Siphoviridae (53%), Podoviridae (22%), Myoviridae (20%), Inoviridae (1%), Leviviridae (0.04%), Tectiviridae (0.01%), and Microviridae (0.01%). A diverse range of vertebrate viruses were detected, of which posavirus 3, mink bocavirus, gyroviruses, and avian‐associated viruses were most abundant. Additionally, sequences from nonvertebrate viruses with water and soil‐associated amebal and algal hosts were also highly prevalent. The results of this study show that viruses shed in the fecal matter of healthy commercial mink are highly diverse and could be closely associated with diet, and that more research is necessary to determine how the detected viruses may impact mink health.Recent viral metagenomic studies have demonstrated the diversity of eukaryotic viruses and bacteriophage shed in the feces of domestic species. Although enteric disease is a major concern in the commercial mink farming industry, few etiologic agents have been well characterized. This study aimed to identify viruses shed in the fecal matter of clinically healthy commercial mink from 40 southern Ontario farms. Viral RNA was extracted from 67 pooled fecal samples  and amplified for Illumina sequencing on the NextSeq platform, and the resulting contigs were trimmed and assembled using Trimmomatic 0.36.0 and Spades 3.8.0 in iVirus  and SeqMan NGen 12 . Identification of assembled sequences >100 bp (Geneious 10.1.3) showed an abundance of bacteriophage sequences, mainly from families  Thirty‐seven pooled kit fecal samples and 30 pooled adult female fecal samples were collected from under three pens, representing up to three adult female mink per sample or up to 15 mink kits per sample. Information on farm location, recent history of antimicrobial use, and mink coat color was collected for each farm. Samples were collected in plastic bags and stored at −80°C until processed. To prepare a 10% fecal sample dilution, the samples were thawed and mixed thoroughly in the bag, and then 1 g of fecal matter was added to 9 ml of phosphate‐buffered saline. The sample was then centrifuged at 10,000× 2.2To reduce nonviral nucleic acids, 200 μl of filtered supernatant was treated with a nuclease mixture of 7 μl TURBO DNase , 3 μl Baseline‐ZERO DNase , and 1 μl of diluted RNase T1  in 7 μl 1× DNase buffer (Ambion). This mixture was incubated at 37°C for 90 min  for 2 min at 85°C. cDNA synthesis was performed using reverse transcriptase from the QuantiTect Reverse Transcription kit  according to manufacturer's instructions. For pre‐PCR amplification enrichment of viral cDNA and DNA, 10 μl of the cDNA synthesis product was incubated with 50 pmol of the previously described random primer at 92°C for 2 min, 4°C for 2 min, then with 5 U of Klenow fragment with 1× Klenow Buffer  at 37°C for 1 h  with a 0.8:1 ratio of beads to sample. Eighty percent ethanol was used for the ethanol wash and 32 μl of elution buffer was used to extract purified DNA fragments from the beads.Klenow products were PCR amplified using KAPA 2G HotStart ReadyMix . Five microliter of the Klenow product was mixed with 1 μl of 2.5 mM a primer containing only the 18 bp fixed portion (GCCGACTAATGCGTAGTC) of the previously described primer. An additional 1 μl of 25 mM of MgCl2.5n = 37, adult female n = 30) were prepared for NGS (next generation sequencing) using Nextera XT DNA Sample Preparation Kit . Samples were sequenced using Illumina NextSeq500 V2 chemistry on a 2 × 125 cycle , and reads were demultiplexed by Donnelly software. Low quality reads were filtered using Trimmomatic 0.36.0 in iVirus  using default parameters. Trimmomatic output was used for de novo assembly in Spades 3.8.0 (CyVerse) using kmer size 65, and SeqMan NGen 12    Figure . The mos) Figure .2.6n = 76) and 2017 (n = 78) were used for antimicrobial resistance (AMR) testing of Escherichia coli and Salmonella isolates. The pooled fecal sample was thoroughly mixed in the collection bag, then 1 g was aliquoted into a sterile bag for AMR testing at Public Health Agency of Canada  using the culturing and testing methods described in the Canadian Integrated Program for Antimicrobial Resistance Surveillance  was used to conduct one‐way nonparametric Wilcoxon tests to compare the relative abundances of top phage and mammalian viral sequences between adult female mink and mink kits. For all statistical tests conducted, a 33.1Bacillus, Clostridium, Enterococcus, Escherichia, Lactobacillus, Lactococcus, Proteus, Pseudomonas, Salmonella, Shigella, Staphylococcus, and Streptococcus bacterial hosts, with the top three bacteriophage species from each group listed in Table Escherichia and Enterococcus‐associated bacteriophage sequences had the highest identities to GenBank reference sequences (84%–94%).A total of 308,817,457 sequences  were used for trimming and de novo assembly. After assembly, 345,127 contigs (>700 bp) were extracted for comparison to GenBank's viral database. Contigs with ≥100 bp  of detectable similarity to a reference genome were used for further analysis. Ninety‐eight percent of reads ≥100 bp aligned to bacteriophage sequences . Phage sequences were compiled and analyzed based on bacterial host. The most prevalent sequences were identified to have Siphoviridae (53%), Podoviridae (22%), Myoviridae (20%), Inoviridae (1%), Leviviridae (0.04%), Tectiviridae (0.01%), and Microviridae (0.01%). An additional 4.8% of detected bacteriophage sequences were unclassified, with the majority belonging to the order Caudovirales. Pseudomonas phage sequences were found to be significantly higher in adult female mink samples (p = .02), but no other significant differences were found in other detected phage sequences between age groups.Seven viral families were identified in the top 12 most prevalent bacteriophage groups , including 3.2E. coli was successfully isolated from 22 samples of the 154 pooled 2016 and 2017 fecal samples for AMR testing (12 from 2016 and 10 from 2017), no Salmonella isolates were obtained. Seven E. coli isolates were found to have intermediate resistance or were resistant to at least one of the tested antimicrobials (three from 2016 and four from 2014), with six of seven isolates found to be resistant to tetracycline  to previously reported viruses include posavirus 3, mink bocavirus, chicken anemia virus, avian gyrovirus 2, avian adeno‐associated virus strains DA‐1 and ATCC VR‐865, gyrovirus 4, gyrovirus GyV3  aligned to vertebrate viruses. The most prevalent of the vertebrate viruses were from viral families Cafeteria roenbergensis virus BV‐PW1, Aureococcus anophagefferens virus isolate BtV‐01, Chrysochromulina ericina virus isolate CeV‐01B, and Acanthocystis turfacea Chlorella virus 1 are water‐associated viruses with algae as hosts. White spot syndrome virus strain CN01 affects shrimp, whereas Culex pipiens densovirus and Melanoplus sanguinipes entomopoxvirus are associated with mosquitos and grasshoppers, respectively.In addition to vertebrate viruses, 51%  of nonphage sequences were associated with nonvertebrate hosts, including environment‐associated viruses , and plant, insect, fungal, or crustacean‐associated hosts. The 10 most prevalent of the nonvertebrate viral sequences include algae, ameba, insect, and crustacean‐associated viruses, with 18%–63% prevalence in samples (Table 3.4AF083424), but was distinct from varicella‐zoster virus (AH002362.2) , showed segments with 100% and 76% identity to genes encoding replication‐associated proteins in chimpanzee feces‐associated circular DNA virus 1 CPNG_29286 and chimpanzee feces‐associated circular DNA virus 1 CPNG_29268 (KR704711). Figure GQ351272–GQ351278, KR704912), but was distinct from human stool‐associated circular virus NG13 (GQ404856). Figure FR751463) compared to HCBI8.215 virus (LK931483) and HCBI9.212 virus (LK931484). Sequences with highest similarity to Desmodus rotundus parvovirus strain DRA25  were used in de novo assembly, resulting in chapparvovirus 2014‐ON_consensus. This consensus sequence was not closely related to the Desmodus rotundus parvovirus strain DRA25 genome or three other parvoviruses with similar structure   had segments with 100% identity to gyrovirus Tu243 VP1 and VP2 genes, as well as 63% identity to the VP1 gene of gyrovirus 4. The longest sequence had 64% identity over 76%  of the gyrovirus Tu243 genome. Phylogenetic analysis shows that assembled sequence (gyrovirus 2014‐ON_consensus) clustered most closely with gyrovirus Tu243 and gyrovirus 4 strain D137 (JX310702), and was also closely related to gyrovirus GyV3 (JQ308210), avian gyrovirus 2 (HM590588), human gyrovirus 1 strain 915 F 06 007 FD (FR823283), gyrovirus Tu789 (KF294862), and chicken anemia virus (M55918)  (parvovirus 2014‐ON_consensus) was highly related to the genomes of chicken parvovirus ABU‐P1 and turkey parvovirus 260 (GU214706), which clustered separately from turkey parvovirus 1,078 (GU214705)  encoded for capsid and replication‐associated proteins with 100% and 95% identities to chicken‐associated smacovirus strain RS/BR/2015/4 and human smacovirus (AJF23075). In phylogenetic analysis, smacovirus 2014‐ON_consensus sequence clustered most closely with chicken‐associated smacovirus strains RS/BR/2015/1–RS/BR/2015/4 (KY086298–KY086301), but were also closely related to bovine feces‐associated smacovirus strain GP3_46075_cow (KT86222) and human smacovirus 1 isolate Virginia/2/2012/Chesapeake/J23 (KP233186)  to the reference sequences of vertebrate viruses. The average identities of detected sequences, their prevalence in samples, as well as query‐encoded proteins are listed in Table ) Figure . Sequenc) Figure . Segment) Figure . Sequenc4Enterococcus, Lactobacillus, Lactococcus, Clostridium, Escherichia, Streptococcus, and Pseudomonas species were also prevalent in the mink microbiome (unpublished data). Bacillus, Salmonella, Shigella, Staphylococcus, and Proteus bacterial populations were not found to be highly prevalent in the mink fecal microbiome study (unpublished data). In a fecal microbiome study conducted on samples from mink in Northeast China, Zhao et al. , but since the detected sequences may not represent colonization by Pseudomonas species, the implications of these results remain unclear. Although previous studies have shown that lytic phage therapies may be useful in controlling Pseudomonas bacterial populations . Yang et al. (This study found the highest number of vertebrate viral sequences from the families g et al.  found noDesmodus rotundus parvovirus strain DRA25, or three other parvoviruses with similar not closely related to Eidolon helvum parvovirus 2 isolate Parvo_th_node176_9_9_893755, rat parvovirus 2 strain 9 or turkey parvovirus TP1‐2012/HUN, indicating that it could be a novel mink parvovirus. Aside from mink bocavirus, the other prevalent vertebrate viruses identified in this study have been previously isolated in other species. HCBI8.215 virus was first isolated from the serum of healthy cattle, and gyrovirus Tu243 and GyV3 were isolated from human fecal samples (Lamberto, Gunst, Muller, Hausen, & de Villiers, Viruses with low average identity were used in de novo assembly and the resulting consensus sequences were compared to closely related viruses. Most consensus sequences found to be closely related to the initial best BLASTn hit of the individual sequences, with the exception of HCBI8.215‐like virus 2014‐ON_consensus and chapparvovirus 2014‐ON_consensus. HCBI8.215‐like virus 2014‐ON_consensus was found to be more closely related to torque teno virus strain TTV‐HD14a. Chapparvovirus 2014‐ON_consensus did not cluster with the initial best BLASTn hit, In conclusion, this viral metagenomic study provides a preliminary overview of the commercial mink fecal virome, showing a diverse range of bacteriophage and eukaryotic virus sequences, including a potentially novel chapparvovirus. It is not known whether the detected bacteriophage and eukaryotic virus sequences represent commensal species, or if these viruses are capable of influencing bacterial populations and causing disease in mink. Further research is required to clarify the phylogeny of low‐identity sequences identified in this study and to determine the role of these prevalent viruses in mink health.P.V.T., B.T., and J.S.W. conceived of the work and prepared the grant; X.T.X., B.T., A.K., and J.S.W. conducted the work and analyzed the data; X.T.X. and P.V.T. co‐wrote the paper, and all authors contributed to manuscript review.The authors declare no conflicts of interest. Click here for additional data file. Click here for additional data file.


