these experiments. The changes in the Al(III) concentration with time was followed to obtain the adsorption curves. The amount of adsorbed Al(III) per dry magnetic beads was calculated by using the concentrations of the Al(III) in the initial solution and in the equilibrium.3 solution. In a typical desorption experiment, 50 ml of the desorption agent was recirculated through the magnetically stabilized fluidized bed containing dye-affinity magnetic beads for 1 h. The concentrations of the Al(III) ions in the desorption medium were measured by using a graphite furnace atomic absorption spectrophotometer. The desorption ratio was calculated from the amount of Al(III) adsorbed on the magnetic beads and the final Al(III) concentration in the desorption medium. In order to test the reusability of the dye-affinity magnetic beads, Al(III) adsorption-desorption procedure was repeated ten times by using the same magnetically stabilized fluidized bed.In all cases adsorbed Al(III) ions were desorbed using 0.1 M HNOAdsorption of Al(III) from aqueous solution was also investigated in batch experiments. Aqueous Al(III) solution (50 ml) was treated with the magnetic dye-affinity beads at room temperature, in the flasks agitated magnetically at an agitation speed of 600 rpm for 2 h. The suspension was brought to pH 5.0 by adding sodium hydroxide and nitric acid. The pH was maintained in a range of ± 0.1 units until equilibrium was attained. Polymer amount was kept constant at 100 mg per 50 ml. Al(III) determination was performed in water sample in an atomic absorption spectrophotometer coupled to a graphite furnace atomiser. Adsorption values (mg/g) were calculated as the difference in Al(III) ion concentration of the pre- and post adsorption solutions divided by the weight of dry magnetic affinity beads.3. The magnetic beads are crosslinked hydrogels. They do not dissolve in aqueous media, but do swell, depending on the degree of cross-linking and on the hydrophilicity of the matrix. The equilibrium swelling ratio (the ratio of the volumes of the microbeads before and after swelling) of the beads used in this study is 34%. The simple incorporation of water weaken the secondary bonds within the hydrogels. This enlarges the distance between the polymer chains and causes the uptake of water. It should be mentioned that the water uptake properties of the mPHEMA beads did not change after Alizarin Yellow attachment.mPHEMA beads (in the size range of 80–120 μm) carrying alizarin yellow were prepared as a specific affinity adsorbent for removal of Al(III) from the water which was used for preparation of dialysis solution. mPHEMA beads used in this study were prepared and characterized in our earlier study . The maiAfter the attachment of the dye  the size of the swollen beads did not change, but the colour became dark yellow, which is a clear indication of the incorporation of the dye molecules in the structure of the mPHEMA microbeads. As shown in our previous paper, the dye molecules were attached to the mPHEMA beads by covalent bonding via hydroxyl groups . The mPHThe adsorption capacity at different flow-rates are given in Figure Figure It should be noted that the nonspecific adsorption (adsorption on plain mPHEMA beads) of Al(III) ions was relatively low (0.63 mg/g). mPHEMA beads do not contain ion exchange or chelating groups. Preferred coordination structure and preferred coordinating ligand atom may be utilized for this adsorption. Al(III) ions may interact with Oxygen atoms as the ligand. Diffusion of Al(III) ions into the swollen polymeric structure and retention in the pores may also contribute to this nonspecific Al(III) adsorption.An adsorption isotherm is used to characterize the interactions of each molecule with the adsorbent. In this case it provides a relationship between the concentration of the Al(III) ions in the solution and the amount of Al(III) ions adsorbed on the solid phase when the two phases are at equilibrium. The Langmuir adsorption model assumes that the species are adsorbed at a fixed number of well-defined sites, each of which is capable of holding only one molecule. These sites