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Tick-Borne Encephalitis Virus Infects Rat Astrocytes but Does Not Affect Their Viability

Tick-borne encephalitis virus (TBEV) causes one of the most dangerous human neuroinfections in Europe and Asia. To infect neurons it must cross the blood-brain-barrier (BBB), and presumably also cells adjacent to the BBB, such as astrocytes, the most abundant glial cell type. However, the knowledge about the viral infection of glial cells is fragmental. Here we studied whether TBEV infects rat astrocytes. Rats belong to an animal group serving as a TBEV amplifying host. We employed high resolution quantitative fluorescence microscopy to investigate cell entry and cytoplasmic mobility of TBEV particles along with the effect on the cell cytoskeleton and cell survival. We report that infection of astrocytes with TBEV increases with time of exposure to TBEV and that with post-infection time TBEV particles gained higher mobility. After several days of infection actin cytoskeleton was affected, but cell survival was unchanged, indicating that rat astrocytes resist TBEV-mediated cell death, as reported for other mammalian cells. Therefore, astrocytes may present an important pool of dormant TBEV infections and a new target for therapeutic intervention.

Introduction
Tick-borne encephalitis virus (TBEV) is medically important human pathogen that causes one of the most dangerous neuroinfections in humans in Europe and in Asia [1][2][3]. TBEV is a member of the genus Flavivirus, family Flaviviridae [4]. Mature virus particles are enclosed within lipidic envelope which carry attachment molecules (E protein) for the host cell receptors (heparan sulfate) [1,[5][6][7]. The first and the most important host cells infected by TBEV are likely epidermal Langerhans cells (i.e. dendritic cells; [8]) which transport the virus to the lymph nodes and initiate the spread of infection to lymphoid compartments [3]. In some vertebrate species the virus is neurovirulent and crosses the blood-brain barrier (BBB), which isolates and protects the brain tissue from fluctuations in nutrients, hormones, metabolites, and from the direct influences of many endo-or exogenous compounds circulating in the blood [3,9]. The mechanism by which TBEV invades the central nervous system (CNS) is not clear, and may include cytokine-mediated entry, entry of TBEV-infected cells of the immune system and infection of endothelial cells [10,11]. After entering the CNS, neurons are primary targets, but other brain cells may also be infected [3]. Such cells are astrocytes which are positioned between synapses and endothelial cells, playing a role in neurovascular coupling [12,13]. The infection of astrocytes may have unfavourable consequences for brain functioning. In general, astrocytes have many leading roles in the brain, including integrating neuronal functions, neuronal support, and the maintenance of BBB inter-endothelial tight junctions in normal and pathologic conditions [14][15][16][17][18][19][20][21][22][23][24][25][26]. It has been shown recently that brain TBEV infection alters the permeability of the BBB in mice [11], and astrocytes may be implicated in this process, since these cells regulate blood flow in the brain [12,13]. In addition, the effects of neurovirulence can be observed relatively rapidly, compared to the clinical course of the disease [3,27], which likely indicates that a reservoir of TBEV may exist in the CNS.
The aim of this study was to test whether astrocytes, the most numerous glial brain cells [17], can be infected by TBEV and to measure cytoplasmic TBEV particle dynamics in the initial phases after the infection, along with the astrocyte viability. The susceptibility to TBEV infection would make astrocytes a potential TBEV reservoir. We used rat astrocytes as a model cell, since rodents of several species are known to be TBEV amplifying hosts and may maintain TBEV through latent persistent infections [3,28].
The results show that TBEV infects rat astrocytes and that the infection of a single cell, which progresses in time-dependent phases, is associated with changes in actin cytoskeleton, but astrocyte viability is unaffected. We propose that astrocytes represent an important reservoir of TBEV brain infection, which makes these cells a new target for therapeutic intervention. Given their tight morphological association with blood vessels, infected astrocytes may possibly affect the BBB and neurons.

Ethics Statement
The care for experimental animals and the euthanization of animals was carried out in strict accordance with the following ethical codes and directives:

Cell Cultures
Astrocyte cultures were prepared from cortices of 3 days old Wistar rats as described [29]. Cells were maintained in highglucose Dulbecco's modified Eagle's medium (Invitrogen, Life Technologies, Carlsbad, NM, USA) containing 10% fetal bovine serum (FBS), 1 mM pyruvate, 2 mM glutamine and 25 mg/ml penicillin/streptomycin at 37uC, 95% air/5% CO2. After reaching confluence, cells were manipulated as described [30]. Prior the experiments cells were sub-cultured onto Lab-Tek TM chambered coverglass (Thermo Scientific) or onto 22 mmdiameter poly-L-lysine-coated coverslips and used within 6 days after plating. Vero E6 cells were used for the preparation of virus stocks and as productively infected control cell type after infection with TBEV. Cells were maintained in Dulbecco's MEM with high glucose (DMEM GlutaMAX TM ; Invitrogen) supplemented with 10% FBS. Cultures were incubated at 37uC/5% CO2.) Unless stated otherwise all chemicals for maintaining cell cultures and experimental procedures were obtained from Sigma-Aldrich (Diesenhofen, Germany).

TBEV Labelling and Cytotoxicity Test
Viral strains and isolates of TBEV can be classified into three subtypes: the European, the Siberian and the Far Eastern [31]. In this study we used the European TBEV strain Ljubljana 1 [32]. TBEV was grown 7 days on Vero E6 cells. Supernatant was collected and centrifuged twice at 4uC (10 min at 3200 6 g and 5 min at 20800 6 g in Eppendorf 5804R centrifuge). Pellet was

TBEV RNA Concentration
The concentration of TBEV RNA was measured using one-step quantitative real time RT-PCR [34]. Vero E6 cells were infected with TBEV and the virus concentration was measured in the supernatant collected from Vero E6 cells 7 days post infection (p.i.) before and after labelling TBEV with fluorescent lipophilic VybrantH DiD labelling solution. Further on, in the experiments, we have used the TBEV in concentration of 10 8 copies/ml, labelled with 50 mM DiD.

Imaging
Imaging of fixed and live cells was performed with an inverted confocal microscope (Zeiss LSM 510 META, Carl Zeiss) using oilimmersion objective 636/NA 1.4. For excitation of DiD dye He/ Ne laser was used (633 nm), the emission light was filtered with long pass filter, with the cut off below 650 nm. The conjugate Alexa Fluor 488 was excited by argon laser (488 nm) and the emission light was collected through the band pass filter (505 to 530 nm). The conjugate Alexa Fluor 546 was excited by He/Ne laser and the emission light was filtered with long pass filter 560 nm. To eliminate possible bleed-through, the green and red emission fluorescence was acquired sequentially. In live cells the mobility of vesicles that expressed DiD fluorescence of labelled TBEV was recorded. Time series images were recorded in 2 s intervals for 2 min of total recording time. Experiments were conducted at 37uC (Heatable universal mounting frame, Carl Zeiss).

Immunocytochemistry
The cells were washed with the phosphate buffered saline (PBS), fixed in 2-4% formaldehyde (prepared from paraformaldehyde) in PBS for 5-15 min at room temperature (RT) and permeabilized with Triton X-100 for 10 min at RT. The non-specific back- ground staining was reduced by incubating cells in blocking buffer, containing 3% bovine serum albumin (BSA) and 10% goat serum in PBS, at 37uC for 1 h. The cells were then stained with primary antibodies, diluted into 3% BSA in PBS and incubated at 37uC for 2 h or at 4uC overnight. When two primary antibodies (raised in different species) were used, the staining was done sequentially. Afterwards, the cells were rinsed in PBS and stained with secondary antibodies at 37uC for 45 min. At the end of the staining protocol the cells were mounted onto glass slides using Slowfade Gold antifade reagent (Molecular Probes, Invitrogen).

Analysis
The mobility of fluorescently labelled vesicles was analysed by ParticleTR software (Celica, Slovenia). To describe vesicle mobility the parameters were calculated as described [36,37]: step length (displacement of a vesicle in the time interval of 2 s), track length (TL, the total length of the analysed vesicle pathway), velocity and maximal displacement (MD; [36]). Vesicle mobility was analysed in cells from three independent astrocyte cultures. The analysis of the vesicle mobility was performed for epochs of 30 s.
Vesicle size was analysed with ImageJ software (available at National Institute of Health, USA, http://rsbweb.nih.gov/ij/). Fluorescent particles above the threshold level 8 pixels 2 (0.1460.14 mm 2 ) were determined as vesicles, corresponding to the vesicle size area above 0.1568 mm 2 to cover a broad span of imaged vesicles with different fluorescence intensities at slightly different z- positions within optical slice, similarly as described [38]. The extent of the co-labelled vesicles by fluorescent probes was determined by manually counting the observed fluorescent probes in the green and red channels.
Statistical significance was determined with the Mann-Whitney Rank Sum test. Values presented on graphs are expressed as mean 6 s.e.m.

Identifying Initial Steps of TBEV Infection in Astrocytes
To image intracellular localization of TBEV particles, we prelabelled them with 50 mM DiD lipophylic dye and afterwards infected cultured astrocytes with conc. of 10 7 RNA copies/ml. Infection with TBEV was confirmed by immunolabelling with antibodies against the viral large envelope protein E [5] (Figure 1A, inset). TBEV labelling of cells increased with the time p.i. as assessed by counting TBEV particles per cell ( Figure 1A,B). On one hand, the screening of several time periods following the   Figure 1A). We analyzed up to 31 cells from three different animals. On the other hand, we also determined the fraction of cells in which at least 3 DiD-TBEV particles were observed in a coverslip with cultured astrocytes. Clearly, the percent of infected cells increased as a function of post-infection time: 6869% (4 h), 9262% (18 h; P,0.05; Figure 1B).