Mullite and combined mullite-alumina  tubular low-cost ceramic supports were fabricated, and their influence on membrane performance was compared to a commercial α-alumina support. The membrane preparation parameters were different ceramic supports and the concentration of chitosan solution (varying from 2 wt.% to 4 wt.%). The supports and hybrid membranes were characterized by field emission scanning electron microscopy (FE-SEM) and contact angle measurements. The results show, with increasing chitosan concentration, the permeability decreases, and selectivity increases. It was also found that the separation factor decreases with increasing feed temperature and feed water content, while the permeation flux increases. The membrane that was coated on α-alumina support with a 3 wt.% chitosan concentration exhibited the best pervaporation performance, leading to a permeation flux and separation factor of 352 g·m Dehydration of alcohols, particularly ethanol, due to the formation of an azeotrope is one of the applications of the pervaporation process. Pervaporation in comparison with azeotropic distillation or extractive distillation consumes less energy and does not require a third component ,2,3,4,5.2 [2 [There are some restrictions on the use of polymeric membranes such as low mechanical and thermal stability, loss of surface integrity and poor processability. In addition, one of the other difficulties in polymeric membranes is the stretching of polymer chains and the formation of voids in aqueous solutions, known as swelling. To overcome these problems for chitosan membranes, blending with other polymers, for example PVA ,15,16, a2 , TiO2 [22 [2 , etc., h2 [2 ,30,31.Despite the fact that numerous ceramic supports such as α-alumina , zirconiIn this study, mullite and combined mullite-alumina tubular supports were prepared. The chitosan selective layer was coated on the outer surface of the supports by the simple dip-coating technique. The objective of this work was to compare the effect of the prepared mullite and mullite-alumina low-cost ceramic supports with commercial α-alumina supports in the pervaporation process. In addition to the support type, the influence of chitosan concentration on membrane performance was investigated at three levels. The performance of the synthesized membranes was assessed by the pervaporation dehydration of ethanol and their characterization was achieved using both FE-SEM and the contact angle measurement. The effect of operational conditions on the pervaporative ethanol dehydration was also examined by using different feed concentrations as well as feed temperatures.Chitosan (medium molecular weight) was obtained from Aldrich . Acetic acid and absolute ethanol were of reagent grade and manufactured by Merck . Deionized water was produced in the laboratory by ELGASTAT . Kaolin powder, which used in the preparation of supports, was purchased from the Iran China Clay Industries Company . An aluminum oxide with a purity of 99.6 wt.%, was supplied by the Fibrona Company . Chemical analysis of kaolin and aluminum oxide are presented in 2O3 powder. As presented in our earlier work [In this work, different ceramic supports, mullite, mullite-alumina, and α-alumina were used. Mullite and mullite-alumina tubular supports were fabricated from kaolin clay and Alier work , tubularTo prepare the polymeric coating layer, chitosan was dissolved in a 2 wt.% acetic acid aqueous solution under stirring at 50 °C. The duration of stirring to form a homogenous solution depends on the solution concentration. Three different chitosan concentrations of 2, 3, and 4 wt.%, were used to coat the polymer on the different supports. The solution was then filtered through a mesh No. 250 to remove undissolved residue particles. The resultant solution was coated on the polished outer surface of ceramic supports by dipping them in the solution for about a minute. The coated supports were dried at room temperature for about 5 h under the slow rotation of 60 rpm and then annealed at 60 °C for 8 h in an oven to achieve complete evaporation of the solvent.The pore size of fabricated ceramic supports was determined using mercury porosimetry. The porosity of the supports was measured by the water absorption method. The morphology and structure of surface and cross-section of supports and membranes were studied by field emission scanning electron microscopy (FE-SEM) using Hitachi S4160 . The hydrophilicity of the used supports was evaluated by a contact angle measurement system made in the Nanotechnology Laboratory of the Institute of Petroleum Engineering of the University of Tehran, Tehran, Iran. In this system, the contact angle of a decane drop in contact with three different points of the ceramic supports surface was measured. The preconditioning of ceramic supports was done by polishing of outer surface of supports using various sandpapers  while the tubular support was rotating in order to obtain a homogeneous smooth surface and no visible hole or bump was seen on the surface. The contact angle measurement error was less than 5 percent.2 for membranes coated on the fabricated mullite and mullite-alumina supports and on the commercial α-alumina support, respectively. The downstream pressure of the module was maintained at 10 Torr using a vacuum pump. The test membrane was immersed in the feed mixture for about 24 h at the process temperature to equilibrate. The permeate was collected in the liquid nitrogen cold trap. The obtained permeate was weighed on the digital laboratory balance. The permeate composition was estimated by measuring its refractive index within an accuracy of 0.00001 using an ABBE refractometer AR4  and by comparing it to the calibration graph of refractive indices of known ethanol/water mixtures. Refractometry is a straightforward, fast, inexpensive, and accurate way of ethanol concentration determination being widely used in the food and beverage industries. Although the obtained refractive indices could be rather dependent on the operator, it is a suitable analytical method giving an accurate determination when it is used by a researcher to measure the alcohol content by comparing the value of the refractive index of a solution to that of a standard curve. All the experiments were carried out three times and the average results are reported.Pervaporation dehydration of ethanol was performed using a laboratory-scale setup shown in J), separation factor (α), and the pervaporation separation index (PSI). These were calculated using the following equations respectively:Q is the weight of the permeate (g); A represents the effective area of membrane (m2); t is the experiment time (h); Y and X are the mass fractions of permeate and feed, respectively; subscripts w and eth, denote water and ethanol respectively.The pervaporation performance of the membranes was assessed in terms of permeation flux , so ethanol molecules can pass through the membrane. Therefore, at higher temperatures, ethanol has a better chance to pass through the membrane and the selectivity decreases. The dependence of the pervaporation process on feed concentration was investigated in the range of the ethanol weight fraction of 50–90% and at a feed temperature of 60 °C. A reduction in ethanol fraction  in the feed for the S6 sample, as shown in Mullite and mullite-alumina tubular supports were fabricated from kaolin clay and alumina powder and successfully coated by the polymeric chitosan layer. The hydrophilicity and pore uniformity of ceramic supports affect the pervaporation performance of the hybrid membranes. The best pervaporation performance belonged to the membranes coated on commercial α-alumina supports due to uniform macropores and more hydrophilicity. Increasing the chitosan concentration increased the membrane layer thickness, which led to a higher separation factor and lower permeation flux. The pervaporation tests showed an optimum of 3 wt.% for chitosan concentration. The permeation flux increased remarkably with increasing either water content or the feed temperature, whereas the separation factor decreased. However, a comparison between the fabricated membranes in this study and the reported chitosan membranes  modified


In Vitro Fertilization (IVF) in couples with infertility.Infertility is estimated to affect approximately 9–30% of reproductive-aged couples. Several conditions involving one or both partners may contribute to infertility. The aim of this study is to evaluate the role of asymptomatic genital tract infections in the outcome of A total of 285 infertile couples were enrolled in the study. Vaginal/endocervical swabs and semen samples were collected and subjected to microbiological analysis. Spermiograms were carried out on semen specimens, and lactobacilli were quantified in vaginal swabs. Data were associated with IVF results and analysed by using non parametric tests and multivariate analysis.Enterococcus faecalis was the most prevalent species (11.6%) in positive semen samples and was found to negatively affect both sperm morphology (p = 0.026) and motility (p = 0.003). Analysis of genital swabs from females showed that the presence of E. faecalis (p<0.0001), Escherichia coli (p = 0.0123), Streptococcus agalactiae (p<0.0001), and Gardnerella vaginalis (p = 0.0003) was significantly associated to reduced levels of vaginal lactobacilli. Association of microbiological data with IVF outcome showed that 85.7% of IVF+ couples was microbiologically negative, while IVF was successful in just 7.5% of couples infected with E. faecalis and/or U. urealyticum and/or M. hominis (p = 0.02).Microbiological analysis showed that 46.3% of couples presented with an asymptomatic genital tract infection. Spermiogram results showed a significantly diminished motility of sperm cells in samples positive to microbiological testing compared to negative specimens. E. faecalis on sperm quality and the association of definite bacterial pathogens with reduced levels of vaginal lactobacilli. The presence of E. faecalis and/or U. urealyticum and/or M. hominis in genital samples of infertile couples is predictive for