are also assumed to be energetically equivalent, and distant from each other so that there are no interactions between molecules adsorbed on adjacent sites.Adsorption isotherms were used to evaluate adsorption properties. The Langmuir adsorption isotherm is expressed by Equation 1. The corresponding transformations of the equilibrium data for Al(III) gave rise to a linear plot, indicating that the Langmuir model could be applied in these systems and described by the equation:max. b . Ceq / (1 + bCeq)     (1)Q = Qeq is the equilibrium Al(III) concentration (mg/mL), b is the Langmuir constant (mL/mg) and, Qmax is the maximum adsorption capacity (mg/g). This equation can be linearized so thatwhere Q is the adsorbed amount of Al(III) (mg/g), Ceq/Q = 1/(Qmax. b) + Ceq/Qmax.     (2)Ceq versus Ceq/Q was employed to generate the intercept of 1/Qmax.b and the slope of 1/Qmax.The plot of Cmax) data for the adsorption of Al(III) was obtained from the experimental data. The correlation coefficient (R2) was 0.989. The Langmuir adsorption model can be applied in this affinity adsorbent system. Maximum adsorption capacities determined using Langmuir isotherms show that dynamic capacity (25.3 mg/g) was somewhat higher than the batch capacity (12.6 mg/g). The dissociation constants for Al(III) were determined using the Langmuir isotherm equation to be 27.3 mM (MSFB) and 6.7 mM (batch system), indicating medium affinity, which was typical for pseudospecific affinity ligands.The maximum adsorption capacity  adsorption of this alizarin yellow-attached mPHEMA beads is also shown in Figure Competitive adsorption of the metal ions from tap water in Ankara and dialysis water (reverse osmosis) was also investigated. The water containing different amounts of each metal ion was treated with dye beads in MSFB. Table The World Health Organization (WHO) and the European Community (EC) guide values for Al(III) ions for tap water is 200 ng/ml ,27. Al ions are disrupted and subsequently Al(III) ions are released from the solid surface into the desorption medium. Therefore, we conclude that HNO3 is a suitable desorption agent for the dye adsorbents, and allows their repeated use. In order to show the reusability of the dye-attached mPHEMA beads, adsorption-desorption cycle was repeated ten times by using the same sample of affinity adsorbent. As can be seen from Figure Desorption ratios were very high (up to 97.6%) with the eluant system and under conditions used. When HNOAs can be seen in Figure The medical relevance of aluminium has stimulated the development of cost and time effective separation techniques including polymeric carriers. Magnetic adsorbents have several potential advantages over conventional adsorbents -32. The


Acanthamoeba polyphaga Mimivirus), with particle sizes of 0.2 to 0.6 μm, genomes of 300 kbp to 1.200 kbp, and commensurate complex gene contents, constitute an evolutionary mystery. They challenge the common vision of viruses, traditionally seen as highly streamlined genomes optimally fitted to the smallest possible -filterable- package. Such giant viruses are now discovered in increasing numbers through the systematic sampling of ocean waters as well as freshwater aquatic environments, where they play a significant role in controlling phyto- and bacterio- plankton populations. The 4th algal virus workshop showed that the study of these ecologically important viruses is now massively entering the genomic era, promising a better understanding of their diversity and, hopefully, some insights on their origin and the evolutionary forces that shaped their genomes.Giant double-stranded DNA viruses (such as record breaking  Though marine ecology rather than basic virology was the main focus of this meeting, exciting new results on the genomics of large/giant viruses kept turning up in many talks. In the context of a comparative study, Corina Brussaard (in collaboration with the US DoE) is herself sequencing a variety of Micromonas pusilla and Phaeocystis globosa dsDNA viruses some of them estimated to have a genome sizes up to 460 kb.The 4th Algal Virus Workshop In his overview, Curtis Suttle  pointed