With Increasing p.i. time DiD-TBEV Particles Acquire Increased Directionality
Large vesicles with fluorescence area above 1.078 mm 2 were immobile. On the other hand, small vesicles were mobile and were subjected to mobility analysis. Speed, track length (TL) and maximal displacement (MD) were calculated as described previously [36]. In 30 s of recording time vesicle pathways appeared predominantly clumped, meaning that vesicles exhibited nondirectional mobility (NDM). However, some vesicles displayed directional mobility (DM), as evident from elongated trajectories ( Figure 4A). The percent of DM periods (Figure 4, MD.1 mm, (dashed line)) remained below 20% during all incubation times, although the percent of small vesicles increased from 41% (2 h) to 72% (18 h, Figure 3). MD and TL of DM periods and NDM periods were distinct ( Figure 5). Mean MD of DM periods significantly increased: from 1.6960.27 mm (2 h), to 2.6860.23 mm (4 h; P,0.05) and 2.4260.09 mm (18 h; P,0.01) ( Figure 5C). And mean TL of DM periods significantly increased from 3.7460.35 mm (2 h) to 5.0060.29 mm (4 h; P,0.05, Figure 5D) and to 4.

TBEV Induces Disintegration of Actin Filaments While Cell Viability Remains Unchanged
Cytoskeleton rearrangements of host cells are reported for several viral infections (Herpesvirus, [43]; TBEV, [27]). To examine whether TBEV infection triggered changes of the rat astrocyte cytoskeleton, molecular motor-associated microtubules and actin filaments were labelled in astrocytes at several p.i. times (4 h, 18 h, 48 h, 3 days and 6 days). No obvious alterations of cytoskeleton were noticed in rat astrocytes until day 2 p.i. Then, after day 3 p.i. significant reorganization of actin filaments was observed, while microtubules appeared unaffected ( Figure 6).
Finally, to asses whether TBEV triggers the cytopathic effect (CPE) in primary rat astrocytes, we recorded fields of view of TBEV infected primary rat astrocytes and control Vero E6 cells and measured their viability for 14 days (Figure 7). The CPE (morphologically altered cell shape, detached cells) was observed in Vero E6 cells at day 3 p.i, whereas in astrocytes no CPE was recorded ( Figure 7A). Morevover, the results of the trypan blue exclusion viability test show that the viability of primary rat astrocytes was not affected by TBEV infection (Figure 7 B). On the other hand, in the control Vero E6 cells, which were used to multiply TBEV, the viability was significantly affected already after 18 h (6662%, P,0.001) and was further reduced to 1662% (P,0.001) at day 14 p.i., compared to non-infected cells (9562%) ( Figure 7B). Successful replication of TBEV in both cell types was confirmed by measuring the virus load (TBEV copies/cell) at different times p.i. (Figure 8). Moreover, the supernatant collected from astrocytes at the end of the experiment (14 days) still reduced the viability of Vero E6 cells (39%) at day 7 p.i. and triggered CPE (Figure 8), confirming the presence of infective TBEV. From these data we conclude that TBEV infection does not significantly affect the viability of rat astrocytes, implying that these cells could act as a potential TBEV reservoir.

Discussion
Despite of the high clinical importance of TBEV neuroinfection (long-term neurological disabilities, over 1% mortality rate, growing incidence in Europe), nothing is known about the TBEV infection of the most abundant neuronal supportive glial cells, astrocytes, and their possible implication in active or dormant TBEV infections. Recent data indicate that TBEV can be detected in rodent organs for longer periods of time and that the brain tissue stands out by high virus load [34]. In this report experimental evidences show that astrocytes, the most abundant mammalian glial cells become infected with TBEV, which makes them a potential mediator of brain infection and a reservoir of brain TBEV in rodents.
TBEV internalization into astrocytes increased with longer postinfection time. Although the mechanism of internalization remains to be elucidated, a number of different proteins have been identified as potential flavivirus receptors on the cell surface, however there is no direct evidence for a role of any of these during TBEV entry into host cells, and some of the results are controversial [44]. Glycosaminoglicans, such as heparan sulphate (HS), are expressed on different tissues [45]. Frequently, HS appears to serve as a primary attachment molecule that concentrates viral particles on the cell surface and may facilitate the subsequent binding to more specific receptor molecules [7,46]. An involvement of HS during attachment and entry of TBEV has been demonstrated, although it appears that more than one single type of host-cell molecule is involved [3,5,7,47]. In astrocytes mimicking injury response, upregulation of HS and higher sulfation of heparan sulphate proteoglycans occurs [48]. Therefore, one possible explanation of increased time-dependent TBEV entry into astrocytes could be attributed to an increased expression of HS on astrocyte membrane of infected astrocytes. However, this was not monitored in our experiments. The internalization of TBEV particles into astrocytes was consistent with confirmed clathrin-dependent entry of several members of Flaviviridae family: West Nile virus (WNV) [2], Dengue virus [49,50], Hepatitis C virus [51] and Bovine Viral Diarrhoea virus (BVDV) [52]. And their localization in late endosomes/lysosomes was consistent with other flaviviruses: BVDV, [52]; WNV, [53]; DIL-labelled dengue virus, [50].
TBEV-loaded vesicles observed following 18 h post-infection time were predominantly smaller or comparable in size to early endosomes (300 nm, [2]; 300-400 nm, [54]); and much smaller from late endosomes which have on average 700 nm in diameter [55]. Taking into account the point-spread function related overestimation of vesicle diameter [38], the majority of TBEV vesicles exhibited diameters between 150-400 nm. This is in line with WNV vesicles, which have 100 nm in diameter until they start to fuse and become approximately the size of 500 nm [2]. A minor portion of DiD-TBEV particles (6% at 2 h p.i., 5% at 4 h p.i. and 20% at 18 h p.i.) was even comparable in apparent size to peptidergic and glutamatergic vesicles monitored in astrocytes (50-100 nm, [38]; [56]). The rest of TBEV-loaded vesicles were between 500 or 600 nm (diameter), which is in the range of late endosomes and lysosomes [54,55]. With longer p.i. time the average size of large vesicles decreased, which indicates changes in vesicle dynamics, such as attenuated formation or enhanced cleavage of very large vesicles.
Despite the increase in the percent of smaller highly mobile TBEV loaded vesicles recorded with longer time p.i. (up to 72%), the percent of directional mobility periods remained below 20% at all times p.i. This is in line with the mobility properties of other vesicles which travel along the cytoskeleton [37,57] and with the restricted availability of host cell trafficking apparatus to be used by internalized TBEV viruses (reviewed in Greber [58,59]). These vesicles apparently move along both molecular motor-associated filaments. Analysis of vesicle speed revealed that the speed of DiD-TBEV vesicles generally corresponded to speeds of processive human myosins (Va, VI, and X) which travel as fast as 0.3-0.9 mm/s in vitro and 0.1-0.4 mm/s in live cells (references within [60]). These values correlate with DENV particle trafficking towards endosomes [61]. On the other hand, maximal recorded speeds above 1 mm/s confirm also the involvement of kinesin motors along microtubules [62]. Increased mobility at longer periods p.i. may reflect the changes in local protein synthesis affecting vesicle trafficking, as it was observed in axons infected by pseudorabies virus [63].
It is possible that viruses are transported on the account of innate cell vesicles, therefore we assessed if this impairs cell viability. In general, little is known about how various cell types in the CNS react to viral infections causing encephalitis [64]. In TBEV infected rat astrocytes we observed alterations of actin cytoskeleton, and no apparent change in the arrangement of microtubules. This is in contrast from the observation in human glioblastoma cells, where TBEV caused a substantial microtubule rearrangement already after 48 h p.i. [27]. The cell viability of primary rat astrocytes remained unaltered during monitored time p.i. (14 days). We did not observe any TBEV-triggered necrosis in rat astrocytes in 14 days p.i. (95.060.9% viable cells in the noninfected control and 97.061.0% viable cells after 14 days p.i., P = 0. 267). It appears that astrocytes are much more resilient to TBEV infection than the control Vero E6 cells (monkey kidney epithelial cell lineage) that showed reduced viability already after 18 h (Figure 7) and than porcine kidney cells, where almost all cells were dead at 50 h p.i. [65]. The shape of astrocytes was unaltered in 14 days p.i., the cells were not rounded, which is also in contrast to glioblastoma cell lines where 20% of cells were apoptotic already after 48 h p.i.; determined by rounding of cells and TUNEL assay [27]. These data suggest that in contrast to human glioblastoma cells rat astrocytes are more resilient to TBEV infection. Thus, rat astrocytes may serve as a reservoir for spreading the viral infection or maintaining TBEV reservoir in wild rodents. It has been reported that the virus likely remains dormant in the brain tissue for long time in wild rodents, while they show no clinical symptoms of TBEV infection [28,34,66]. We propose that the cell reservoir of dormant virus may consist of astrocytes. Vesicle traffic in astrocytes can be modified by therapeutics, such as fingolimod [67], which has been recently introduced to treat multiple sclerosis. Therefore, a similar strategy may be used to prevent astrocyte TBEV infection and possibly other brain cells. Recently, it was reported [11] that TBEV entry is independent on the breakdown of the BBB, but that the BBB breakdown is a consequence of TBEV brain infection. Whether astrocytes play a role in this process remains to be investigated, especially in the light of astrocyte role in compromising the permeability of the BBB.


Immunocytochemical Localisation of Phospholipid Hydroperoxide Glutathione Peroxidase in Bull’s Spermatogenic Cells

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein, which protects biomembranes from oxidative damages, and it also accounts for almost the entire selenium content of mammalian testis. The present investigation was performed to localise PHGPx in the testis and in epididymal and ejaculated spermatozoa of the bull by using light and electron immunomicroscopy. The study also aimed to further clarify the possible functions of the protein in bull fertility. In the testis, spermatogenic cells of the adluminal tubular compartment showed cytoplasmatic immunostaining; whereas, in the epididymal and ejaculated spermatozoa immunostaining was specifically localised at the level of the head and mid-piece. Ultrastructural data revealed the presence of signals for PHGPx in different subcellular compartments of maturing and mature sperm (mitochondria, chromatin, nuclear envelope, acrosomes, cytoskeletal structures) suggesting that this enzyme plays versatile and important biological roles during spermatogenesis. The final localisation of the immunostaining at acrosomal level puts forward a new role of the protein which further emphasises its relevance in male reproduction: it is reported to anchor substrate of the sperm acrosome to the oocyte zona pellucida during the fertilisation process.