a negative outcome of IVF.The results show the negative impact of  Infertility is a medical condition that is appraised to affect between 9% to 30% of reproductive-aged couples worldwide . In 2009Neisseria gonorrohoeae, Chlamydia trachomatis, Treponema pallidum, human immunodeficiency virus (HIV), and mumps are relevant for infertility. In particular, N. gonorrohoeae can influence both male and female fertility, while C. trachomatis can affect sperm motility and viability, but it is particularly dangerous for the female where it can lead to tubal infertility [Enterococcus faecalis has been associated with oligozoospermia and teratozoospermia [Escherichia coli has been shown to induce apoptosis in sperm cells and reduce their motility [Ureaplasma urealyticum and Mycoplasma hominis are more commonly isolated from the genital tract of women and are considered as relevant for female infertility [M. hominis has been associated with abnormal sperm morphology [U. urealyticum has been shown to damage nuclear chromatin with possible implications for embryo development [Mycoplasma genitalium nor Ureaplasma parvum could be correlated to male infertility based on a recent meta-analysis [M. genitalium has lately been appraised in relation to cervicitis and pelvic inflammatory disease [Various genital pathogens have been implicated in infertility with different degrees of statistical significance. Infections caused by ertility ,4,9. Botospermia , whereasmotility ,10. Ureaertility ,11,12. Nertility ,10,13; arphology  and U. uelopment . On the analysis , while M disease .Lactobacillus spp., is reduced and replaced by mostly anaerobic microorganisms. BV is regarded as a dysbiosis of the vaginal microbiota and is associated to a heterogeneous cluster of pathogens rather than a single etiologic agent. The list of BV agents continues to enlarge and includes Gardnerella vaginalis, Atobopium vaginae, M. hominis, and different species of Prevotella, Porphyromonas, Mobiluncus, Sneathia, Peptoniphilus, Anaerococcus and Clostridium [Lactobacillus-based microbiota shifts to a microbiota dominated by enterobacteria, staphylococci, streptococci, and enterococci [The health status of the female genital tract is largely related to the presence of a normal vaginal microbiota . The comstridium . Recent stridium . Severalstridium . In conterococci . These derococci ,23–25.in vitro handling of human oocytes and sperm cells or embryos with the purpose of establishing a pregnancy [in vitro fertilization (IVF) approaches, introduction of the intracytoplasmic sperm injection (ICSI) procedure in the early 1990s represented a major breakthrough in reproductive medicine. ICSI, initially preferred to other techniques to overcome male factor infertility, is now also employed for advanced maternal age and idiopathic infertility. Currently, ICSI reckons for approximately 70–80% of total ART cycles, thus representing the most commonly used ART treatment [Assisted Reproductive Technology (ART) consists of all procedures that include regnancy . Among treatment .Despite the fact that genital tract infections are recognized to affect human fertility, there are still no consensus guidelines available on the microbiological management of infertile couples undergoing IVF treatment. In the present study, 285 infertile couples were tested for the presence of asymptomatic infections of the genital tract before being subjected to IVF, and results were associated with the outcome of IVF.A total of 285 couples, consecutively attending for 3 years the Centre for Diagnosis and Treatment of Couple Sterility at Siena University Hospital, were enrolled in the study. All couples presented with fertility disorders and were subjected to different medical examinations prior to undergoing IVF procedures. Among the medical tests, the presence of genital tract pathogens in semen and vaginal/endocervical swabs and the quality of semen were evaluated. None of the couples had signs or symptoms of genital infection. Written informed consent was obtained from each patient. The local ethical committee CEAVSE (Comitato Etico Area Vasta Sud Est) approved conduction of the study.Semen and vaginal/endocervical specimens were obtained about two months before IVF procedure. Semen was collected by masturbation after 3–5 days of sexual abstinence and subjected to spermiogram according to WHO guidelines . Male pa3 cfu/ml according to WHO guidelines recommending 103 cfu/ml as the cut-off value for ‘significant bacteriospermia’ [5 cfu/swabs.Standard bacteriological culture methods were used to detect genital pathogens from semen specimens and vaginal swabs. Semen was used directly upon arrival at the laboratory, while vaginal swabs were immersed in 1 ml of saline solution (0.9% NaCl) for 15 min prior to testing. Vaginal swabs were employed to assess for the presence of vaginal pathogens along with lactobacilli (see below). Selective and differential solid media  were used for cultivable microorganisms, including gram-positive cocci, gram-negative bacteria, lactobacilli, anaerobes, and fungi. Species identification was carried out by using Matrix Assisted Laser Desorption Ionisation-Time Of Flight (MALDI-TOF) VITEK MS  coupled with the Myla software v2.0 with a cut-off identification ≥ 99%. Semen samples were considered positive if bacterial concentrations were ≥ 5x10spermia’ ,28. Vagi4 cfu/swab, and ‘low’ at values < 104 cfu/swab. Assay detection limit was 102 cfu/swab.Quantitative analysis of lactobacilli in vaginal swabs was carried out on selective media for lactobacilli  and anaerobes . Presence of lactobacilli was regarded as ‘normal’ when cfu counts were ≥ 10C. trachomatis, U. urealyticum, M. hominis, N. gonorrhoeae, Trichomonas vaginalis and HSV, were searched out both in semen and vaginal/endocervical swabs by Real-Time PCR. Endocervical swabs were the specimen of choice for searching U. urealyticum, C. trachomatis and N. gonorrhoeae, whereas vaginal swabs were assessed for the presence of M. hominis, T. vaginalis and HSV. As abovementioned, swabs were immersed in 1 ml of saline solution (0.9% NaCl) for 15 min prior to analysis. All genital samples were heat-inactivated (85°C for 10 min) and subjected to automated DNA extraction using the MagNA Pure LC DNA Isolation Kit III  and the MagNA Pure LC machinery (Roche Diagnostics) according to the manufacturer’s instructions. For PCR reactions, 2 μl of DNA, 10 pmol of each primer  was added to 4 ml of benzidine stock solution . Ten μl of seminal fluid were mixed with 20 μl of freshly prepared benzidine-H2O2 solution. After 5 min, 160 μl of phosphate buffered saline (PBS) were added, and peroxidase-positive (round brown cells) and peroxidase-negative (unstained) cells were counted using a Mackler chamber and a phase-contrast microscope.Specimens were analysed according to WHO guidelines . After lg to WHO . Leukocyg to WHO . BrieflyMultiple follicle growth was obtained by using recombinant Follicle Stimulating Hormone (rFSH) at a dose of 150–300 IU based on the ovarian response as evaluated by hormone serum levels and ultrasound examination. As soon as the dominant follicle reached 14 mm in diameter, a Gonadotropin Releasing Hormone (GnRH) antagonist was administered daily. Ovulation was induced by injection of human chorionic gonadotropin (hCG) when at least three follicles of size > 16 mm were present in the ovaries. Oocyte pick-up was scheduled 34–36 h after hCG injection. Oocytes were fertilized by the ICSI procedure, and embryo transfer (ET) was performed 3–5 days after IVF. Serum levels of hCG were determined at day 14 post-ET, and the presence of a gestational sac was evaluated by transvaginal ultrasound examination at week 7 of gestation.Candida species and Enterobacteriales other than E. coli which were analysed as two microbial groups. Results of spermiogram and microbiological analysis of semen samples were analysed by using the Mann-Whitney test. The Chi square (χ2) test (with Yates’ correction or Fisher exact test) was employed to assess the association between the presence of genital pathogens and vaginal lactobacilli in vaginal/endocervical swabs. The association of genital tract pathogens in infertile couples with the outcome of IVF was analysed by using the χ2 test (with Yates’ correction or Fisher test). Multivariate analysis was chosen to identify single infectious agents or groups associated as independent risk factors to IVF outcome. A p value lower than 0.05 was regarded as significant.Statistical analysis was performed using the software GraphPad Prism 5.0. Analysis of microbiological results was carried out by evaluating each infectious agent as a distinct unit, except for E. faecalis represented the most common finding with a prevalence of 24.1% (47/195). Other frequently identified microbial species included S. agalactiae (15.9%), E. coli (15.4%), M. hominis (10.8%), Candida spp. (8.2%), and U. urealyticum (5.1%)  . Co-presE. faecalis (32.6%), E. coli (22%), S. agalactiae (20.5%), M. hominis (13.6%), Candida spp. (9.8%), Enterobacteriales other than E. coli (6.8%), and U. urealyticum (6.8%)  of males and 26.3% (75/285) of females were found positive to at least one genital pathogen. A total of 132 couples (46.3%) was positive for at least one pathogen in at least one of the two partners. Of the 132 positive couples, the male partner only was positive in 57 cases, the female only was positive in 49, and both partners were positive in 26 couples . Out of m (6.8%) . M. homi females .6/ml), 65 (23.8%) showed asthenozoospermia , 62 (22.7%) exhibited leukocytospermia (leukocyte counts ≥ 106 cell/ml) and 28 (10.3%) presented with teratozoospermia . Percent sperm motility in samples with leukocytospermia  was significantly lower compared to that of specimens with normal counts of seminal leukocytes , (p = 0.0003).A total of 285 semen specimens were analysed, of which 12 samples were azoospermic. Out of 273 samples, 72 (26.4%) had oligozoospermia (concentration < 15x10p = 0.012) and progressive (p = 0.0098) motility were significantly diminished in samples positive to microbiological testing compared to negative specimens  