out that viruses  constitute a significant part of the biomass in ocean coastal waters  where they play a dominant role in the control of phyto- and bacterio-plankton populations, and hence on the production of oxygen and atmospheric dimethylsulphide, an important factor in climate regulation. Most of these viruses are uncharacterized .freshwater unicellular green alga that the best characterized large DNA virus Paramecium bursaria chlorella virus (PBCV-1), the prototype of the Phycodnaviridae, was isolated more than 20 years ago in Jim van Etten's laboratory [Paramecium bursaria chlorella viruses: NY-2A (infecting PBCV-1 host Chlorella species NC64A) and Chlorella Pbi virus MT325. NY-2A genome contains 368,683 bp, making it the largest chlorella virus sequenced to date. Despite a 10% difference in size, the NY-2A genome and PBCV-1 genome (330 kb) exhibits a near perfect colinearity. With 314,335 bp the MT325 genome is slightly smaller and does not exhibit long range colinearity with the PBCV-1 and NY-2A genomes. As in previously sequenced phycodnaviridae, new unexpected functions turned up to be encoded is these two new genomes, such as the first aquaglycerolporin the activity of which has been experimentally verified. A detailed comparative proteomics of the three viral particles (each of them exhibiting about 120 virus-encoded polypeptides) is also under way in the same laboratory .Ironically, this is in a Feldmannia were infected by large dsDNA viruses (phaeovirus) coming in two genome sizes: 158 kbp and 178 kbp [Feldmannia sp. The final sequence size is 153,259 bp (51.8 G+C). About 50% of the 161 predicted ORFs have their best matching homologues in Feldmannia irregularis virus (FirrV-1) or Ectocarpus Siliculosus virus (EsV-1). Nicolas Delaroque  reported on the difficulty to reach full closure in sequencing the genome of Feldmannia irregularis virus (FirrV-1), most probably due to the presence of long repeats. The current FirrV genome sequence data consists of 191,667 bp (in 16 contigs), encoding 156 putative proteins [It was known for some time that filamentous marine brown alga of genus  178 kbp . Prof. Tproteins . His mor356 kbp-genome sequence of HcV01, a dsDNA virus infecting dinoflagelate Heterocapsa circularisquama, and of the 294 kbp-genome sequence of Heterosigma akashivo infecting virus (HaV01). The same laboratory is also finishing the sequencing of the 145 kb-genome of T4-like looking cyanophage Ma-LMM01, infecting the toxic cyanobacterium Microcystis aeruginosa.Dr Keizo Nagasaki and his collaborators  announced the near completion of the Emiliania huxleyi  and encodes 472 putative protein coding regions. Only 66 (14%) of them have recognizable homologues in the public databases. As other giant viruses, EhV-86 exhibits its share of unexpected genes and functions, most notably a number of enzymes involved in the biosynthesis of sphingolipids. Albeit phylogenetically branching at the root of the Phycodnaviridae (e.g. PBCV-1 or EsV), EhV-86 does encode it own DNA-dependent RNA polymerase complex, thus filling the gap with the other Nucleo-Cytoplasmic Large DNA virus families  that all exhibit virally-encoded RNA-polymerases. Pending approval by ICTV, EhV-86 might become the prototype of the coccolythovirinae, a new subfamily of phycodnaviridae.Finally, Willie Wilson's group  claimed the bronze medal in the fierce competition for genome size . They preyi Fig. . The genIn his closing lecture, Jim Van Etten, reminded the new comers in the field of algal viruses that reports of very large icosahedral virus-like particles in various aquatic and marine organisms can be traced back to the 50's, but failed to elicit much interest outside of the community of marine biologists. The discovery and genome characterization of the large freshwater chlorella viruses , and morThe 4th Algal Virus Workshop made it clear that these giant algal viruses are now entering the genomic era at full speed. The amount of surprises that we can expect while deciphering their genomes will be as big as their diversity, and more dogma on what a virus should look like will probably be shattered along the way.