Introduction
Selenium (Se) deficiency has been linked to reproductive problems and reduced sperm quality in rats, mice, chickens, pigs, sheep, and cattle (Combs and Combs, 1986;Olson et al., 2004;Baiomy et al., 2009). Selenium supplementation to diets has been reported to improve reproductive performance in mice, sheep and cattle (Sanders, 1984;Tang et al., 1991;Van Ryssen et al., 1992;Aréchiga et al., 1998), while others have not found association between herd milk and blood Se concentrations and fertility parameters (Ropstad and Refsdal, 1987;Jukola et al., 1996).
The phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an enzyme working as the deposit of almost the entire Se content in the mammalian testis. The enzyme is a member of the large subfamily of the glutathione peroxidase (GPx) selenoproteins, most of which are antioxidant enzymes that reduce hydroperoxides at the expense of glutathione (GSH). However, PHGPx has a number of unique features compared to other family members, making the role of this enzyme in the testis worth of particular interest. Among GPx, PHGPx is the least specific (Ursini et al., 1995(Ursini et al., , 1997Maiorino and Ursini, 2002) reducing a broad spectrum of hydroperoxides and accepting different thiols, including protein thiols as reductants. By catalysing hydroperoxide reduction, PHGPx plays a key antioxidant function, controls inflammatory responses (Schnurr et al., 1996;Imai et al., 1998;Sakamoto et al., 2000), and inhibits apoptosis (Nomura et al., 2001). Additionally, in spermatogenesis, PHGPx plays a major role in oxidising specific protein thiols. Phospholipid hydroperoxide glutathione peroxidase is also represented by glutathione peroxidase 4 (GPx4) and three different isoforms are identified (Brigelius-Flohé and Maiorino, 2013): cytosolic (cGPx4), mitochondrial (mGPx4) and sperm nuclear GPx4 (snGPx4). Indeed, it is associated with the cytoplasm, mitochondria and nucleus of spermatogenic cells in rodent testis, suggesting that this enzyme plays versatile roles during the various phases of spermatogenesis (Tramer et al., 2002;Haraguchi et al., 2003). However, PHGPx -abundantly expressed in early spermatogenic cells as an active peroxidase -is transformed into an enzymatically inactive insoluble structural protein during the late phase of spermatogenesis (Ursini et al., 1999). Therefore, it makes up the keratin-like material, constituting the mitochondrial capsule that surrounds the helix of mitochondria in the sperm mid-piece. Hence, this function may explain the Se deficiency dependent alterations in sperm cells, suggesting a new approach to investigate male fertility dependency on Se. Previously, a reduced PHGPx content was reported in both infertile human males (Foresta et al., 2002) and bulls with impaired seminal characteristics and the normal motility and morphology of mature spermatozoa were demonstrated to be associated with a high PHGPx content (Stradaioli et al., 2009).
The aim of this study was to localise subcellular PHGPx in spermatogenic cells and epididymal and ejaculated spermatozoa of the bull. Our results should help to clarify the biological significance of PHGPx in the fertilisation process, its clinical relevance and whether it has any role in improving bovine reproductive efficiency.

Immunohistochemistry in light microscopy
Immunostaining was performed on bull testes and isolated epididymal and ejaculated spermatozoa. Samples of testes were obtained, immediately after slaughter, from 5 Chianina bulls clinically healthy aged 32±8 months. They were fixed by immersion in Bouin's fixative for 24 h, dehydrated and embedded in paraffin wax. The paraffin sections were cut to 5-6 µm of thickness. The epididymides were rapidly isolated from the testes and divided into caput, corpus and cauda in order to collect the spermatozoa separately from the distinct regions as described by Seligman et al. (1991). In brief, pieces of tissue were shaken in 0.1M phosphate buffered saline (PBS) at pH 7.4 and 37°C for 15 min to allow the release of spermatozoa and the epididymal fluid. The ejaculated spermatozoa were collected by means of an artificial vagina from 5 Chianina bulls of proven fertility aged 40±19 months. Epididymal and ejaculated spermatozoa were washed twice by centrifugation for 10 min at 600 g in 0.1M PBS at pH 7.4 and then smeared on double glass slides, air-dried and stored at-70°C until analysis. A series of smear preparations were treated with 6M guanidine-HCl, 10 mM GSH, 10 mM dithiothreitol (DTT) and 10 mM 2-mercaptoethanol (2-ME) for 30 min at room temperature (RT) and fixed in 4% paraformaldehyde for 30 min (Haraguchi et al., 2003). Subsequently, the testis sections and smear preparations, with and without the prior abovementioned treatments, were subjected to immunohistochemical procedures as previously described (Zerani et al., 2012(Zerani et al., , 2013a(Zerani et al., , 2013bColitti and Parillo, 2013;Parillo et al., 2013Parillo et al., , 2014aParillo et al., , 2014b. Briefly, the testis sections were incubated with 3% H2O2 for 20 min to block the endogenous peroxidase activity. After rinsing in PBS containing 0.2% Triton X-100 and 0.1% bovine serum albumin (BSA), background labelling was prevented by incubating the slides with normal goat serum (NGS), diluted 1:10, for 30 min at RT. Then, the slides were incubated overnight in a humid chamber at 4°C with the primary antiserum, rabbit anti-PHGPx (supplied by Prof. Ursini, University of Padova), diluted 1:400. The next day, the slides were washed in PBS and incubated with biotin goat anti-rabbit IgG conjugate (Zymed 81-6140) diluted 1:200 for 30 min. After PBS washes, the slides were exposed to avidinbiotin complex (ABC kit; Vector Labs, Burlingame, CA, USA) for 30 min. The slides were rinsed again and the immunoreactive cells were visualised using a freshly prepared solution of 3,3'-diaminobenzidine tetrahydrocloride-hydrogen peroxidase (DAB-H2O2 kit; Vector Labs). Finally, after washing in tap water, the slides were dehydrated through an ethanol series, cleared in xylene, and mounted in natural Canada balsam (BDH Chemicals, Radnor, PA, USA). Slides on which the primary antibody was omitted or substituted with rabbit IgG, were used as negative control of unspecific staining.

Postembedding immunoelectron microscopy
Samples of testes, isolated epididymal (from caput, corpus and cauda epididimys) and ejaculated sperms were also processed for immunogold microscopy. Samples of testes were fixed with 4% paraformaldehyde and 0.25% gluteraldehyde in 0.1M cacodylate buffer (pH 7.4) for 1.30 h at 4°C. Aliquots of epididymal and ejaculated spermatozoa from each bull were diluted in PBS, centrifuged twice (600xg) and the resulting pellets were fixed as above. The samples were washed several times in a cacodylate buffer, dehydrated in a graded series of ethanol up to absolute and embedded in Bioacryl resin (Bio-optica, Milan, Italy) (Parillo et al., 2003(Parillo et al., , 2005. The ultrathin sections (90 nm) were mounted on 200-mesh nickel grids, treated with 1% BSA in 0.1M tris buffered saline (TBS) pH 7.4 for 5 min at RT, and then incubated overnight in a humid chamber at 4°C with a solution of 0.1M TBS containing 1% BSA, 1% NGS, 0.1% Tween-20 and anti-PHGPx rabbit polyclonal antibody (diluted 1:400). After several washes in TBS to remove the excess antibody, the sections were incubated for 20 min at RT with the goat anti-rabbit IgG secondary 10 nm gold-conjugated antibody (Sigma Chemicals, St. Louis, MO, USA) diluted 1:10 in 0.1M TBS pH 7.4 plus 1% BSA and 0.05% Tween 20. As controls, some grids were treated with the incubation mixture without the primary antibody and then processed as described above. Finally, after counterstaining with uranyl acetate, the sections were examined with a Philips EM 208 electron microscope (Philips, Amsterdam, The Netherlands).

Immunohistochemistry in light microscopy
In the testis sections, specific immunostaining by antiserum against PHGPx was evident in the apical compartment of the seminiferous tubules where it was localised in the cytoplasm of maturing sperm cells; whereas there was any apparent immunostaining in the basal compartment of the tubules (Figure 1). In epididymal (caput, corpus, cauda) and ejaculated spermatozoa, weak staining was localised in the head and mid-piece portion (Figure 2a), whereas the tails resulted constantly negative. Treatment with 6M guanidine-HCl, 10 mM DTT as well as with 10 mM 2-ME strongly enhanced the reactivity of the sperm mid-piece (Figure 2b), while treatment with 10 mM GSH strongly increased immunostaining of the sperm head (Figure 2c). The positive staining was missing in control sections (Figure 2d). Results are summarised in Table 1.