and progressive (p = 0.003) motility and in typical morphology (p = 0.026) of sperm cells between semen cultures positive and negative for E. faecalis were statistically significant  between reduced amounts of vaginal lactobacilli (< 104 cfu/swab) and the presence of genital tract pathogens was found in the female study population , E. coli (p = 0.0123), S. agalactiae (p<0.0001), and G. vaginalis (p = 0.0003), , . It is wE. faecalis, U. urealyticum, M. hominis, G. vaginalis, E. coli) were more prevalent in unsuccessful (IVF-) than successful (IVF+) couples, however, no significant differences were calculated when each pathogen was tentatively associated with IVF outcome. Therefore, analysis was performed by examining couples positive for groups of genital tract pathogens after sequential exclusion of the pathogens that seemed not to affect IVF outcome. The microbial group constituted of E. faecalis, U. urealyticum, M. hominis, G. vaginalis, and T. vaginalis was more prevalent in IVF- than IVF+ couples, but differences were not significant . Elimination of T. vaginalis showed that prevalence of the microbial group was significantly higher in IVF- (36.3%) compared to IVF+ (16.7%) couples . Finally, by further excluding G. vaginalis, the smallest infectious group significantly associated with IVF failure included E. faecalis and/or U. urealyticum and/or M. hominis  obtained a successful IVF  the presence of E. faecalis, E. coli, S. agalactiae, and G. vaginalis in females was significantly associated to reduced levels of vaginal lactobacilli, (iv) the presence of the group E. faecalis, U. urealyticum, and M. hominis in infertile couples was significantly associated to IVF negative outcome.The present study focused on the impact of asymptomatic genital tract infections on couple fertility. Main findings are: (i) approximately half of the couples was diagnosed with a genital tract infection, (ii) E. faecalis, S. haemolyticus, Group F Streptococcus) or females . E. faecalis and M. hominis/S. agalactiae were the most frequently detected microbes in males and females, respectively. M. hominis is a common endosymbiont of T. vaginalis, which acts not only as a protective niche but also as a ‘Trojan horse’ to transmit the bacteria to the human genital tract [T. vaginalis-positive vaginal swabs also contained M. hominis. Out of all pathogens detected in infected couples, E. faecalis was the most common (32.6%) and was shown to adversely affect couple fertility.All the infertile couples enrolled in the study were asymptomatic for genital tract infections, but nearly half (46.3%) resulted positive to microbiological testing. As to our knowledge no data are available in the literature on the prevalence of asymptomatic genital tract infections in naturally fertile couples, it is difficult to estimate the impact of asymptomatic infections on fertility. The microbial species identified in our study are mostly colonizers of the male anterior urethra and coronal sulcus or the vaginal milieu ,32, but al tract . In our E. faecalis, which was significantly associated to reduced motility and altered morphology of spermatozoa, suggesting that enterococci may have a direct negative influence on semen quality as previously published [E. coli- and S. agalactiae-positive samples, the second and third most commonly isolated species. Some authors have shown that E. coli induces alterations in human spermatozoa, resulting in reduced motility, altered acrosomal function, and decreased vitality [in vitro studies based on large bacterial concentrations that will unlikely be reached during in vivo infection, may explain our negative result on E. coli. Leukocytospermia is generally considered as a marker of inflammation with a poor diagnostic value for genital tract infections [p = 0.09), as evidenced by the fact that 79% of leukocytospermic samples were microbiologically negative as reported before [Genital infection and inflammation can impact on male fertility in several ways, including deterioration of spermatogenesis, impairment of sperm functions, generation of ROS leading to DNA fragmentation or oxidative protein modification, production of sperm antibodies, and obstruction of the seminal tract . The relublished ,6,36. Invitality ,35,40. Nfections ,41. Sevefections ,8. In oud before . This fid before ,43, probd before .E. faecalis, E. coli, S. agalactiae, and G. vaginalis was significantly associated with reduced levels of vaginal lactobacilli. G. vaginalis is strongly linked to BV [E. coli, S. agalactiae, and E. faecalis are not typical BV-defining microorganisms but are instead common agents of UTI and AV. Absence of lactobacilli, besides being a key feature of BV, has also been correlated to AV [E. coli [M. hominis, another recurrent species in BV, was not significantly associated to decreased numbers of vaginal lactobacilli in this study population. It should also be noted that a small percentage (18.6%) of females negative to microbiological testing had low amounts of lactobacilli, indicative of either a healthy microbiota not dominated by Lactobacillus spp. [Lactobacillus-dominated microbiota for a positive outcome of IVF.BV, AV and abnormal vaginal flora (decrease/absence of lactobacilli) have been reported to affect pregnancy rate and outcome . A recened to BV , whereased to AV ,48 and r[E. coli  and othe[E. coli , underlircentage .6% of felus spp. . The outlus spp. , and a mlus spp. . In our E. faecalis and/or U. urealyticum and/or M. hominis) was identified that significantly correlated with IVF failure (p = 0.02). It should be noted that G. vaginalis was initially included in the group (p = 0.031), in accordance with a recent prospective study reporting the association of G. vaginalis and A. vaginae with low pregnancy rates in IVF patients [M. hominis has been shown to synergistically cooperate with G. vaginalis in BV [T. vaginalis in trichomoniasis and BV [E. faecalis can be detected in polymicrobial urogenital and biofilm-based infections [E. coli, E. faecalis, S. agalactiae, and Staphylococcus spp. [Mollicutes in couples undergoing IVF was also associated to reduced pregnancy rates [M. hominis had a negative IVF outcome. The present work shed light on three specific genital tract pathogens as predictive infectious markers for poor outcome of IVF, emphasizing the importance of microbiological testing of infertile couples for E. faecalis, U. urealyticum, and M. hominis prior to IVF procedures.In the current study, IVF negative outcome was slightly more elevated in infected than non-infected couples, but differences were not significant. However, specific pathogens were more frequently found in IVF- couples, and hence a definite infectious group Click here for additional data file.


Müllerian anomalies are very common, and a frequent cause of infertility. The most used classification system untilnow, proposed by the American Society for Reproductive Medicine in 1988, categorizes comprehensively uterineanomalies but fails to classify defects of the cervix or vagina. This is based on a developmental theory that postulatesthat müllerian duct fusion is unidirectional, beginning caudally and extending cranially, which does not account forisolated cervical or vaginal defects. More recently, the European Society of Human Reproduction and Embryologyhas developed a consensus, which allows for independent cervical anomalies. We present a case of a 39-year-oldwoman with secondary infertility, found to have a cervical duplication in an anteroposterior disposition, which putsinto question the principles of embryology formerly known, but supports the theory that development happens in asegmentary fashion. Female genitourinary tract malformations are extremelycommon, being found in around 5.5% of the generalpopulation and 8% of infertile women, specifically affectingas many as 25% of women with infertility due tomiscarriage . The rea A 39 year-old woman was referred to our institution dueto secondary infertility. Menarche was at 14 years of age,with regular cycles and slight dysmenorrhoea. She hadexperienced a term caesarean section 8 years prior dueto failure to progress, and had been trying to get pregnantfor 3 years. Her past medical history was unremarkable.On gynaecological examination external genitalia and vaginawere normal; two cervical orifices in an anteroposteriordisposition were clearly visualized -this wasAn extremely rare müllerian malformation is described,which questions classical knowledge of developmentalembryology. An extensive literature search was conductedrevealing only a few similar cases , 4, one The aetiology for most of the congenital anomalies ofthe female genital tract is unknown. The importance ofnormal embryological development lays in its reproductiveconsequences, and also in concomitant urological abnormalities and in quality of life (for possible dysmenorrhoeaor dyspareunia in obstructive defects). In 1988 the ASRMattempted to classify these anomalies , howeverTwo main theories, both described in 1960s, are thefoundations for the classification system of the ASRM.The first theory, described by Crosby and Hill , suggestAcién et al.  advocateMore recently, the European Society of Human Reproductionand Embryology (ESHRE) developed ananatomy-based consensus on congenital anomalies of thefemale genital tract and its related clinical significance,in a comprehensive and accessible system. Here, cervicaland vaginal anomalies are categorized into independentsub-classes , and a nEmbryology of the female genital tract is not completelyunderstood, as the mechanism of müllerian developmentis more complex than previously described. Acién’ssegmentary theory is currently the one that best explainssegment malformations as the one presented here. Thistheory puts into question our decade-long understandingof embryological development of the female reproductivesystem, and supports the classification system of mülleriananomalies of ESHRE. The fact that in our case thetwo cervices were displayed in an anteroposterior fashionalso calls into question the fact that development may notalways occur in a side-by-side manner.