Whole genome sequence data is a step towards generating the 'parts list' of life to understand the underlying principles of Biocomplexity. Genome sequencing initiatives of human and model organisms are targeted efforts towards understanding principles of evolution with an application envisaged to improve human health. These efforts culminated in the development of dedicated resources. Whereas a large number of viral genomes have been sequenced by groups or individuals with an interest to study antigenic variation amongst strains and species. These independent efforts enabled viruses to attain the status of 'best-represented taxa' with the highest number of genomes. However, due to lack of concerted efforts, viral genomic sequences merely remained as entries in the public repositories until recently.VirGen is a curated resource of viral genomes and their analyses. Since its first release, it has grown both in terms of coverage of viral families and development of new modules for annotation and analysis. The current release (2.0) includes data for twenty-five families with broad host range as against eight in the first release. The taxonomic description of viruses in VirGen is in accordance with the ICTV nomenclature. A well-characterised strain is identified as a 'representative entry' for every viral species. This non-redundant dataset is used for subsequent annotation and analyses using sequenced-based Bioinformatics approaches. VirGen archives precomputed data on genome and proteome comparisons. A new data module that provides structures of viral proteins available in PDB has been incorporated recently. One of the unique features of VirGen is predicted conformational and sequential epitopes of known antigenic proteins using in-house developed algorithms, a step towards reverse vaccinology.. Various steps in the curation and annotation of the genomic data and applications of the value-added derived data are substantiated with case studies.Structured organization of genomic data facilitates use of data mining tools, which provides opportunities for knowledge discovery. One of the approaches to achieve this goal is to carry out functional annotations using comparative genomics. VirGen, a comprehensive viral genome resource that serves as an annotation and analysis pipeline has been developed for the curation of public domain viral genome data  Haemophilus influenzae in 1995 [SV-40 was the first virus for which the complete genome  sequence was obtained in late 70s [The emergence of high throughput technologies for genome sequencing, microarrays and proteomics transformed biology into a data-rich information science. Sequencing the complete genome of an organism is the first step in generating the 'parts list' of life. One of the first efforts involved sequencing of  in 1995 . Enormou in 1995 . Viruses in 1995 ,5. The g in 1995 ,7. SV-40late 70s . About ~genome 5,24 bp seqMimivirus [Polydnavirus [Whole genome sequence data of viruses offer unlimited opportunities for data mining and knowledge discovery . The comimivirus  and Polydnavirus  substantdnavirus . Howeverdnavirus ,13. Mostdnavirus -18.VirGen is developed and maintained at the Bioinformatics Centre, University of Pune and is available on-line ,20. VirGThe major focus of this paper is towards sharing with the research community the issues involved in curation of viral genomic data apart from demonstrating the utility of the derived data in understanding the biology, a pre-requisite for development of viral diagnostics, vaccines and drugs.The release  contains genomic data of 25 viral families  of genomes and proteomes▪ Reconstruction of phylogeny using genome/proteome data▪ Prediction of sequential and conformational B-cell epitopes▪ Curation and compilation 3D structures of viral proteins available in PDBFigure Genome sequences deposited in the public domain sequence repositories were retrieved using well-defined search strategies. The queries were formatted using keywords and MeSH terms to ensure that none of the complete genome sequences were missed. The entries were curated with respect to taxonomic hierarchy as per the guidelines provided by the ICTV . For manHepatitis C Virus (HCV), the curation of genomic records for assignment of genotypes called for exhaustive sequence analyses. Hepatitis C virus is a member of family Flaviviridae, genus Hepacivirus and is a major causative agent of liver diseases. The assignment of Hepatitis C virus genotypes is essential as molecular epidemiology differs from subtype to subtype. Also, due to the absence of significant cross-protection among different HCV subtypes, the exact subtype identification becomes a prerequisite in determining the suitability of present antiviral