Postembedding immunoelectron microscopy
Only at electron microscopy it was possible to detect the presence of gold particles for PHGPx in different subcellular structures of sperm cells at various phases of the spermatogenic cycle.
In spermatogonia, PHGPx labelling was evidenced in the matrix of the mitochondria, cytosol and euchromatin (Figure 3).
In round spermatids, characteristic PHGPx reactivity appeared in the acrosomal vesicles; the number of gold particles gradually increased as the acrosomes developed ( Figure  4a,b). Furthermore, PHGPx signals were observed in the matrix, in the outer membrane of the mitochondria and the cytosol ( Figure  4c). In the nuclei of these cells, PHGPx positivity was localised in the heterochromatin and over the nuclear envelope (Figure 4a-c).
In elongated spermatids, the mitochondria surrounded the flagellar axoneme to form a spiral structure; PHGPx labelling was located in the matrix and mainly in the outer membrane (Figure 5a,b). Signals for PHGPx were also observed in the condensed chromatin and over the nuclear envelope, in the residual cytoplasm ( Figure 5a) and the acrosome (Figure  5b,c). At this stage of germ cell development, a few gold particles for PHGPx were also noted in the outer dense fibres of mid-piece cytoskeletal structures (Figure 5a).
Epididymal and ejaculated spermatozoa showed very similar immunogold signals; in particular, gold labelling for PHGPx was detected in the acrosome, in the condensed chromatin and the nuclear envelope. In the midpiece, PHGPx signals were closely associated with the outer mitochondrial membrane and concentrated in the outer dense fibres, which surround the flagellar axoneme ( Figure 6a). An immunocytochemical response was also seen in the main piece of the tail at the level of both the outer dense fibres and the fibrous sheet, which underlies the plasma membrane ( Figure 6b). In addition, gold particles were observed in the cytoplasmic droplets of spermatozoa (Figure 6c). There was no immunostaining in the control sections.

Discussion
Specific immunostaining for PHGPx was revealed in the apical region of the seminiferous tubules with a light microscope. In epididymal and ejaculated sperm, immunostaining for PHGPx was more evident if the sperm smears had been treated with chaotropic or reducing agents. The enhancement of immunoreactivity following some pre-treatments suggests that the access of antibody to PHGPx in ejaculated sperm is hindered. It is possible, as suggested by Roveri et al. (1992), that in spermatozoa the protein could be embedded in a complex matrix, hampering its interaction with the antibody. It is clear that only chaotropic treatments are capable of rendering more protein from the heads and mitochondria of the midpiece accessible.
More detailed information on the presence of PHGPx signals in bull testes and isolated spermatozoa were obtained with immunogold electron microscopy, which allows also some considerations about the functional role of this protein. phospholipid hydroperoxide glutathione peroxidase immunostaining was evident at the mitochondria, chromatin, cytosol and acrosome levels; indeed, these localisation sites were similar to those previously described in the rat (Tramer et al., 2002;Haraguchi et al., 2003) and the mouse (Nayernia et al., 2004).
Mitochondria resulted positive throughout the differentiation process although changes in the localisation of PHGPx labelling during the various phases of sperm cell maturation were evidenced. In fact, PHGPx signals were localised in the matrix of the mithocondria during spermatogonia developmental stage, evidenced both in the matrix and in the mitochondrial outer membrane during round spermatids stage, and were strongly associated to the outer membrane in elongated spermatids and in mature sperm. The translocation of PHGPx signals from the matrix to the peripheral region of the mitochondria was previously reported in the rat (Haraguchi et al., 2003). It has been hypothesised that the re-localisation of PHGPx in the mitochondrial outer mem-

Without treatment With treatment
Cytoplasm of germ cells present in the apical compartment of seminiferous tubules ++ Cytoplasm of germ cells present in the basal compartment of seminiferous tubules -Head of epididymal and ejaculated sperm + +++ (after treatment 10 mM GSH) Mid-piece of epididymal and ejaculated sperm + +++ (after treatment with 6M guanidine-HCl, 10 mM dithiothreitol, and 10 mM 2-ME) Tail of epididymal and ejaculated sperm -PHGPx, phospholipid hydroperoxide glutathione peroxidase; GSH, glutathione; DTT, dithiothreitol; 2-ME, 2-mercaptoethanol. °+ and -indicate staining intensity on a subjective scale attributing -to negative reaction and +++ to a strong one.
brane may indicate the switch of PHGPx from a soluble active enzyme, involved in the reduction of H2O2 generated in the mitochondria, to an enzymatically inactive, oxidatively crosslinked and insoluble protein that performs a structural role in forming the mitochondrial capsule (Ursini et al., 1999). Cytosolic PHGPx positivity was also evident in all maturation stages; in fact, the enzyme plays a crucial role in reducing intracellular increases in the concentration of H2O2 dispersed in the cytoplasm matrix, reflecting the need to protect these cells from oxidative damage caused by hydroperoxides (Arai et al., 1999).
The aforementioned localisation is likely due to the change in substrate availability of the enzyme; in fact, the most striking observation related to PHGPx substrate specificity was that protein thiols can take over the function of GSH as reductants when the latter becomes limited. This has been shown for chromatin (Godeas et al., 1996), for sperm mitochondria associated cysteine-rich protein  and even for GPx4 itself (Ursini et al., 1999;Mauri et al., 2003). Thus, depending on the availability of GSH, GPx4 can act either as a GSH peroxidase or as a thiol peroxidase.
Chromatin positivity could indicate an important role of PHGPx in the maturation process of sperm cell nuclei by protecting them from mutagens and chromatin from autolysis and, in general, by protecting the nucleus from external stress before fertilisation. In fact, it is well known that sperm maturation in testes ends with nucleus compaction achieved by protamines replacing nuclear histones. Through this, the sperm haploid genome is compacted to ca. 1/10 the size of the nucleus of other somatic cells (Miller et al., 2010). This process does not end with the entry of spermatozoa into epididymal lumen when they are still functionally immature, namely, they are not yet able to move appropriately in female genital tract and to recognise, bind to and penetrate Parillo et al.   nuclear envelope (a,b, arrows). Gold particles are also detected in the matrix, the outer membrane of mitochondria (c) and the cytosol (c, arrows). Bars=0.5 µ.
oocytes. Indeed, it has been demonstrated that, apart from a protective role against DNA peroxidative damage, PHGPx acts as a protamine thiol peroxidase, responsible for the formation of cross-linked protamine disulfide (Pfeifer et al., 2001), thus contributing to the structural stability of sperm chromatin (Conrad et al., 2005). The positivity of the spermatid's and mature sperm's nuclear envelope may have a role as a structural protein similar to the PHGPx associated with the mitochondrial outer membrane (Haraguchi et al., 2003). In fact, it has been recently demonstrated that mGPx4 knockout male mice are sterile as they produce spermatozoa with structural abnormalities, such as bent sperm, sperm heads detached from the mid-piece, sliding mitochondria along the mid-piece, i.e. with charac-teristics reminding of long-term Se deficiency (Schneider et al., 2009).
In addition, the fact that PHGPx has also been localised in the acrosomal granules and membranes of both spermatids and mature spermatozoa leads us to hypothesise an additional effect of male gamete PHGPx, released during acrosomal reaction and the early steps PHGPx in bull sperm  of fertilisation. We suggest that acrosomal PHGPx could be part of the insoluble matrix which persists after acrosome reaction allowing stabile binding of the sperm head to the oocyte zona pellucida during the early steps of the fertilisation process (Bedford, 2014). This could be a further functional explanation of Se deficiency related sterility.
Finally, the presence of PHGPx in the outer dense fibres and fibrous sheet seems to suggest that PHGPx plays an important function in stabilising cytoskeletal structures.

Conclusions
Our immunocytochemical study clearly evidenced the presence of PHGPx in different subcellular compartments of bull sperm together with dynamic changes occurring to the protein in the cells during the maturative processes. These findings confirm biochemical data suggesting that PHGPx is required for a correct development and function of mammalian spermatozoa (Ursini et al., 1999). The structural function ascribed to the protein may therefore explain the Se-deficiency-dependent alterations in sperm cells. Indeed, ultrastructural analyses of spermatozoa from Se deficient animals have identified structural irregularities in the mid-piece mitochondria where the enzyme is amply present (Olson et al., 2004). In addition, in both humans and bulls failure of the expression of mitochondrial PHGPx in spermatozoa has been reported to be one of the possible causes of oligoasthenozospermia (Foresta et al., 2002;Stradaioli et al., 2009).


extfieldsofstudy: Biology Medicine

text:
In- silico exploration of thirty alphavirus genomes for analysis of the simple sequence repeats

The compilation of simple sequence repeats (SSRs) in viruses and its analysis with reference to incidence, distribution and variation would be instrumental in understanding the functional and evolutionary aspects of repeat sequences. Present study encompasses the analysis of SSRs across 30 species of alphaviruses. The full length genome sequences, assessed from NCBI were used for extraction and analysis of repeat sequences using IMEx software. The repeats of different motif sizes (mono- to penta-nucleotide) observed therein exhibited variable incidence across the species. Expectedly, mononucleotide A/T was the most prevalent followed by dinucleotide AG/GA and trinucleotide AAG/GAA in these genomes. The conversion of SSRs to imperfect microsatellite or compound microsatellite (cSSR) is low. cSSR, primarily constituted by variant motifs accounted for up to 12.5% of the SSRs. Interestingly, seven species lacked cSSR in their genomes. However, the SSR and cSSR are predominantly localized to the coding region ORFs for non structural protein and structural proteins. The relative frequencies of different classes of simple and compound microsatellites within and across genomes have been highlighted.