Influenza viruses can cause severe life threatening infections in high-risk patients, including young children, the elderly and patients with compromised immunity due to underlying medical conditions or immunosuppressive treatment. The impaired immunity of these patients causes prolonged virus infection and combined with antiviral treatment facilitates the emergence of viruses with resistance mutations. The diverse nature of their immune status makes them a challenging group to study the impact of influenza virus infection and the efficacy of antiviral therapy. Immunocompromised ferrets may represent a suitable animal model to assess influenza virus infection and antiviral treatment strategies in immunocompromised hosts. Here, ferrets were given a daily oral solution of mycophenolate mofetil, tacrolimus and prednisolone sodium phosphate to suppress their immune system. Groups of immunocompromised and immunocompetent ferrets were inoculated with an A/H3N2 influenza virus and were subsequently treated with Oseltamivir or left untreated. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed on the throat and nose specimens to study virus replication during the course of infection. All immunocompromised ferrets had prolonged presence of viral RNA and a higher total amount of virus shedding compared to the immunocompetent ferrets. Although Oseltamivir reduced the total amount of virus shedding from the nose and throat of treated ferrets, it also resulted in the emergence of the neuraminidase R292K resistance substitution in all these animals, as determined by mutation specific RT-PCR and next-generation sequencing. No additional mutations that could be associated with the emergence of the R292K resistance mutation were detected. The immunocompromised ferret model can be used to study A/H3N2 virus shedding and is a promising model to study new antiviral strategies and the emergence of antiviral resistance in immunocompromised hosts. Influenza viruses cause infections in millions of individuals worldwide every year. In most cases, infected patients develop mild clinical symptoms associated with modest virus shedding that usually resolves within one to two weeks. However, high risk groups, including young children, the elderly, pregnant woman and individuals with chronic medical conditions such as obesity, asthma, diabetes and neurological disorders are more likely to develop severe and complicated influenza, with increased risk of mortality , 2. ReceThe primary way to prevent influenza virus infection, especially in high-risk groups, is through the use of influenza vaccines . UnfortuThe overall efficacy of antiviral drugs to resolve clinical symptoms caused by influenza virus infection is relatively modest . AccordiFerrets have been widely uses as a model to investigate influenza virus infection, because the susceptibility to infection and the range of clinical signs seen in infected ferrets are very similar to those observed in humans. Moreover, the anatomy of the airways and the distribution patterns of sialic acid receptor expression in the respiratory tract of ferrets are comparable to those in humans . FerretsGiven that besides A/H1N1pdm09 viruses also A/H3N2 influenza viruses are circulating in nearly every winter season , 29, theet al. 2014). Biological clones were obtained by plaque purification. A clone with a genome sequence identical to the consensus sequence of the original specimen was used as the inoculum.An Oseltamivir-sensitive influenza A/H3N2 virus A/Netherlands/16/1998 (A/NL/16/98) was used in this study. This virus was isolated from a respiratory specimen upon inoculation of a Madin-Darby Canine Kidney (MDCK) cell culture  and Dutch Legislation . An independent animal experimentation ethical review committee, Stichting Dier Experimenten Commissie Consult, approved the animal studies . Eleven-month-old purpose-bred male ferrets were used, which were seronegative for Aleutian disease and circulating influenza viruses . The animals were kept in standard housing until the start of the experiment. Four days before inoculation, the animals were transferred to negatively pressured isolator cages at animal biosafety level 3. Animals were sedated during animal handling using a mixture of ketamine (4 to 8 mg/kg)  alone or in combination with medetomidine (0.1 mg/kg) , which was antagonized by the administration of atipamezole (0.1 mg/kg)  afterwards. Euthanasia under anesthesia was performed by exsanguination. Health status and animal behavior was checked twice daily and included the following parameters: weight loss, loss of appetite, degree of gastroenteritis, breathing problems and strong abnormal behavior. Commercial food pellets and water were provided The following immunosuppressive drugs were used to suppress the immune system of the animals: Mycophenolate mofetil (MMF) powder for infusion (20mg/kg) , tacrolimus concentrate for infusion 5 mg/ml)  and oral solution of prednisolone sodium phosphate (5 mg/ml) . This regimen is similar to the immunosuppressive treatment of patients undergoing solid organ transplantation. To prevent opportunistic infections, all animals received an antibiotic prophylaxis of amoxicillin supplemented with oral suspension of clavulanic acid  . Oseltamivir phosphate  was added to the regimes of ferrets receiving antiviral therapy and was provided by Hoffman-La Roche LtD. . Preparation of the drugs was done as previous described [ mg/ml (PTwenty-four ferrets were randomly assigned into four groups (n = 6) . A scheme of the experiment is depicted in 4 TCID50 of A/NL/16/98. Nose and throat swabs were collect daily in 3 ml virus transport medium [At day 0, all ferrets were inoculated intranasally and intratracheally with 10t medium . RNA wast medium . An elect medium .RNA was extracted from throat and nose samples taken 2, 4, 6, 8 and 10 days post inoculation (dpi), from immunocompromised and immunocompetent ferrets treated with Oseltamivir (groups 3 and 4), using the QIAamp Viral RNA Mini Kit . Subsequently, RNA sample volumes were reduced by evaporation in a SpeedVac at 65°C and first-strand cDNA was made using SuperScript III reverse transcriptase  and random primers  according to the manufacturer’s protocol. Second strand cDNA was synthesized in NEBnext second strand synthesis reaction buffer with second strand enzyme mix  and nuclease-free water. The cDNA was concentrated using the Genomic DNA Clean & concentrator kit  according to the manufacturer’s instructions. The DNA products were quantified using the Qubit dsDNA BR Assay kit .et al. [The enrichment, hybridization, Illumina library preparation of the HA and NA gene targets were performed according to the protocol previously described by Eckert et al. . SequencSequence reads were imported into CLC genomic Workbench version 8 (Qiagen) and trimmed using a quality score cut-off of Q30. The trimmed reads were then mapped against the HA and NA sequences of influenza virus A/Netherlands/5/1998 . Nucleotide substitutions that resulted in an amino acid substitution relative to the reference sequence of A/Netherlands/5/98 were called if the nucleotide position was covered in more than a 100 reads, with an average quality score of ≥ 30 and a forward and reverse balance of ≥0.4. The substitutions that varied within the samples and had a frequency above 1% were reported in the results tables.P value was below 0.05.All data is reported as the mean ± standard error of the mean (s.e.m.). The area under the curve (AUC) of the virus load determined by qRT-PCR was calculated for each ferret group using the trapezoid rule with graphpad prism. The student t-test was used to compare AUC values between the groups. Data were considered significant if the 4 TCID50 A/NL/16/98 (H3N2) influenza virus. Viral RNA was detected from 1 dpi onwards in the nose and the throat of the ferrets, reaching a peak around 3 dpi  was calculated for all groups . The totIn a separate experiment, immunocompetent ferrets inoculated with influenza virus A/NL/16/98 were also treated with Oseltamivir, but only for 7 days. The levels of virus shedding from the nose and throat of these animals were similar to those of immunocompromised ferrets treated with Oseltamivir, as were the total amounts of viral RNA as calculated from the AUC .Samples from all inoculated animals  were ana2 = 0.94, P<0.0001). For some ferrets, the resistance mutation was already detected at 4 dpi at a low frequency by Illumina NGS, when it was not yet detected by msRT-PCR  of Oseltamivir [50 [50 associated with the E119V mutation. This could explain why the E119V mutation was not detected in the present study.Oseltamivir treatment led to the emergence the R292K substitution in immunocompromised and immunocompetent ferrets inoculated with A/H3N2 virus. The resistant R292K genotype of the A/H3N2 virus was detected initially as a minor variant by msRT-PCR. With Illumina NGS, resistance mutation R292K was already detected at 4 dpi in Oseltamivir treated ferrets. None of the immunocompetent and immunocompromised ferrets had acquired the E119V, although this is also a common mutation associated with Oseltamivir treatment that is found in NA. The E119V mutation was found to cause a 20 to 1000-fold increase in the 50% inhibitory concentration  infection and that the virological outcome observed in these ferrets mimicked the increased virus loads and sustained virus infection seen in humans [Individuals with suppressed immunity due to age, pregnancy, disease or treatment with immunosuppressive drugs are susceptible to various virus infections beyond influenza, which can lead to high morbidity and mortality , 16, 50.n humans . MoreoveIn conclusion, the present study demonstrated that the immunocompromised ferret model can be used to study virus shedding of influenza A/H3N2 virus. The immunocompromised ferret model is a promising model to study the duration of virus shedding and the efficacy of novel mono and combination antiviral treatments for immunocompromised humans. In combination with msRT-PCR and Illumina NGS, this model may also be used to evaluate the chance of developing and selecting mutations associated with drug resistance, direct and compensatory, in immunocompromised hosts.S1 Fig4 TCID50 A/NL/16/98 (H3N2) influenza virus. One day after virus infection, OSP (10 mg/kg) was administered to ferrets from group 3 and 4. Ferrets from groups 1, 2 and 3 were euthanized 18 days post infection and the once from group 4 were euthanized 7 days post infection.Four days before virus inoculation, all ferrets were given a cocktail of antibiotics (10 mg/kg amoxicillin and 2.5 mg/kg clavulanic acid) once daily till the end of the experiment. The ferrets assigned to the immunosuppressive groups (group 2 and 3) were given a mixture of immunosuppressant  starting 3 days prior to virus inoculation twice daily till the end of the experiment. All ferrets were inoculated on day 0 with 10(PDF)Click here for additional data file.S2 FigSerum influenza antibody titers of the untreated immunocompetent and immunocompromised ferrets against the influenza homologous strain (A) A/NL/16/98 and (B) the heterologous strain A/NL/271/95 of immunocompetent and immunocompromised ferrets were determined by hemagglutinin inhibition (HI) assay. The antibody titer of each animal are depicted as individuals points. The horizontal bars represent the mean ± S.E.M.(PDF)Click here for additional data file.S3 FigImmunocompetent  and immunocompromised  ferrets were inoculated with influenza virus A/NL/16/98 (H3N2) and subsequently treated with oseltamivir  or left untreated . Virus RNA load in samples of the throat (A) and nose (C) was determined by qRT-PCR daily for 7 days. The area under the curve of panels A and C was used to estimate the total amount of viral RNA shedding from the throat (B) and nose (D) of the inoculated ferrets. The line and bar graphs depict the mean ± S.E.M. The asterisks indicates a statistically significant P value .(PDF)Click here for additional data file.S4 FigThe emergence of oseltamivir resistant viruses was monitored in throat and nose samples from untreated immunocompetent ferrets (A), untreated immunocompromised ferrets (B), oseltamivir treated immunocompromised ferrets (C) and treated immunocompetent ferrets (D) by mutation-specific RT-PCR for the oseltamivir-resistance mutation R292K. The green bars depict the presence of the wildtype genotype (292R) and the magenta the presence of the resistant genotype (292K). If both genotypes were present in one sample, the proportions of the genotypes are stacked.(PDF)Click here for additional data file.S5 FigThe relative proportion of the R292K resistance mutation detected in samples of all oseltamivir treated immunocompromised (grey circles) and immunocompetent (open squares) ferrets inoculated with A/NL/16/98 (H3N2) as determined by mutation specific RT-PCR was compared to the relative proportion determined by Illumina NGS.(PDF)Click here for additional data file.S6 FigThe percentage of the R292K variant was determined in pooled throat/nose samples collected at 2, 4, 6, 8 and 10 dpi from immunocompetent ferrets that received oseltamivir treatment. The graph depicts the frequency of the R292K mutation over time for each ferret separately, with circles, squares, triangles, down triangles, diamonds and open circles representing ferrets 1–6 respectively.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.