therapy as well as designing new antiviral compounds and vaccines [In case of vaccines . Isolativaccines  is rathevaccines . Howevervaccines . Furthervaccines . Given tIt was observed that there were many sequences in the public domain repositories, which were not explicitly annotated with keywords such as full or whole or complete genome even though their sequence lengths were in the typical range of the complete genome sequence for a given species. Such sequences, which are not explicitly annotated as the complete genome entries have been referred to as 'putative genomes' in VirGen .As there exist multiple genomic entries for every viral species, a well-annotated and characterized entry has been identified as the 'representative genomic entry' for a given species. The representative entries provide a non-redundant set of viral genome sequences, which are subsequently used for annotation of genomic records, alignment of genomes and proteomes and to study phylogenetic relationships.VirGen uses sequence-based Bioinformatics approaches and stringent cutoffs for annotating the viral genomes and proteomes. Entries are annotated with respect to the genome organisation, typical to a given family. The annotations are further refined to accommodate genus and species-specific organisation. Using the representative genomes and the program BLAST , the entJapanese encephalitis virus strain JaOArS982.One of the issues in curation of molecular data is lack of standard nomenclature. VirGen addresses this issue by implementing sequence-based searching to compile a dictionary of synonyms for viral proteins ,28. SuchBovine Viral Diahorrea Virus 1 (BVDV-1) virus is shown in the Figure Bos taurus, with statistically significant scores, indicating that the sequence was acquired by BVDV-1 from its host, the bovine, through a process of horizontal transfer. BVDV is known to acquire such heterologous and homologous inserts [The schematic representation of the viral genome/proteome organisation using scalable vector graphics (SVG) has enabled visualization of individual gene/protein in the context of the entire genome. This display also facilitates retrieval of gene/protein sequence data in FASTA format, a procedure which may otherwise require a lot of pre-processing. The genome organisation of the  inserts .Comparative genomics inadvertently includes multiple sequence alignment (MSA) in a major way, as the inferences drawn are very informative. Viruses being one of the smallest replicating species are under severe selection pressure not only to evade host immune response but also to sustain its survival in the vector. Availability of genome data of viruses offers opportunities to study variations at molecular level.Bioinformatics tools like MSA can be used to identify such variations, which when mapped on phenotypic properties like antigenicity, immunogenicity etc. may provide a rationale for the observed strain and species-specificity. MSA data and predicted epitopes for HN protein along with the predicted 3D structures were used to study strain specificity of mumps virus .MSA module in VirGen is computed using the parallel version of ClustalW . MultiplFlavivirus is discussed. Variability Index, calculated using the formulae proposed by Wu & Kabat [As an example, multiple sequence alignment of NS3 protein for the 26 representative species of genus  & Kabat  for MSA  & Kabat  revealedFlavivirus. However, due to availability of limited annotations and NS3 sequence being part of polyprotein entries in the public domain databases, the curation process could not be automated completely and required manual intervention. Thus, the entire procedure of generating a curated dataset for calculation of sensitivity and specificity of any pattern is time-consuming and calls for inclusion of steps that are specific for a given data set.It must be mentioned that these calculations require a curated non-redundant data set that includes NS3 protein sequences of all the known species belonging to genus Japanese encephalitis virus (JEV) is shown in Figure 279M play a deterministic role in maintaining neurovirulence [Multiple sequence alignment can also be used to understand the strain-specific variations. A block of multiple sequence alignment of envelope glycoprotein (Egp) of 47 strains of irulence .Furthermore, the 399-RGD-401 sequence motif, present in domain III is unique to the mosquito-borne Flaviviruses and was proposed to form a part of receptor binding site . The mutMolecular phylogenetic studies help to decipher the evolution of viruses and offer a mechanism to understand the origin and spread of infectious viral diseases. Phylogenetic trees are usually reconstructed using a single gene/protein or a region