Introduction
According to International Committee on Taxonomy of Viruses (ICTV) report (King et al., 2012), the family Togaviridae has two genera namely Alphavirus and Rubivirus. Rubivirus genus has a solo member, Rubella virus whereas the Alphavirus includes thirty small, enveloped, plus-strand RNA viruses. These are responsible for human or animal diseases and are classified antigenically into six complexes (Garmashova et al., 2007;Powers et al., 2001). Further, the species are categorized into either the New World or Old World group based upon their prevalence.
Alphaviruses form icosahedral spherical particles of 65-70 nm in diameter and are considered a model system for structural studies of enveloped viruses. The species herein are known to cause several human diseases such as chickengunya, encephalitis and salmon pancreas diseases. Each alphavirus genome is a single-strand positive sense~11.5Kb RNA molecule with terminal un-translated regions and, in between them, a single open reading frame (ORF) that is translated into a poly-protein. The poly-protein is cleaved after translation into four non-structural (virus replication and pathogenesis) and five structural proteins (virion synthesis) by proteinases that are a part of the polyprotein (Strauss and Strauss, 1994).
Repeat sequences have been established as highly divergent but ubiquitously present genome components across both prokaryotic and eukaryotic genomes. However, the viral genomes are now being actively pursued for their presence and significance. Tandem repeats of short motifs of DNA are called simple sequence repeats (SSR). On the basis of presence of interruptions in SSR; they are classified as interrupted, pure, compound, interrupted compound, complex and interrupted complex (Chambers and MacAvoy, 2000). These SSRs are termed compound SSR (cSSR) if two SSR are separated by a defined distance. dMAX is the maximum difference between the two SSRs of a cSSR as discussed later. The repeat number, length, and motif size influence microsatellite mutability. For instance, more the number of repeats; higher is the mutability (Pearson et al., 2005). Moreover, variations in copy number due to strand slippage and unequal recombination highlight the instability of the microsatellites (Tóth et al., 2000); which in turn makes it a predominant source of genetic diversity and a crucial player in viral genome evolution (Deback et al., 2009;Kashi and King, 2006). Their role in gene regulation, transcription and protein function has also been elucidated (Kashi and King, 2006;Usdin et al., 2008).
The presence and possible functional significance of SSRs in viruses have been recognized only recently (Alam et al., 2013a(Alam et al., , 2013b(Alam et al., , 2014a(Alam et al., , 2014bChen et al., 2009Chen et al., , 2011Chen et al., , 2012Xiangyan et al., 2011). Concerted efforts are required to identify and confirm the presence, distribution and variations of SSRs in human infecting viruses. Here, we systematically analyzed the occurrence, size, and density of different microsatellites in different species of alphaviruses, which can serve as a model for understanding functional aspects and evolutionary relationships.

Genome sequences
Complete genome sequence of 30 alphaviruses species were assessed form NCBI (http://www.ncbi.nlm. nih.gov/) and analyzed for simple and compound microsatellites. Genome size of these species ranged from 11245 nt (Acc No-HM147990) to 12052 nt (Acc No-HM147991). The accession numbers, genome size and GC content of studied alphaviruses genomes have been summarized in Table 1.

Microsatellites identification
The microsatellite search was performed using the IMEx software (Mudunuri and Nagarajaram, 2007). Earlier studies on eukaryotes and E. coli genomes have focused on microsatellites with lengths of 12 bp or more (Tóth et al., 2000) but due to smaller size of alphaviruses genome, simple and compound microsatellite search using these parameters did not yield any results. Therefore, the search for simple and compound microsatellites was accomplished using the 'Advance-Mode' of IMEx using the parameters as used for HIV (Chen et al., 2012). The parameters were set as follows: Type of Repeat: perfect; Repeat Size: all; Minimum Repeat Number: 6, 3, 3, 3, 3, 3; Maximum distance allowed between any two SSRs (dMAX) is 10. Other parameters were used as default. Compound microsatellites were not standardised in order to determine real composition.

Statistical analysis
The simple mathematical calculations were performed using Microsoft Office Excel 2007. However, the Pearson correlation coefficient (r) was calculated using GraphPad Prism Software, version 5 (La Jolla, CA, USA) to evaluate the influence of genome size and GC content, if any, on SSRs and cSSRs. A P-value b0.05 was considered to be significant.

Number, relative abundance and relative density
Genome-wide scan of thirty alphavirus genomes using IMEx revealed a total of 1160 SSRs distributed across all the species with varying incident frequencies ranging from 27 in A8 (Acc No-HM147986) to 58 in A2 (Acc No-U73745) ( Table 2, Supplementary Table S1 and Fig. 1a). Further, the relative abundance values lied between a minimum of 2.41 for A30 to a maximum of 5.05 bp/kb for A2. (Table 2, Fig. 1b). Comparatively, relative density of SSRs varied from 18.58 bp/kb in A14 to 29.62 bp/kb for A18 (Table 2, Fig. 1c).
IMEx scan for alphavirus genomes revealed a total of 39 cSSRs (Table 2 and Supplementary Table S2). In contrast to the ubiquitous presence of SSRs, seven alphavirus genomes lacked any cSSR. Further, relative abundance varies from 0.00 bp/kb in 7 alphavirus sequence (A1, A5, A11, A14, A15, A19, A21) to 0.35 bp/kb in A2(Acc No-U73745) ( Table 2, Fig. 1b) whereas relative density varies from 0.00 bp/kb in 7 alphavirus sequence to 5.83 bp/kb in A2 (Acc No-U73745) ( Table 2, Fig. 1c). The correlation of cSSR incidence in a genome to SSR abundance led to few interesting observations. cSSR% is the percentage of individual microsatellites   being part of a compound microsatellite and it has been summarized in Fig. 2. It was 'nil' in C8 (Acc No-AJ620300) which was harbouring 46 SSRs as compared to 10.71% in A30 (Acc No-HM147993) with just 28 SSRs.
Varying dMAX and incidence of cSSR dMAX is the maximum distance between any two adjacent microsatellites and if the distance separating two microsatellites is less than or equivalent to dMAX, these microsatellites are classified as cSSR (Kofler et al., 2008). To determine the impact of dMAX, compound microsatellites from A5, A10, A15, A20, A25 and A30 were chosen at random to determine the variability of compound microsatellite with increasing dMAX by analyzing cSSRs%. It is noteworthy that the dMAX value can only be set between 0 and 50 for IMEx (Mudunuri and Nagarajaram, 2007). Our analysis revealed an overall increase in cSSRs% with increasing dMAX in all the six alphavirus genomes analysed (Fig. 3).

Motifs types in analysed genomes
Mononucleotide repeats were observed in all the analyzed alphavirus genomes. Poly (A/T) repeats were significantly more prevalent (82.62%) than poly (G/C) repeats in each of the studied genomes (Supplementary  Table S1) which may be attributed to the high (A/T) content of the genomes (Karaoglu et al., 2005). However, the (A/T) content being only slightly higher than G/C content in each of the analyzed sequences suggests a weak influence on the occurrence of poly (G/C) repeats. Similar to eukaryotic and prokaryotic genomes with reportedly more abundant poly (A/T) tracts (Gur-Arie et al., 2000;Karaoglu et al., 2005;Tóth et al., 2000) in the analyzed sequences poly A or poly T mononucleotide repeats prevailed over poly G or poly C (Fig. 5a).
A maximum of 64 mononucleotide A repeats was observed in genome of Una virus (A27). Further, the genomes had di-nucleotide repeats of six types: AG/GA, GT/TG, AC/CA, CT/TC, AT/TA, and CG/GC with variations in occurrence across genomes. AG/GA repeats were the most prevalent whereas, CT/TC was least represented (Fig. 5b). The average occurrence of GT/TG repeats was~4 times more than CG/GC. The distribution of di-nucleotide repeats in the studied genomes has been summarized in Supplementary Table S1.
Tri-nucleotide repeats are the third most abundant SSRs within the alphavirus genomes. Of the 64 triplet repeat types, the density of AAG/GAA coding for lysine/glutamic acid was the most abundant followed by AGA coding for arginine respectively. Though differences exist in the abundance of trinucleotide repeats amongst alphavirus species but AAG/GAA showed highest prevalence in most species (Fig. 5c; Supplementary  Table S1). Ten different types of tetra-nucleotide repeat motifs were distributed in 14 alphaviruses species while pentanucleotide repeat motif ATTTT was present in two genomes A7 and A27 only. There were no hexanucleotide repeats in the analysed genomes.

Comparative distribution across coding and non-coding regions
The distribution of SSRs and cSSR in coding/non-coding region revealed a clear bias of incidence in the coding regions. The coding regions accounted for 90% of the SSRs (Non-structural proteins ORF: 58%; Structural Proteins ORF: 32%) whereas residual 10% contribution was from the non-coding region of the genomes (Fig. 6a). The cSSR distribution also exhibited similar results with only 15% incidence being attributed to  the non-coding regions. Their distribution across structural and non structural ORFs has been summarized in Fig. 6b. Also, the actual distribution of SSRs and cSSRs across coding and non-coding regions has been summarized in Table 3 for representative genomes.
Further, we assessed the relative composition of repeat motifs. The structural proteins ORF had predominantly trinucleotide repeats (~50%) whereas tetranucleotide motif constituted almost eight out of every ten repeats present in the non structural proteins ORF. Contrastingly, the non coding region had mononucleotide motifs as the most prevalent one (Fig. 7).