No locus ceruleus AMG neurons were seen in 38 individuals aged under 20 years. 47% of the 190 adults  had AMG locus ceruleus neurons. The proportion of adults with locus ceruleus AMG neurons increased during aging, except for a decreased proportion in the 90-plus years age group. No differences were found in the proportions of locus ceruleus AMG neurons between groups with different neurological, psychiatric, or other clinicopathological conditions, or among various causes of death. Elemental analysis with laser ablation-inductively coupled plasma-mass spectrometry was used to cross-validate the metals detected by AMG, by looking for silver, gold, bismuth, cadmium, chromium, iron, mercury, nickel, and lead in the locus ceruleus of ten individuals. This confirmed the presence of mercury in locus ceruleus samples containing AMG neurons, and showed cadmium, silver, lead, iron, and nickel in the locus ceruleus of some individuals. In conclusion, toxic metals stained by AMG (most likely inorganic mercury) appear in locus ceruleus neurons in early adult life. About half of adults in this study had locus ceruleus neurons containing inorganic mercury, and elemental analysis found a range of other toxic metals in the locus ceruleus. Locus ceruleus inorganic mercury increased during aging, except for a decrease in advanced age, but was not found more often in any single clinicopathological condition or cause of death.Damage to the locus ceruleus has been implicated in the pathogenesis of a number of neurological conditions. Locus ceruleus neurons accumulate toxic metals such as mercury selectively, however, the presence of toxic metals in locus ceruleus neurons of people of different ages, and with a variety of disorders, is not known. To demonstrate at what age toxic metals are first detectable in the locus ceruleus, and to evaluate whether their presence is more common in certain clinicopathological conditions, we looked for these metals in 228 locus ceruleus samples. Samples were taken at coronial autopsies from individuals with a wide range of ages, pre-existing conditions and causes of death. Paraffin sections of pons containing the locus ceruleus were stained with silver nitrate autometallography, which indicates inorganic mercury, silver and bismuth within cells (termed autometallography-detected toxic metals, or AMG The locus ceruleus supplies noradrenaline to the central nervous system, by which it influences neuronal and glial function and maintains the blood-brain barrier . The locThe underlying cause of damage to the locus ceruleus that contributes to these various disorders remains unknown, though it has been hypothesised that a selective uptake of environmental toxicants into the locus ceruleus could be the culprit, possibly combined with genetic susceptibility to these toxicants . MercuryAlthough toxic metals have been found in locus ceruleus neurons in patients with Alzheimer disease  and amyo™-detected toxic metals in the human locus ceruleus has previously been estimated in amyotrophic lateral sclerosis [http://www.coroners.justice.nsw.gov.au). Our primary aim was to obtain locus ceruleus samples for analysis of toxic metals from at least 10 people in each decade of life, with a mixture of clincopathological conditions and causes of death within each decade. Our second aim was to see if any groups of people numbering 10 or more, with various clinicopathological conditions or causes of death (cases), were more likely to have toxic metals in their locus ceruleus than groups with no known pre-mortem conditions, or undetermined and sudden causes of death (controls). A third aim was to look for any common factors among people who had high levels of locus ceruleus toxic metals to try to understand why these people had a particular predilection to accumulate toxic metals in their locus ceruleus.The prevalence of AMGclerosis , Alzheimclerosis  and auticlerosis , and in clerosis ,21. ThesAutopsy reports between 1997 and 2014 from the New South Wales Department of Forensic Medicine were examined to find individuals in whom the locus ceruleus had been examined microscopically. To ensure that a varied sample of the coronial population was studied, these reports were filtered to select a variety of clinicopathological conditions, such as a history of incarceration, cancer, neurodegenerative disorder  or psychiatric disorder . Further samples were chosen from individuals who had died from sudden non-medical causes of death, eg, drowning, suicide or trauma. Cases were collected until ethics-approved numbers for age decades, individual clinicopathological conditions, and causes of death were reached. Neurodegenerative disorders were confirmed on neuropathological examination of brains. Brain and body weights, and body heights, were recorded when available. A total of 228 individuals were studied, with numbers of individuals in successive age ranges being 28 (0–9 years), 10 (10–19 years), 27 (20–29 years), 45 (30–39 years), 32 (40–49 years), 11 (50–59 years), 18 (60–69 years), 16 (70–79 years), 14 (80–89 years) and 27 (90-plus years). Adults were defined as those aged 20 years and over.This study, "The role of the locus ceruleus in disorders of the nervous system" (X14-029), was approved by the Human Research Committee, Sydney Local Health District (RPAH Zone), and by the Office of the New South Wales Coroner. The institutional review board waived the need for written informed consent from relatives of individuals studied since this was a de-identified retrospective study of autopsy tissue.Seven μm paraffin sections of pons containing the locus ceruleus from all 228 individuals, and in 65 of these individuals (including 39 adults) the midbrain containing the substantia nigra, were stained for inorganic mercury, silver and bismuth bound to sulphides or selenides, with silver nitrate autometallography (AMG), an amplification technique that can detect only a few atoms of these metals within tissues, including the human brain ,24. BrieNumbers of locus ceruleus AMG neurons were counted using a 10x10 eyepiece grid viewed at 400x magnification, with right and lower exclusion margins, and stepped sequentially throughout one transverse section of the locus ceruleus . An AMG-Seven μm paraffin sections of ten pons samples containing the locus ceruleus  were deparaffinised and subjected to LA-ICP-MS analysis  for the GraphPad Prism 7 software was used for unpaired t-tests to look for differences between means of normally distributed continuous variables, chi-square 2x2 analyses with Fisher’s exact tests to look for associations between two categorical variables when all cell numbers were ≥5, and chi-square tests for trend to analyse locus ceruleus toxic metals in relation to age. Significance was assessed at the 0.05 level.Locus ceruleus neurons, most containing neuromelanin, were found in the posterolateral aspect of the rostral pons. At least one large thin-walled blood vessel was usually present within or adjacent to the locus ceruleus . CapillaNormal locus ceruleus neurons without AMG (grade 0) contained yellow-brown granules of neuromelanin . In gradSmall dense particulate AMG was seen associated with capillaries in about 20% of adult locus ceruleus samples. These particles were adjacent to cell nuclei that had either oval  or round (probably pericyte) profiles . Some AMpontine reticular formation and dorsal raphe nuclei contained AMG, usually in sections in which locus ceruleus grade III AMG was present. No AMG was seen in other pontine neurons, eg, in the mesencephalic nucleus of the trigeminal nerve immediately dorsolateral to the locus ceruleus , nor in any of the 26 individuals aged less than 20 years. Perivascular neurons in pontine basal nuclei contained non-specific  multiple small dark-brown birefringent particulate cytoplasmic bodies in some sections, of undetermined nature.No AMG was seen within glial fibrillary acid protein-immunostained astrocytes in the locus ceruleus. Occasional scattered neurons in the ceruleus . One indNo locus ceruleus AMG neurons were seen in the 38 infants and adolescents under the age of 20 years. In the 190 adults aged ≥20 years, a total of 53% had grade 0, 13% grade I, 18% grade II, and 16% grade III AMG. All four grades of AMG were present in each adult age decade .The proportion of people with any locus ceruleus neuronal AMG increased from none between the ages of 0 and 19 years, to a low level in the third decade (22%), then plateaued at a moderate level between the ages of 30 and 60 years (44–50%), followed by a higher plateau between the ages of 60 and 90 years (56–67%), before falling back to a moderate level from the age of 90 years on (44%) . Two of the 10 individuals aged 10–19 years  had a history of epilepsy.Comparisons between low and high category locus ceruleus AMG among various causes of death in adults can be seen in N = 20), trauma (N = 5) and drowning (N = 3). In the 10 individuals aged 10–19 years causes of death were trauma (N = 4), undetermined (N = 2) and one each of drowning, drug overdose, suicide and respiratory.Causes of death in the 28 individuals aged <10 years were the sudden infant death syndrome  were on average younger than adult females  . The proportions of adults with different grades of locus ceruleus AMG did not differ between males and females on Fisher’s exact testing. Proportions for males and females respectively were grade 0: 53.2% and 53.0% (p = 0.99), grade I: 15.3% and 7.6% (p = 0.17), grade II: 19.4% and 16.7% (p = 0.70) and grade III: 12.1% and 22.7% (p = 0.06). The tendency for Grade III to be more common in females was probably because there were more older females.Adult males  and high category  locus ceruleus AMG groups (p = 0.58). Adult body mass index did not differ significantly between low category  and high category  locus ceruleus AMG groups (p = 0.92). There are fewer numbers in these groups since body heights were not always available to calculate body mass index.Brain weights of adults did not differ significantly between low category  were detected in about half of adults from a mixed clinicopathological and cause of death population. The toxic metals started to appear in locus ceruleus neurons after the age of 20 years, and the proportion of people with locus ceruleus toxic metals increased until a decrease in late old age. No clinicopathological condition or cause of death was statistically associated with the presence of locus ceruleus AMG-demonstrable toxic metals. Elemental analysis of a subset of individuals confirmed that inorganic mercury was likely to be the metal responsible for the AMG staining. For the purposes of discussion, it will therefore be assumed that in most individuals the toxic metal demonstrated by AMG was inorganic mercury bound to sulfides and selenides, this being the form of mercury recognised by AMG.An increase in inorganic mercury in the locus ceruleus with aging could be due to accumulated mercury from mercury-containing dental amalgam restorations, occupational exposure to inorganic mercury, as well as exposure to organic mercury since methylmercury from fish, the commonest human source of mercury, is slowly demethylated in the human brain to inorganic mercury ,28. InorThe proportion of people with locus ceruleus inorganic mercury declined in the 90-plus years age group, decreasing from its heights in the previous three decades. This may be because those who survived to this advanced age were exposed to insignificant amounts of mercury throughout their lives and therefore avoided the many diseases associated with mercury toxicity . The lowOlder adults with a high cognitive reserve have higher locus ceruleus neuroimaging signal intensities than those with a low reserve , and argInorganic mercury was taken up by the locus ceruleus in only some people in each of the different clinicopathological and cause of death groups. A clue as to why this is so may be that various stressors upregulate locus ceruleus activity for prolonged periods of time . So, in It is not clear why the locus ceruleus accumulates toxic metals selectively. Rhesus monkeys have an extensive capillary blood supply to the locus ceruleus , but in Several lines of evidence link neurodegenerative disorders with mercury and the locus ceruleus. Locus ceruleus neuronal loss is marked in many neurodegenerative disorders in which associated losses of noradrenaline have been described . The locInorganic mercury in our study did not appear to be markedly more frequent in the locus ceruleus of people with neurodegenerative conditions. However, locus ceruleus neuronal loss is severe in Alzheimer and Parkinson diseases, and the remaining neurons available for autopsy study could be the ones that never contained inorganic mercury (a "survivor" effect).Damage to the locus ceruleus has been implicated in psychiatric conditions , but we The finding of inorganic mercury in the locus ceruleus may be a clue as to why incidences of several neurological disorders have been either increasing or decreasing over time. This may be because human exposure to the two major sources of environmental mercury, fish consumption and mercury-containing dental restorations, are going in different directions. Rising atmospheric mercury, mostly from burning fossil fuels , is convincrease noradrenaline output from the locus ceruleus by binding to and damaging cysteine-rich enzymes that normally deactivate noradrenaline, such as coenzyme S-adenosylmethionine; this is a mechanism proposed for an increase in noradrenaline output from mercury-containing adrenal medulla ganglion cells that can cause arterial hypertension [Mercury within the locus ceruleus could affect noradrenaline output in two opposite ways. First, oxidative stress caused by mercury  could dertension . Increasrtension .Elemental analysis of a subset of individuals showed that other neurotoxic metals apart from mercury can be taken up selectively by the locus ceruleus. Cadmium was found in the locus ceruleus of all ten individuals examined, which may not be surprising since cadmium exposure is common from widespread environmental sources , includiLA-ICP-MS did no show any consistent pattern of metals in two individuals who had locus ceruleus capillary-associated AMG. Greater numbers of cases with capillary-associated AMG will need to be analysed for more metals to assess the chemical makeup of these collections.Limitations of the study are: (1) Full medical histories and complete results of investigations were not available, since these were not hospital autopsies. However, major pre-mortem diagnoses and medications were listed, and full autopsy pathology results were available from all individuals. (2) The study aimed primarily to look at age as a factor in the uptake of toxins into the locus ceruleus, so numbers of people with specific diagnoses or causes of death were often limited. Further investigations of locus ceruleus toxic metals within specific disease subgroups would best be performed using tissue from disease-specific brain banks. (3) We did not quantitate the metals found within the locus ceruleus, but this is planned as a future project. (4) Coronial autopsy populations, aimed largely at investigating unnatural deaths, are unlikely to precisely replicate conditions found in general living populations. We attempted to minimise the differences by studying people with a wide range of disorders, as well as those without known medical conditions who died suddenly and unexpectedly. Of note, the proportion of people in this study who had locus ceruleus AMG staining was similar to that of smaller age-matched brain donor populations who had no neurological disorders ,21. (5) Toxic metals in the locus ceruleus can be found at autopsy in about half of an adult population with diverse clinicopathological conditions and causes of death. The toxic metal detected by autometallography is most likely to be inorganic mercury, which has the potential to damage locus ceruleus neurons and affect noradrenaline output to the brain. This has potential consequences in particular for neurodegenerative and psychiatric disorders. The proportion of locus ceruleus neurons containing toxic metals appears to increase throughout adulthood, except for a decrease in advanced age. This may underlie why some neurodegenerative disorders have reduced incidences at advanced ages, and why mortality rates appear to be stable in the older old. No particular medical condition or cause of death was associated with the amount of inorganic mercury in the locus ceruleus, so other reasons for the variable uptake of this toxic metal remain to be found. One possibility is that toxins are taken up by a stressor-activated locus ceruleus.