encompassing them . UTRs haFlaviviridae generated using whole genome data is shown in the Figure Flavivirus, Pestivirus and Hepacivirus clearly depicts the clustering of viruses as per their assigned genus. The tree also shows grouping of Flaviviruses into the mosquito-borne, tick-borne and no known vector clades. The mosquito-borne viruses show further separation into two clades namely, those which are transmitted through Aedes and through Culex. As can be seen from the figure 7, viruses transmitted by Aedes form two paraphyletic clades; one clade is made up of Yellow fever virus and Yokose virus and the other clade contains  Dengue virus type I, II, III and IV. Cell fusing Agent virus (CFAV) and Kamiti River virus (KRV), though transmitted by Aedes form a separate clade. Even though they share common ancestor with other mosquito-borne viruses, the fact that they are known to be insect-only and fail to replicate in the vertebrate cells or mice explains the observed branching pattern [Culex mosquito form a monophyletic clade. The branching pattern of the tick-borne Flaviviruses is gradual as compared to the dispersed branching of mosquito-borne clades. This could be attributed to the duration of life cycle of their respective vectors and vectors association with host [Tamana Bat virus (TABV) branches out separately from rest of the Flavivirus members possibly due to its sequence divergence. The tree also shows that the Pestiviruses form a polyphyletic clade with the mosquito-borne Flaviviruses. Border Disease virus (BDV), Pestivirus Reindeer (PERSE) and Classical Swine Fever virus (CSFV) form a group where as Bovine viral diarrhea virus-1 (BVDV-1) and Pestivirus Giraffe (PESGI) group separately. Bovine viral diarrhea virus-2 (BVDV-2) branched out prior to the branching of other Pestiviruses. The branching of all six genotypes of Hepatitis C virus is also clearly seen. The tree shows that the unassigned members of Flaviviridae family form a polyphyletic clade with the Hepaciviruses, the outermost branch of which is GB virus B (GBV-B). The latest report of ICTV documents inclusion of GBV-B as a member of genus Hepacivirus. Phylogenetic tree reconstructed using the polyprotein data showed similar branching topology.Reconstruction of a phylogenetic tree of family  pattern . Virusesith host . Tamana in silico design of vaccines, a field popularly known as 'reverse vaccinology' [in silico vaccine design is the availability of a curated data set of antigenic proteins and a set of programs for prediction of epitopes. As a step towards reverse vaccinology, VirGen stores predicted B-cell epitopes of antigenic proteins using sequence-based and structure-based algorithms developed in-house [Availability of genomic sequences has paved way for inology' . One of in-house -52.Kolaskar & Tongaonkar's method is a sequence-based approach for prediction of B-cell epitopes. The algorithm is based on antigenic propensity, which is assigned to each of the twenty amino acids depending on their frequency of occurrence in experimentally determined B cell epitopes. Parameters like hydrophilicity, accessibility and flexibility are averaged for every overlapping heptapeptide from N to C terminii and assigned to the central residue of every segment. The residues having average antigenic propensity ≥ 1.0 are termed as potential antigenic determinants. The accuracy of this method is ~75% and has been implemented in major sequence analysis packages viz, GCG  and EMBODengue virus II (PDBID:1OAN) [Conformational Epitope Prediction (CEP) algorithm developed in-house implements structure-based approach for prediction of sequential and conformational epitopes ,51. CEP ID:1OAN) . This coID:1OAN) . The epiSARS [Poxvirus [Japanese encephalitis virus have been predicted. These models were used to design candidates for peptide vaccine for Japanese encephalitis and helped to gain an insight into strain-specific variations [Three-dimensional structural data is essential to understand function of proteins at molecular level. The 3D structures of proteins are known to be conserved and can accommodate sequence variation up to 80%. Structures of ~1082 viral proteins and viral assemblies have been solved and 3D coordinates are available in the PDB . StructuSARS  and PoxvPoxvirus . VirGen riations ,38.Analyses of curated data of whole genomes and proteomes reveals molecular variations, which when correlated with the observed phenotype provide a rationale for species-specific properties. As an attempt towards this, molecular mechanism of polyprotein cleavage at NS3-NS2B in JEV was modeled using the experimental data available for related flaviviruses since NS3 is a candidate for anti-viral therapy.Japanese Encephalitis Virus (JEV), a member of Flaviviridae is a major causative agent of encephalitis in South-east Asia [cis cleavage of NS2A/NS2B and NS2B/NS3 as well as trans cleavage of NS3/NS4A and NS4B/NS5 [Dengue type II [Hepatitis C virus [West Nile virus [Alkhurma virus [ast Asia , the 11 ast Asia ,62. The NS4B/NS5 ,64. Mole type II -67, Hepa C virus , West Nile virus  and Alkhma virus . Similarma virus -73. A deJapanese Encephalitis virus (Virulent SA-14-14-2 strain) was predicted using knowledge-based homology modeling approach. Two models of NS3 serine protease of JEV were built using the crystal structures of NS3 of Dengue virus II (DEN) PDB_ID: 1BEF [Hepatitis C Virus (HCV) PDB_ID: 1JXP [The 3D structure of NS3 serine protease domain of ID: 1BEF  and HepaID: 1JXP . These tDengue virus II (1BEF) was accepted and used for subsequent studies , an integral membrane protein, serves as a cofactor for NS3 ,72. Howele index  helped tle index , where NIn the initial phase, docking of NS3 serine protease with the cofactor was carried out to study their interactions at molecular level. The cofactor (NS2B) was first docked onto NS3, followed by docking of substrate with the complex of NS2B-NS3.The Kabsch & Sander secondary rendering of predicted structure of NS3 and NS2B complex is shown in the Figure The exquisite selectivity of serine proteases for particular substrate is a result of the existence of specific binding sites on the enzyme for amino acid side chains of the substrate . The subNS3 of JEV cleaves the polyprotein at specific locations having the sequence KR-GR (site between NS4B and NS5) apart from other known cleavage sites. In the absence of structural information of the substrate, the structure of six residues (LKRGRP) flanking on either side of the NS4B/NS5 cleavage site was predicted using the MD strategy described for NS2B. The ternary complex was obtained by docking of substrate to NS3-NS2B complex using the docking protocol mentioned above. Of the 100 docked structures, 23 were selected based on geometry and the interactions with NS3. It was observed that the van der Waal interactions contributed maximally towards the overall interaction energy. The active site residues His51 and Ser135 were found to interact with Leucine of the substrate via the formation of hydrogen bonds  was placed near the binding site.▪ The residues of protein lying within 5Å radius from the center of ligand were defined as 'movable atoms' whereas the rest of the molecule was kept rigid during docking and was defined as 'bulk set'.▪ Ligand was flexible with respect to  torsional angles. Hydrogen bond donor and acceptor atoms were defined for both the protein and the ligand. In order to avoid displacement of the ligand far away from the active site residues, the ligand was confined to 3Å radius.▪ Initial coarse search was carried out using Quartic_vdw_no_Coulb method in which coulombic terms are not calculated thereby excluding electrostatic interactions. This allows sampling of larger conformational space in a shorter time interval. 100 docked conformations were collected during this phase in which the ligand is subjected to random combinations of translational and rotational movements followed by 1000 steps of minimization using conjugate gradients. The conformations lying within the energy range of 100 kcal and 1Å RMS deviation were selected.▪ The conformations collected in previous step were filtered using various parameters like orientation of ligand, distance from the active-site residues and bad contacts, if any.▪ Twenty conformations satisfying above criteria were further refined in the second stage using Group_based method in which non-bond interactions were calculated using van der Waals and coulombic cutoffs of 15 and 10Å respectively. The conformers so obtained were refined by minimization of 100 steps of conjugate gradient followed by 50 steps of simulated annealing starting at an initial temperature of 500 K up to a final temperature of 300 K with temperature leap of 4 K at each step. The Newton's equations of motion were integrated using the Verlet algorithm  with a t▪ The conformers were evaluated based on the interaction energy, which is a sum total of van der Waal and coulombic interactions. The least energy conformer having favorable interactions with the residues in the binding pocket was selected for further analysis.UKK: Design and coordination of the study, analyses and interpretation of data, drafted the manuscriptSGB: Development of database & query interface, docking studiesGSM: Whole genome phylogeny, docking studies and drafted manuscriptMJ: Homology modeling of JEV NS3SB: MSA and derivation of signature sequencesASK: Conceived the study, critical evaluations and useful suggestions