Discussion
In this study, we screened 30 alphavirus genomes for the presence, abundance, and composition of SSR tracts. A total of 1160 SSRs were revealed ranging from mono-to penta-nucleotide motifs. A higher prevalence of di-nucleotide repeats over tri-nucleotide repeats may be attributed to instability of the former because of higher slippage rate (Katti et al., 2001) and suggests a possible role of host in the evolution of di-nucleotide repeats within alphavirus (Alam et al., 2013a(Alam et al., , 2013b(Alam et al., , 2014a(Alam et al., , 2014b. The repetitive sequences are hypothesized to provide for a molecular device for faster adaptation to environmental stresses (Kashi et al., 1997;Li et al., 2004) and hence, diverse repeats observed in alphavirus genomes might accelerate their evolution.
The compound microsatellites are reportedly involved in regulation of gene expression and at functional level of proteins in several species (Chen et al., 2011;Kashi and King, 2006). Though their significance in alphavirus is not clear, it suggests presence of a possibly complex regulation at the functional level. Further, varying dMAX (10 to 50) in 6 analyzed species lead to a higher cSSR% with increasing dMAX though, not in a linear way. This further adds to the significance of differential cSSRs distribution across genomes. In alphaviruses compound microsatellites were composed of a maximum of two SSRs whereas, in prokaryotes it is four and more than eight in eukaryotic species. Interestingly, cSSRs% varied between 0-11.42% in alphavirus genome; 0 to 15.5% in Human Papilloma Viruses; 0-15.15% in potyvirus genome; 0-11.43% in tobamovirus genome; 0-12.5% in carlavirus genome; 0-11.76% in potexvirus genome; (Alam et al., 2013a(Alam et al., , 2013b(Alam et al., , 2014a(Alam et al., , 2014bSingh et al., 2014), 0-24.24% in HIV-1 genomes (Chen et al., 2012); 4-25% in eight eukaryotic genomes (Kofler et al., 2008) and 1.75-2.85% in E. coli genomes (Kruglyak et al., 2000). Distribution of microsatellite in the viral genome is organism specific rather than host specific. This is supported by the fact that the taxonomy of alphavirus shows no comparable congruence with host taxonomy, and species from the same lineage may have quite unrelated hosts (Gibbs et al., 2008). Accordingly, we observed that cSSR from viruses infecting common host does not possess similar number and type of cSSR motifs in their genome (data not shown). Surprisingly, seven of the alphavirus species did not possess even a single compound microsatellite this may be due to less number of strains present in these species wherein non availability of cSSR which might be restricting their variation and evolution.
It has been widely accepted that genetic RNA recombination is crucial for the emergence of new viral strains or species (Tan et al., 2005) and genomes are known to have recombination hot spots for the same (Jeffreys et al., 1998). The repeat sequences are possibly facilitating these recombination events (Nagy and Burjarski, 1996). Moreover, the unique composition of repeat motifs across structural/non structural proteins ORF and non coding regions as illustrated in Fig. 7 suggests that different parts of the genome might be evolving independent of one another in a dynalic manner.
A complete understanding of the functional and evolutionary role of tandem repeat sequences in viruses is still elusive. However, their ubiquitous presence though with varying frequency and complexity across species as well as coding and non-coding regions is suggestive of them being involved in the already established roles of gene regulation recombination hot spots. But owing to the smaller genome size of viruses as compared to prokaryotes and eukaryotes these tandem repeats are probably more influential in guiding the evolution of viruses. The diversity of microsatellites in alphavirus genomes may be useful for better understanding of viral genetic diversity, evolutionary biology, and strain/species demarcation

Conclusion
Genome-wide scan of thirty alphavirus genomes using IMEx revealed a total of 1160 SSRs and 39 cSSRs. The SSRs showed ubiquitous presence across the species albeit with varying incidence rates while seven alphavirus genomes lacked any cSSR. There was a bias towards occurrence of SSRs and cSSRs in the coding region as compared to the non-coding region which accounted for 10% and 15% contribution respectively. We postulate that such repeats in viral genomes could be involved in recombination, leading to sequence diversity that drives host adaptation. More detailed study of compound microsatellites in alphavirus genomes may be useful for understanding complex biological features such as changes in virulence and their emergence as new epidemics.


s2fieldsofstudy: Biology Medicine

text:
Resveratrol and Calcium Signaling: Molecular Mechanisms and Clinical Relevance

Resveratrol is a naturally occurring compound contributing to cellular defense mechanisms in plants. Its use as a nutritional component and/or supplement in a number of diseases, disorders, and syndromes such as chronic diseases of the central nervous system, cancer, inflammatory diseases, diabetes, and cardiovascular diseases has prompted great interest in the underlying molecular mechanisms of action. The present review focuses on resveratrol, specifically its isomer trans-resveratrol, and its effects on intracellular calcium signaling mechanisms. As resveratrol’s mechanisms of action are likely pleiotropic, its effects and interactions with key signaling proteins controlling cellular calcium homeostasis are reviewed and discussed. The clinical relevance of resveratrol’s actions on excitable cells, transformed or cancer cells, immune cells and retinal pigment epithelial cells are contrasted with a review of the molecular mechanisms affecting calcium signaling proteins on the plasma membrane, cytoplasm, endoplasmic reticulum, and mitochondria. The present review emphasizes the correlation between molecular mechanisms of action that have recently been identified for resveratrol and their clinical implications.

Effects and Mechanisms of Action of Resveratrol as the Basis for Its Therapeutic Potential in Various Diseases
Resveratrol is a stilbenoid commonly found in the roots of Japanese Knotweed and the skin of red grapes. Plants produce phytoalexin as a response to harmful stimuli such as UV radiation, infection, or other pathogenic threats [1,2]. Increased curiosity about resveratrol and its potential health benefits began with a phenomenon termed the "French Paradox" [3] that became a fascination of health professionals and researchers. The "French Paradox" describes the extremely low incidence rate of cardiovascular disease in France compared with other European countries despite a high fat diet with little to no exercise [4]. Epidemiological studies indicated that polyphenols present in red wine could be responsible for the cardioprotective effects experienced by the French [4][5][6]. Resveratrol then initially became a subject of study for potential cardiovascular benefits but additional benefits are becoming apparent in recent studies [7]. Some of the proposed mechanisms include antioxidant, anticarcinogenic, anti-inflammatory, anti-aging [8], and anti-nociceptive functions indicating the potential of resveratrol as therapeutic agent for preventing and ameliorating a wide range of pathologies [9][10][11][12][13][14], including neurodegenerative diseases such as Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS) [8,15,16]. Cardiovascular diseases that may benefit from resveratrol treatment include hypertension, ventricular arrhythmia, myocardial infarction (MI) induced ventricular tachycardia, ventricular fibrillation, arteriosclerosis, arteriolosclerosis, and restenosis particularly after angioplasty [7,13,[17][18][19][20]. In addition, cerebral ischemia, diabetes mellitus, cancer, autoimmune diseases, and other inflammatory-related issues have been discussed as potentially benefitting from resveratrol intervention [13,15,21,22]. Resveratrol also provides protection from UV radiation explaining its potential use in the prevention of age-related macular degeneration (AMD) [23,24]. Potential mechanisms underlying resveratrol's actions are its ability to control protein activity via interaction with transmembrane and intracellular enzymes [25].

Key Signaling Proteins Controlling Cellular Calcium Homeostasis
Resveratrol is a potent regulator of genomic and non-genomic processes including regulation of membrane potential, DNA transcription, enzyme activity, secretion, apoptosis, mitochondrial activity, and intracellular ion homeostasis, including the modulation of the intracellular calcium concentration [3,7,25].

Modulation of Cellular Calcium Signaling Mechanisms by Resveratrol in Excitable Cells
Resveratrol potently modulates the intracellular calcium concentration in excitable cells through a variety of mechanisms that control calcium influx, store filling, release from intracellular stores, and downstream activation of calcium sensitive molecules. In myocytes, resveratrol increases the refractory period and decreases the threshold for membrane depolarization [17]. This results in restored calcium homeostasis evident by a reduction of delayed after depolarization (DAD, occurs with cytoplasmic calcium influx) and triggered activity (TA, ensues via irregular calcium release) in myocytes [37,40,41]. Resveratrol, therefore, can ameliorate cardiac arrhythmia and prevent premature atrial contraction (PAC) [17].
Similar membrane hyperpolarization in response to resveratrol also occurs in smooth muscle cells (SMC) leading to vasodilation, making resveratrol a candidate for the treatment of a wide variety of disorders including hypertension, ventricular arrhythmia, myocardial infarction (MI) induced ventricular tachycardia, and ventricular fibrillation [18,41,42]. Furthermore, it has been suggested that the ability of resveratrol to induce endothelium-dependent hyperpolarization of SMCs may compensate for pathologic absence or dysfunction of endothelium nitric oxide synthase (eNOS) and cyclooxygenase-1 (COX-1) [43]. Endothelial cells line the walls of the lymphatic and cardiovascular vessels and are critical modulators of contractility and vessel dimensions. Vasorelaxation is the most extensively studied effect of resveratrol on vascular endothelia. Most studies focus on two interlinked mechanisms of action, the direct effect on the intracellular calcium concentration and the downstream indirect effects on cellular potassium regulation [44]. A resveratrol dependent increase in nitric oxide biosynthesis was shown to result from calcium mobilization rather than direct agonism of eNOS by the drug [45]. The major plasma membrane targets of resveratrol include the L-type VGCC, which is directly inhibited by resveratrol, and the large conductance calcium-activated potassium (BK) channel, that resveratrol indirectly effects [19,37]. Under physiological conditions, calcium influx via L-type VGCCs stimulates BK channels that facilitate cytosolic potassium ion efflux, which results in membrane hyperpolarization. Resveratrol directly attenuates calcium influx, indirectly decreasing potassium efflux, and leading to a state of vasorelaxation in an endothelium-dependent manner [20,46,47]. Due to these effects, resveratrol may be beneficial in preventing or mitigating the physiological effects of hypertension, arteriosclerosis, arteriolosclerosis, and restenosis (particularly after angioplasty). Interestingly, diabetes mellitus is an important, clinically relevant risk factor for all four conditions [48].
In neuronal cells, resveratrol reduces the action potential threshold and both delays and prolongs calcium entry ( Figure 1) [16]. This effect causes rapid sodium-induced action potentials that increase neuronal conduction. This mechanism of action has the potential to regulate and increase neural impulses which may provide a basis for the prevention of conductance disorders or to retard neurodegenerative processes. Resveratrol has also been shown to protect against cerebral ischemic damage via indirect BK channel activation, thereby preventing neuronal hyperexcitability and the ensuing cell death [15,16]. While resveratrol can prevent neuronal death and provide protection against oxygen-glucose deprivation [49,50], the potential of resveratrol for therapeutic intervention in axonopathy or other conduction block disorders remains to be elucidated [8,51]. The pleiotropic effects of resveratrol with its respective molecular mechanisms, and the pharmacological modulation of those mechanisms, affect separate components of membrane depolarization and repolarization, resulting in a distinct modulation of spatio-temporal properties of electrical signaling by resveratrol in excitable cells.