Varicella-zoster virus (VZV) can produce painful, cutaneous lesions in human beings. Reactivation of this neurotropic virus leads to herpes zoster or shingles: a painful, unilateral, vesicular eruption in a restricted dermatomal distribution. Rarely, reactivation of this virus can lead to cardiac complications, such as myocarditis and even conduction abnormalities. In this case report, we present a patient with a cardiac complication post VZV reactivation and address an unusual question and concern resulting from latent VZV presentation in adults. Varicella-zoster virus (VZV) is a DNA core virus with the ability to produce cutaneous lesions in human beings . ClinicaA 64-year-old female with past medical history of diabetes mellitus presented to the emergency department in November 2017 for left eye pain of two days duration followed by left-sided neck rash. She complained of intermittent, shooting, burning pain along the distribution of this rash. She was examined and found to have nonconfluent grouped vesicles on the posterior neck and diagnosed clinically with herpes zoster reactivation (shingles). Initial electrocardiography (EKG) was unremarkable (no conduction blocks or evidence of pericarditis). She was treated with valacyclovir and oral steroids and advised to seek urgent ophthalmological evaluation at a specialized center. She was readmitted in March of 2018 after presenting with altered mental status. EKG performed at that time showed a new first degree atrioventricular (AV) block as shown in Figure Admission vitals were blood pressure of 96/58, heart rate of 65 beats per minute. Laboratory studies done on admission were unremarkable except for hypoglycemia at 32. Later on that day, she developed bradycardia at 49 beats per minute with blood pressure 117/63 mm Hg. Repeat EKG was performed, this time showing Second Degree AV block (Mobitz I) as seen in Figure She denied any fever, chills, arthralgias, myalgias, vomiting or diarrhea. She also denied any chest pain, palpitations, or shortness of breath. Electrolyte abnormalities were corrected and she was continuously monitored on telemetry and followed with serial EKGs Figures -4.She had a positive chronotropic response with exertion and awakening. Echocardiography done during admission showed preserved left ventricular function (60%) with no wall motion abnormalities and mild grade 1 diastolic dysfunction. Electrophysiology evaluation was obtained, who advised outpatient monitoring within two weeks to evaluate for progression. The patient was not felt to be at high risk of progression due to preservation of the chronotropic response as well as absence of high-grade block.VZV was first recognized as a possible cause of myocarditis in 1953 . This leThe possibility that a conduction disturbance linked to varicella zoster virus reactivation can progress in just a couple of hours or even days is a cause for concern. In our opinion, this warrants cardiac telemetry monitoring for any dangerous findings which may require urgent management should a patient be diagnosed with reactivated varicella. In the end, given the increasing incidence of VZV reactivation in adults the possible complications, especially significant cardiac conduction abnormalities, should be kept in the differentials when treating these patients.


In the Philippines, non-therapeutic genital cutting is viewed as a culturally sanctioned rite of passage from boyhood to manhood. Strong social and peer pressure is exerted on boys aged between 8-16years to submit to destructive genital cutting, despite the fact that many men who have been subjected to genital cutting during infancy or childhood often describe their experiences in the language of violence, torture, mutilation, and sexual assault. Among a group of 505 Filipino boys subjected to ritual genital cutting (Tuli), 69% fulfilled the DSM-IV criteria for a diagnosis of PTSD, while among 1072 boys circumcised by medical operators or their assistants, 51% exhibited PTSD symptoms. Pursuant to ritual genital cutting, almost 3 out of every 4 boys exhibited PTSD-like symptoms. •1072 Filipino boys (11–16 years) from the Batangas had undergone medical circumcision and 505 ritual circumcision (Tuli).•69% of the Tuli group exhibited PTSD symptoms and 51% of boys circumcised by medical operators exhibited PTSD symptoms.•Boys subjected to Tuli exhibited PTSD comparable to Vietnam veteran inpatients at St. Cloud (Minnesota) VA Medical Center..•Both groups reported higher PTSD (p < .0001) than women subjected to “very distressing or terrifying” obstetric procedures.•A limitation of the study was its failure to examine ongoing levels of PTSD in both groups over increasing time intervals. PTSD caDespite ethical concerns about the child's right to bodily integrity , and theSabado de Gloria (Black Saturday) in a remote wooded area from which girls are excluded  deeply respect this fundamental tenet of international human rights law (UNESCO 2005) ,33.The results of this retrospective cohort study provide strong evidence of direct causal relationship between non-therapeutic genital cutting and the subsequent development of PTSD-like symptoms among Filipino boys. Destructive, non-therapeutic genital cutting of defenceless minors is a serious abuse of power enacted on children by attacking the root of their sexual identity. In males, circumcision irreversibly diminishes and damages their sexual organ with the guaranteed result of significantly reducing sexual sensation. Consequently, many young circumcised men suffer from premature ejaculation ,27, whilchildren's bodily integrity is a fundamental human right that must be respected. The societal pressure exerted on young healthy boys in the Philippines to endure destructive non-therapeutic genital cutting is clearly a flagrant abuse of power and violation of children's human rights [This large-scale retrospective cohort study empirically documents the clearcut psychosexual harm caused by both ritual and medical non-therapeutic genital cutting procedures. In this supposedly enlightened twenty-first century, destructive genital cutting of non-consenting minors, carried out as a rite of passage, is patently unethical and the time has come to recognize once and for all that n rights ,35.4.1The lack of a control group of genitally intact boys makes determining a baseline of PTSD in the male population aged between 11 and 16 years uncertain thereby making the observed results somewhat difficult to contextualise. As well, the different sample sizes of the two comparison groups (and the resultant heterogeneity of variance) may well have contributed to higher levels of statistical significance than otherwise might have been observed. Future research should analyse the severity of reported PTSD-like symptoms taking into account changing attitudes and social changes in the Filipino boys’ lives, how this affects their perceptions of genital cutting, and whether as adults they would also subject their own children to these destructive genital-cutting procedures. Replicating the present study among other populations and regions of the Philippines would be instructive in helping to provide external validation. Although both medically-performed and ritual genital cutting is a controversial topic, given widely held subjective views among Filipinos based on unchallenged cultural and religious beliefs, it is time to consider the human rights of Filipino boys and to cease this physically, sexually, and psychologically damaging practice.Not commissioned, externally peer reviewed.Bond University Human Research Ethics Committee.Ethical approval was granted by No sources of fundingNo conflicts of interest.The brief commentary was written entirely by Gregory J. Boyle.Samuel Ramos conducted the study under the supervision of Gregory J. Boyle.ACTRN12619000589189.Registration number: http://www.anzctr.org.au/TrialSearch.http://www.anzctr.org.au/TrialSearchGregory J. Boyle.


Osedax japonicus, called Osedax japonicus RNA virus 1 (OjRV1). This sequence showed the highest amino acid sequence similarity to a virus of the family Togaviridae. However, the phylogenetic position and genome structure of OjRV1 differed from those of viruses in Togaviridae. These results suggest that OjRV1 belongs to a new virus family and that deep-sea animals may associate with new viruses.The deep sea, the largest biosphere on Earth, nurtures a large variety of animals. However, no virus that infects deep-sea animals has been found. We herein report the first full-length RNA viral genome sequence identified from the deep-sea animal,  A virus is a universal genetic element that affects the physiology and evolution of life. Historically, virus discovery started with the identification of “filterable” animal and plant pathogens around 1900. Over the next 100 years, scientists found numerous viruses as causal agents for various diseases, and “illnesses” of organisms were generally required to inspire a motive for virus surveillance . TherefoThe deep sea is the largest biosphere on Earth, and it nurtures a large variety of animals . The ideOsedax are marine invertebrates that belong to the phylum Annelida, family Siboglinidae  Natsushima on March 28 (NT12-07) and April 13 (NT12-09), 2012. O. japonicus were kept in 100-L tanks at 11°C in the laboratory  at 20°C  in dsRNA extraction buffer and phenol-chloroform. dsRNA was purified with cellulose resin and then subjected to agarose gel electrophoresis (de novo exclusively with the CLC Genomics Workbench (CLC Bio) using trimmed reads and Sanger sequencing reads with the following parameters: a minimum contig length of 500, word value set to Auto, and bubble size set to Auto. The major two contigs were manually examined and extended using the Tablet viewer (The purification and sequencing of dsRNA were performed as previously described . In briephoresis . After tphoresis . In highphoresis . Contigst viewer  and Genet viewer .http://tree.bio.ed.ac.uk/software/figtree/).In the phylogenetic analysis, multiple alignments based on the deduced amino acid sequences of putative RNA-dependent RNA polymerase (RdRp) or the helicase domain in the contigs were constructed by MUSCLE  in MEGA5O. japonicus by gel electrophoresis . Therefore, we called this novel bisegmented virus Osedax japonicus RNA virus 1 (OjRV1).Two dsRNA bands were detected from phoresis  and werephoresis . The oriphoresis . The twophoresis  revealedRoss River virus  and the structural polyprotein of Venezuelan equine encephalitis virus , respectively. The results of the BLASTp search are summarized in Togaviridae, and have a non-segmented positive sense ssRNA genome (10–12 k nucleotides) with a poly(A)-tail -tail and was longer  than the known viruses in Togaviridae (9.7–11.8 kb). The RdRp and helicase domains of OjRV1 presented similarities with those in Closteroviridae, Virgaviridae, Bromoviridae, and Idaeovirus in addition to Togaviridae. The phylogenetic analysis based on the amino acid sequences of RdRp and helicase domains confirmed that Togaviridae was the closest known viral family, but was phylogenetically distinct from OjRV1  in these virus groups include viruses with bisegmented genomes; however, these viruses contain three or more ORFs. These results suggest that OjRV1 is a novel bipartite RNA virus that belongs to a new virus family.The known sequences that showed the lowest e-values in the BLASTp analysis  against (A)-tail . This mo(A)-tail . The 5′-om OjRV1 . Five geO. japonicus cells because O. japonicus harbors endosymbionts and the rearing conditions were not aseptic. However, we assumed that the biomass of these associated (micro)organisms was too small to possess dsRNA visible in gel electrophoresis. In addition, RdRp identified in the present study has similarities to viruses that infect eukaryotes, and most eukaryotic reads contained in the FLDS library were derived from Osedax (O. japonicus. To confirm the infection and influence of this virus on the host phenotype, the detection of intracellular viral RNA by FISH technology is necessary. However, it was impossible to conduct a FISH analysis in the present study because the Osedax strain was lost after the experiment and, as a consequence, we lost the virus. The effect of this virus on this loss has not yet been clarified.We were unable to verify whether OjRV1 replicated in m Osedax . These rDeep-sea animals may associate with new viruses that differ from those we have studied to date. A virus similar to OjRV1 has not yet been obtained in large-scale shotgun RNA-seq targeting non-deep sea invertebrates . To date


We also present the latest advances regarding their origin, classification, and definition that have been widely discussed.The last decade has been marked by two eminent discoveries that have changed our perception of the virology field: The discovery of giant viruses and a distinct new class of viral agents that parasitize their viral factories, the virophages. Coculture and metagenomics have actively contributed to the expansion of the virophage family by isolating dozens of new members. This increase in the body of data on virophage not only revealed the diversity of the virophage group, but also the relevant ecological impact of these small viruses and their potential role in the dynamics of the microbial network. In addition, the isolation of virophages has led us to discover previously unknown features displayed by their host viruses and cells. In this review, we present an update of all the knowledge on the isolation, biology, genomics, and morphological features of the virophages, a decade after the discovery of their first member, the Sputnik virophage. We discuss their parasitic lifestyle as  Viruses are the most abundant biological entities in the biosphere ,2,3. ThiGiant viruses were discovered in 2003, and since then, they have sparked sustained interest ,10. TheyMimiviridae, a new taxonomic group created based on its outstanding features . Th. Th30]. Mimivirus genus was isolated from the same sample. This mimivirus belongs to lineage A and has been named Sambavirus . T. T102]. In parallel, a partial genome of a Zamilon virophage designated Zamilon 2 (6716 bp) was assembled from the same metagenome of the bioreactor explored in the previous study  99]. Th. Th99]. In 2016, the genome of Dishui Lake virophage (DSLV) was discovered in the Dishui Lake (China) . PCR andThe same year, Qinghai Lake virophage (QLV) was identified in the Qinghai Lake (Tibetan mountain) and its genome was proposed to be circular, 23,379 bp in length, and with 25 predicted ORFs  104]. E. E104]. Sputnikvirus and the genus Mavirus . B. B119]. Virophages target the virus factory and the replication machinery of their host and also integrate their own genome in that of their viral hosts . It has Bigelowiella natans, a unicellular alga from the chlorarachniophytes group. The size and content of these endogenous elements were extremely heterogeneous and many of them corresponded to truncated genes, but six presumably complete copies flanked with terminal inverted repeats (TIRs) were described. The virophage-like copies identified exhibited a lower GC% compared to flanking host-sequences, which suggested that they had recently been integrated. Interestingly, most of the virophage-like elements were transcriptionally active, some being highly expressed including the morphogenesis elements and one integrase domain. The finding of Blanc et al. reinforced the scenario previously proposed by Fischer et al. of host cell–giant virus–virophage interaction [In an extensive genomic analysis for virophage signatures in eukaryotic genomes ,122, Blaeraction . Under tC. roenbergensis [C. roenbergensis [Recently, Fischer et al. demonstrated that the Mavirus virophage can integrate at several loci in the nuclear genome of its cellular host ergensis . This meergensis . Three nergensis . The retergensis ,123. Thiergensis . ProviroC. roenbergensis adopts an altruistic host-defense strategy mediated by the provirophage against the giant virus CroV [Accordingly, it has been proposed that rus CroV . This parus CroV . In the rus CroV ,127,128.rus CroV . Likewisrus CroV . As for Such a mechanism of virus integration into the host cell genome has long been known for the adeno-associated virus (AAV), a single-stranded DNA dependoparvovirus that requires the presence of a helper virus  to replicate ,131. In Acanthamoeba spp. [Acanthamoeba virophages infect their host cells simultaneously with the giant virus. Therefore, vertical transmission of provirophage could be prevented by cell lysis.Although little is known about virophage genomes endogenized in host cell chromosomes, this mechanism does not seem very widespread in amoebae. In a recent investigation conducted by Chelkha et al., no virophage like-sequences were detected in the genomes of sixteen eba spp. . As descTo repress virophage attacks, it has been proposed that some giant viruses have evolved a defense mechanism that, as CRISPR–Cas systems, involves the collection of genetic information from virophages and their use for specific inhibition of virophage multiplication . Indeed,Necrovirus [Over the past decade, the classification of virophages has been the subject of intense debate within the scientific community . Krupovicrovirus ,156. Thecrovirus . AAV hascrovirus ,158. AAVcrovirus ,161,162.crovirus . Most ofKrupovic et al. suggested that virophages are no more than a subgroup of satellite viruses with a double-stranded DNA genome ,152. TheThe second point of controversy was the specific polyadenylation signals shared between the transcripts of some giant viruses and their virophages. The same is true for some satellite viruses that share specific RNA signals with their helpers. This feature was considered to show the dependence on the transcription–replication complex of the helper/host virus, without supporting the virophage concept.The negative effect of virophages on their viral hosts is probably the strongest support of the virophage concept. However, it has been observed that even satellite viruses could affect the replication of their helpers. Indeed, infection with STNV could reduce the titer of TNV to undetectable levels, resulting in fewer and smaller necrotic lesions than with infection with TNV alone. In addition, co-infection with AAV reduces the production of adenovirus particles by up to 100-fold and the replication of adenovirus DNA by up to 10-fold ,164,165.bona fide viruses than to subviral agents or defective particles , classified virophages in the group of satellite viruses. However, this classification has been criticized on the basis of two major points that question the classification of virophages as subviral agents ,166. Firarticles . FurtherThe second point raised by researchers defending the virophage concept is that virophages are viruses that really infect other viruses . Indeed,Sarthroviridae and five genera have been proposed to allocate satellite viruses of plants and insects. As for virophages, the creation of the family Lavidaviridae (Large virus-dependent or -associated virus), which includes two genera (Sputnikvirus and Mavirus), has been proposed. This taxonomic proposal has been approved by the 10th report of the ICTV, which now considers virophages as a new divergent family of double-stranded DNA satellite viruses infecting protists that are not subviral agents. Recent discoveries related to virophages, including the MIMIVIRE system and the Mavirus mediated host-defense, support the uniqueness of virophages and strengthen the concept of “a virus infecting another virus” [bona fide viruses “with the distinguishing feature that the organisms they infect are viral themselves” [Great debates in science usually end with a compromise solution and the question of the existence of the virophage concept is no exception. In 2015, Fischer and Krupovic proposed to redress the classification of satellite viruses, previously classified as subviral agents, by the creation of two new viral families and seven new genera . The famr virus” ,166. To mselves” ,166.Over the last decade, virophages have emerged as fascinating viruses that require the presence of a giant virus co-infecting the host cell to replicate. While negative consequences of virophage integration into the host cell genome has recently been discussed , they do


The other influenza virus surface protein, neuraminidase (referring to brain lipids from which it was first derived) is a virus receptor-destroying enzyme that removes its substrate, sialic acids, from infected cell surfaces so that newly made progeny viruses are released to infect additional cells. At present, 18 hemagglutinin subtypes (H1–H18) and 11 neuraminidase subtypes (N1–N11) are recognized.In 1941, virologist George K. Hirst discovered that adding influenza virus to red blood cells (erythrocytes) in a test tube caused the cells to agglutinate, mediated by one of the virus surface glycoproteins, hemagglutinin (from the Greek  “glue”) . Alfred