Modulation of Cellular Calcium Signaling Mechanisms by Resveratrol in Cancer and Immune Cells
Resveratrol shows great promise as a therapeutic agent for a variety of cancers [52]. Given its effects on calcium homeostasis, resveratrol may be useful as a latent remedy for the primary stages of some malignant cancers [53]. Calcium signaling pathways are necessary for the motility and invasive characteristics of malignant tumors, as it controls cell restructuring and dedifferentiation [54]. Another potential mechanism of action for resveratrol targets the excessive energy demand of tumor cells, lowers calcium thresholds, and prolongs intracellular calcium surges, all of which ultimately lead to cell death as a result of mitochondrial dysfunction [55]. Previously, resveratrol has been shown to suppress CD4 + CD25 + regulatory T cells (T reg cells) in a preclinical cancer model [56]. T reg are critical mediators of self-tolerance and inhibit the proliferation of CD4 + CD25 − conventional T-cells (T con ) through a recently identified mechanism involving the suppression of intracellular calcium release [57]. Further studies are needed to identify how resveratrol modulates intracellular calcium homeostasis during the early phases of tumor growth.
Resveratrol is a powerful modulator of the immune system response via both pro-and anti-inflammatory pathways [58,59]. Resveratrol has the ability to activate immune cells at low doses while exerting an inhibitory effect at high doses [21,52]. The mechanism of action of resveratrol in immune cells likely involves a combination of proteasome inhibition [60] and modification of the cytosolic calcium concentration through blocking calcium entry and increasing calcium storage [31]. It has been suggested that resveratrol can reversibly modify disturbed system responses such as mast cell granulation [56]. Resveratrol decreases mast cell hypersensitivity by reducing calcium influx thereby reducing granulation (Figure 2) [12]. Resveratrol has also been shown to decrease inflammatory responses in autoimmune disorders, such as multiple sclerosis, through potentiation of active T-cell apoptosis [56,61]. It appears that resveratrol exerts effect on intracellular calcium by modulating sequestration and release of intracellular stores but further studies are needed to elucidate the exact mechanism of action in lymphocytes and other cell types of the immune system.

Modulation of Cellular Calcium Signaling Mechanisms by Resveratrol in Human Retinal Pigment Epithelial Cells
Trans-resveratrol is susceptible to photoisomerization to cis-resveratrol when exposed to ultraviolet irradiation [62]. When present in retinal pigment epithelial (RPE) cells, or any cell subject to UV ray exposure, resveratrol absorbs damaging UV light, thus reducing exposure of other structures to damaging wavelengths and ultimately enhancing the protective function of RPE cells (Figure 3) [23].
One key mechanism in RPE cells is phagocytosis, an important renewal mechanism that is susceptible to UV damage [23]. In experimental studies, resveratrol preserved RPE cell phagocytosis when applied prior to insult but had no protective effect when used as a post-insult treatment [23]. It has been proposed that this protective effect is mediated with BK channels [23]. Chronic resveratrol supplementation, rather than acute administration, would therefore be required for pharmaceutical intervention studies.
Another protection mechanism includes the anti-oxidative effect of resveratrol [63,64]. Pretreatment of RPE cells with resveratrol ameliorated the oxidative stress related damage caused by hydrogen peroxide by preventing oxidation related RPE phagocytic impairment, which may be linked to BK channel activity [23]. Furthermore, the clinical course of age-related macular degeneration (AMD), and potentially other vascular diseases of the retina, may be slowed by resveratrol when given as a nutritional additive [24,65].

Intracellular Calcium Channels
Two main types of intracellular calcium channels (ICC), inositol 1,4,5-trisphosphate receptors (IP 3 Rs) and ryanodine receptors (RyRs), are vital for the release of calcium from the endoplasmic and sarcoplasmic reticulum (ER & SR). Release of intracellular calcium through IP3Rs increases the intracellular calcium concentration which in turn activates RyRs to amplify the calcium signal -a process termed calcium induced calcium release (CICR). ICCs are involved in fundamental cellular processes such as cell proliferation, signaling, excitability, gene expression, and apoptosis [66]. Thus, ICCs are considered a potential target for neuroprotective and cytoprotective strategies for a number of pathologies [28,66,67] although, to date, no studies have been targeted at identifying the specific effects of resveratrol on ICCs (Table 1). Understanding the potential effects of resveratrol on ICCs could identify novel treatment strategies for neurodegenerative diseases such as Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and other neurodegenerative diseases.

Store-Operated Calcium Channel
Calcium-release activated channels (CRACs) are store-operated calcium channels located at the plasma membrane that are activated by low intracellular calcium store concentrations, resulting in a sustained increase of the cytosolic calcium concentration. Immune cells rely on this mechanism and CRACs are potential drug targets for anti-inflammatory diseases [68]. Resveratrol has not been shown to exert a direct effect on CRAC channels [69] (Table 1) although it has been suggested that resveratrol acts indirectly on store-operated calcium entry (SOCE). However, the potency of resveratrol stimulation on CRAC is far lower than that of other known neuroprotective and neuromodulatory compounds including nonsteroidal estrogens [33].

Voltage-Gated Calcium Channels
Plasma membrane voltage-gated calcium channels (VGCCs) are classified into high or low voltage-activated channels. VGCCs are activated by a depolarization of the membrane potential and allow extracellular calcium flux into the cytosol, thereby translating an electric signal into a secondary chemical signal. The class of high VGCCs include Ca V 1 (L-type) and Ca V 2 (P/Q-, N-, and R-type) channels. The low VGCCs includes Ca V 3 (T-type) channels. Ca V 1, Ca V 2, and Ca V 3 have slow, intermediate, and fast inactivation responses, respectively. In excitatory cells, resveratrol decreases cellular excitability by attenuating calcium influx through VGCCs [70]. Diseases related to VGCC dysregulation include neurodegenerative disorders such as AD, PD, cerebral ischemia, cardiovascular diseases, chronic and acute inflammatory diseases, many cancers, and even mood disorders [28,45,71]. The primary mechanism of action of how VGCC dysregulation leads to these disorders is hyperexcitability resulting in a chronic increase in the cytosolic calcium concentration. Therefore, VGCCs, especially the slow inactivating Cav1 channels, have become a focus of neuroprotective strategies [28,72]. Resveratrol has been shown to dose-dependently inhibit both L-type and T-type calcium channels and to increase the time period for reactivation [29]. More specifically, resveratrol inhibits L-type currents during phase 2 of an action potential, leading to vasorelaxation [26]. Also, it has been suggested that resveratrol-induced vasorelaxation is partially due to a stimulation of NOS and the ensuing chronic increase of the nitric oxide concentration [30,41].

Calcium-Activated Potassium Channels
Calcium-activated potassium (K Ca ) channels include three major types of channels-large (BK), intermediate (IK), and small conductance (SK) channels. K Ca channels help regulate the action potential activity of excitable cells via the modulation of membrane hyperpolarization. These channels play a critical role in restricting calcium influx [73]. Resveratrol does not appear to act directly on K Ca channels but rather exerts its effects through other types of calcium channels. This higher level regulation of calcium homeostasis in turn indirectly regulates potassium flux [19,47,74]. While there is no general consensus, most studies suggest that the modulation of K Ca channels by resveratrol's inhibitory effects illustrates an indirect partial reduction of potassium efflux due to moderation of calcium influx [9,75].

SERCA and PMCA
Calcium ATPases are calcium transport proteins that return the cellular calcium concentrations back to the transmembrane electrochemical gradient state prior to renewed depolarization. The two key proteins that mediate this mechanism are the plasma membrane calcium ATPase (PMCA), which pumps calcium out of the cell when present in high concentrations, and the SR calcium ATPase (SERCA), which replenishes the SR calcium store from the cytoplasm. To our knowledge there is no study investigating potential effects of resveratrol on PMCA. However, it appears that resveratrol upregulates SERCA via SIRT1 activation, further replenishing calcium stores and reducing intracellular calcium influx [62]. Resveratrol may prove useful to attenuate diseases related to SIRT1 downregulation [26]. Additionally, resveratrol facilitates apoptosis in cancerous cells by induction of a calpain-dependent PMCA degradation mechanism. This strategy may be further elucidated through experiments targeted at identifying resveratrol's mechanism of action in tumor cells [27].

Mitochondrial Calcium Signaling
Mitochondria are temporary and rapidly releasing calcium stores, capable of generating calcium spikes or waves to trigger second messenger systems. Mitochondria are critical initiators of apoptotic pathways via calcium signaling. Resveratrol has been shown to induce mitochondrial apoptosis by alteration of the mitochondrial membrane potential and eliciting a mitochondrial permeability transition (MPT) pore by lowering the calcium threshold necessary for MPT opening [27,55,76]. This mechanism is currently intensively investigated as a potential for anti-cancer drug therapy [77,78]. For instance, it has been proposed that resveratrol can induce this apoptosis in cancerous cells due to differences in mitochondrial function and that it can be injected directly into a tumor mass to initiate tumor regression prior to resection [79].

Conclusions
Resveratrol is a potent modulator of many cellular calcium signaling pathways ( Table 1). The number of different targets for modulation by resveratrol (Table 1) confound the interpretation of in vivo analyses and result in cell type-and organism-specific responses. While L-type and T-type VGCCs have been identified as direct targets of resveratrol modulation, further studies are required to identify the effects on intracellular calcium channels and other neurotransmitter receptor systems. Yet there is significant evidence to suggest that the beneficial effects of resveratrol cannot be attributed only to its immunomodulatory function but also to a resveratrol dependent lowering of membrane hyperexcitability and cellular calcium toxicity. Thus resveratrol bears great promise for a number of multifactorial pathologies including neurodegeneration, autoimmune and cardiovascular disease, and cancer. Further, resveratrol as should be considered both an immunomodulator and a modulator of intracellular calcium signaling.


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Isolation and Characterization of Bacteriophages Infecting Nocardioforms in Wastewater Treatment Plant

Activated sludge plants (ASP) are associated with the stable foaming problem worldwide. Apart from the physical and chemical treatment methods, biological treatment method has been least explored and may prove to be a novel and ecofriendly approach to tackle the problem of stable foam formation. In ASP Nocardia species are commonly found and are one of the major causes for forming sticky and stable foam. This study describes the isolation and characterization of three Nocardia bacteriophages NOC1, NOC2, and NOC3 for the control of Nocardia species. The bacteriophages isolated in this study have shown promising results in controlling foam producing bacterial growth under laboratory conditions, suggesting that it may prove useful in the field as an alternative biocontrol agent to reduce the foaming problem. To the best of our knowledge to date no work has been published from India related to biological approach for the control of foaming.

Introduction
Activated sludge process (ASP) is the most commonly used process to reduce the toxicity of waste water by processing it microbiologically. In activated sludge system, treatment is given to every type of wastewater using microbial communities for the degradation of organic matter present in water [1]. Microbes use the organic matters as their energy source and degrade them into a less toxic form, but most of the system suffers from the excessive growth of unwanted mycolic acid containing filamentous bacteria or mycolata [2], which lead to the formation of brown and sticky foam [3]. Mycolata like Sphaerotilus spp., Leptothrix spp., Microthrix parvicella, Corynebacterium spp., Dietzia spp., Nostocoida limicola, Gordonia spp., Skermania spp., Mycobacterium spp., Nocardia spp., Rhodococcus spp., Tsukamurella spp., Type 021N, and Type 0041 play a role in foaming. However, among all mycolata the Eikelboom species are mainly responsible for foaming [4][5][6][7].
The foam covers a large area in the aeration tank which create interference in the treatment process and ultimately leads to the major environmental, operational, cosmetic, and health problems [3,8,9]. The outer surface of foam forming bacteria has hydrophobic properties similar to that of fats, oils, and grease which enables the bacterial bulk to float on the surface of liquid in aeration tank. The wastewater containing slowly degradable organic material like lipids, proteins, and fats may favor the growth of filamentous microorganisms like M. parvicella and G. amarae thereby leading to increased foaming [3,6,10]. The presence of unfavorable conditions in the activated sludge system, such as toxic conditions (pH below 6.5 or above 9.0), insufficient dissolved oxygen (DO), nutrient deficiencies, or seasonal temperatures may contribute to the foaming. It has been reported that the formation of scum and stable foam in aeration tank (AT), secondary clarifier (SC), and activated sludge (AS) is a global problem [9,11,12].
Various studies suggest that a range of physical, chemical, and biological [11,[13][14][15] methods is available for the control of foaming in activated sludge process [12], but they need a higher cost of maintenance. Current 2 Biotechnology Research International approaches for controlling foam includes decreasing mean cell retention time [16], use of classifying selectors, and nonspecific measures such as water sprays, steam application [17], polymer addition [18], and chlorination [19]. Dosing with cationic polymer [18] and controlling dissolved oxygen levels in the preoxidation reactor have been reported as useful methods for foam reduction [20].
There are only limited reports related to biological foam control methods especially the application of bacteriophages in AS systems [11,14,15,[21][22][23]. Bacteriophage therapy for the treatment of infectious diseases has shown promise [24]; on the similar lines lytic bacteriophages has the potential to be exploited as an biocontrol for filamentous bacteria which may lead to foam reduction in the AS treatment plant [11,25]. Efforts have been made by Thomas et al. [23] and Petrovski et al. [13][14][15] for the isolation of bacteriophages against filamentous bacteria responsible for the stable foam formation in ASP. The successful application of phage for effective bacterial control relies on the population density which must be sufficient to support phage replication [26].
Bacteriophage based approach may have the potential as an environmentally safe option for tackling worldwide ASP operational problem of foaming. Nevertheless, phagebased foam control approach has certain limitations: (1) high concentration of phages must be applied for the successful application; (2) due to the polyvalent phages, broader host range could lead to the degradation of useful bacteria; (3) specific phage must be identified by the operator to counter specifically the foam forming bacteria without affecting other bacteria; (4) the microbial analysis of the system is a prerequisite to phage application as the bacterial population may vary between wastewater treatment plants (WWTP) [11].
The present research has been done with the objective of developing a biocontrol approach to manage foaming within AS systems in Nagpur city of India. In this study, we report the characterization of three Nocardia phages NOC1, NOC2, and NOC3 isolated from effluent treatment plant (ETP) and dairy ETP Nagpur, India. To the best of our knowledge to date, no work has been published from India related to biological approach for the control of foaming in WWTP.

Materials and Method
2.1. Sampling Sites. The sampling was done from effluent treatment plant (ETP) and dairy ETP around Nagpur, India, considering the foaming status of the treatment plants. As filamentous bacteria, for example, Nocardia, grows well on the slowly degradable organic matters like lipids, oil, and grease, fatty matters [3,5,10] and is found almost in every WWTP. So, in expectation of getting phages against Nocardia spp. the samples were collected from the above mentioned sites. Samples were collected from different points, namely, AT, SC, and AS, in sterile plastic bottles of 200 mL and processed within two days of sampling.

Bacterial Strains and Growth
Conditions. The bacterial strains used in this study are American type culture collection which is listed in Table 1. All the ATCC Nocardia bacterial strains were grown on peptone-yeast-calcium (PYCa) agar [13], R2A agar [27], and Tryptone Yeast Glucose Agar (TYGA) [28]. Three growth media were used to comparatively see the best growth of Nocardia species. The PYCa agar was found to be the best media for supporting the growth of Nocardia species on the basis of plate count assay. Henceforth, PYCa medium was preferred for maintenance of Nocardia cultures at 4 ∘ C and was routinely maintained by subculturing every two weeks. After incubation, all the bacteria were enriched in the PYCa broth and stored at 4 ∘ C as per the method described by Petrovski et al. [13].

Isolation and Purification of Nocardiophage.
Nocardiophages were isolated from wastewater sample collected around Nagpur, India. In brief, 20 mL wastewater sample from each different sampling sites was subjected to centrifugation at 3000 ×g for 20 mins. After centrifugation the supernatant was filtered through cellulose acetate membrane filters (0.22 m) to remove bacterial cells. For enrichment of bacteriophages, 1 mL of each filtered sample was inoculated with the 50 mL of PYCa broth of mixed bacterial culture and incubated at 30 ∘ C [13]. After enrichment the bacterial cells were first centrifuged at 5,000 ×g for 10 min, the supernatant was filtrated through 0.22 m cellulose acetate filter paper. For plaque assay method, 1 mL filtered sample was mixed with 1 mL of host culture of each species and poured onto autoclaved petriplates after mixing the melted PYCa agar [29,30]. Plates were then incubated at 30 ∘ C for 2 days as per the method of Petrovski et al. [13].

Nocardiophage DNA Isolation.
DNA of all the phages NOC1, NOC2, and NOC3 were isolated using SDS-proteinase K method as described by Petrovski et al. [13].

Phage Host Range
Determination. All three Nocardia species were subjected to host range determination by challenging against nocardiophages as per the method described by Petrovski et al. [13]. Phage recovery and purification was achieved with their respective host, namely, N. rhodochrous, N. amarae, and N. pinensis, as described by Petrovski et al. [13]. The phage characterization was achieved by nucleotide sequencing and analysis.

Phage Nucleotide Sequence Analysis.
Nocardiophage DNA was genome sequenced using the Roche GS FLX genome sequencer and titanium chemistry by Genoseq AT: aeration tank, AS: activated sludge, SC: secondary clarifier, − no visual growth; + appearance of visual growth; + + + excellent visual growth. a NOC1. b NOC2. c NOC3.

Isolation and Purification of Nocardiophages.
Petrovski's method was followed for the isolation and purification of bacteriophages. Single plaques were observed after 2 days incubation at 30 ∘ C. The quantitative results of plaque assay are summarized in Table 2 indicating phage growth. A representative culture plate of plaque assay is presented, wherein the plaques were picked and subjected to further isolation and purification of bacteriophages ( Figure 1). On the basis of growth of bacteriophages on N. rhodochrous, N. amarae, and N. pinensis (reclassified as Skermania piniformis) phages were named as NOC1, NOC2, and NOC3, respectively. Further, plaques were purified by four rounds of dilution and reisolation to ensure that each plaque resulted from a single type of bacteriophage. The plates which were showing the excellent visual growth were taken for the purification of bacteriophages.

Phage Host Range Determination.
Phage host range was determined by challenging each of the three Nocardia species against individual phage on PYCa media. The determination of host range of bacteriophages against nocardioforms is summarized in Table 3. Briefly, NOC2 (isolated from ETP, Nagpur) was able to grow on the two species of Nocardia, namely, N. amarae and N. pinensis; notably few plaques of NOC2 were seen against the N. pinensis. NOC1 and NOC3 were able to propagate only on their host, that is, N. rhodochrous and N. pinensis (Skermania piniformis), respectively. NOC2 had relatively broader host range when compared to NOC1 and NOC3. Although NOC3 was able to show slight growth on N. rhodochrous but clear lytic plaques  [31]. The isolated and characterized phages in this study were showing similarity to those phages reported earlier as active against the stable foam forming bacteria under laboratory conditions. This observation implies that the isolated bacteriophages, namely, NOC1, NOC2, and NOC3, may be helpful in reducing the stable foaming problem in WWTP.

Conclusion
In the present study, isolates (NOC1, NOC2, and NOC3) bacteriophages were identified and characterized. All three phages had the ability to inhibit the growth of filamentous bacteria. The host range determination showed that NOC1and NOC3 were inhibitory against their respective host while NOC2 phage has slightly broader host range. Further, characterization and analysis showed that these phages showed similarity and identity with the previously reported phages against filamentous bacteria. Thus, the result obtained suggests that the isolated bacteriophages are potent inhibitor of foam forming bacteria. Isolated bacteriophages could be used potentially as a biocontrol agent for foaming problems in activated sludge treatment plants. Potential application of bacteriophage need much attention for the control of foaming as it is the ecofriendly and cost effective approach. Further work is in progress for isolation and characterization of other phages against other foam forming filamentous bacteria.