Very little has been studied about its normal expression and functions.S100P is a CaWe examined S100P expression in normal human tissues by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. S100P protein expression was also studied in a series of tumors, consisting of 74 ovarian, 11 pancreatic, 56 gastric, 57 colorectal, 89 breast and 193 prostate carcinomas using a novel anti-S100P monoclonal antibody.Among the normal tissues, the highest S100P mRNA levels were observed in the placenta and esophagus. Moderate signals were also detected in the stomach, duodenum, large intestine, prostate and leukocytes. At the protein level, the highest reactions for S100P were seen in the placenta and stomach. Immunostaining of tumor specimens showed that S100P protein is expressed in all the tumor categories included in the study, being most prevalent in gastric tumors.Based on our observations, S100P is widely expressed in both normal and malignant tissues. The high expression in some tumors suggests that it may represent a potential target molecule for future diagnostic and therapeutic applications. These results suggested that S100P functionally participates in the control of the tumorigenic potential in vivo [S100P protein is a relatively small (95 amino acid) isoform of the S100 protein family that was first isolated from human placenta . Overexp in vivo . In colo in vivo , and in  in vivo ,15. HoweIn the present study, we describe a novel monoclonal antibody for S100P protein designated 18-9 and evaluate S100P expression in normal and neoplastic human tissues by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction (RT-PCR). This data could provide valuable information on where S100P is expressed under normal and pathological conditions, and whether it could serve as a tissue- or tumor-specific biomarker.NM_005980). The primers were located in two different exons separated by a 2822 bp-long intron. The sequences were as follows: forward primer: 5'-TCAAGGTGCTGATGGAGAA-3', reverse primer: 5'-ACACGATGAACTCACTGAA-3'. Three housekeeping genes  were used as internal RNA controls to normalize the cDNA samples for differences [The amount of human S100P transcript in different tissues was assessed by quantitative real-time RT-PCR using the Lightcycler detection system . Real-time PCR primers were designed on the basis of the complete cDNA sequences deposited in GenBank , and 0.5 μM of each primer. Amplification and detection were carried out as follows: After an initial 15-min activation step at 95°C, amplification was performed in a three-step cycling procedure: denaturation at 95°C, 15 s, ramp rate 20°C/s; annealing at the temperature determined according to the melting temperature for each primer pair , 20 s, ramp rate 20°C/s; and elongation at 72°C, 20 s, ramp rate 20°C/s for 45 cycles and final cooling step. The melting curve analysis was always performed after the amplification to check PCR specificity. To quantify the levels of transcripts for reference genes and S100P in tissue specimens, a standard curve for each gene was established using five-fold serial dilutions of known concentrations of purified PCR products generated from the same primer sets. Each cDNA sample was tested in duplicate, and the crossing point (Cp) value obtained allowed us to determine the amount of the starting message using the specific standard curve. The geometric mean of the three reference genes was used as a normalization factor for gene expression levels . The copThe monoclonal anti-human S100P antibody 18-9 was produced by hybridoma technology as follows: Six weeks old BALB/c mice were immunized by two i.p. inoculations of GST-S100P fusion protein purified from bacteria transfected with pGEX-3X-S100P plasmid. The plasmid contained the full length S100P cDNA subcloned from the pSG5C-S100P eukaryotic vector described earlier [Cells grown in confluent monolayer were rinsed twice with cold PBS and solubilised in ice-cold RIPA buffer (1% Triton X-100 and 1% deoxycholate in PBS) containing the commercial COMPLETE cocktail of protease inhibitors  for 30 min on ice. The extracts were collected, cleared by centrifugation at 15 000 rpm for 10 min at 4°C and stored at -80°C. Protein concentrations of the extracts were quantified using the BCA protein assay reagent .The extracts were resolved in 12% SDS-PAGE and transferred to PVDF membrane . After blocking in 5% non-fat dry milk with 0.2% Nonidet P40 in PBS, the membrane was probed with the MAb 18-9, washed and treated with secondary anti-mouse HRP-conjugated swine antibody diluted 1/7500 . The protein bands were visualized by enhanced chemiluminescence using the ECL kit (Amersham Pharmacia Biotech).Total RNA was isolated from the cell monolayers using the INSTAPURE solution  according to the protocol of the manufacturer. Reverse transcription was performed with Mo-MuLV reverse transcriptase  as described before . RT-PCR The normal tissue and tumor specimens were collected at Oulu and Tampere University Hospitals. The tumor materials included 74 ovarian, 11 pancreatic, 56 gastric, 57 colorectal, 89 breast and 193 prostate carcinomas. All tissue samples were obtained during surgery according to the guidelines of the Declaration of Helsinki and processed for routine histopathological evaluation. Collection of specimens was approved by the local ethics committees. The specimens were first fixed in 4% neutral-buffered formaldehyde, dehydrated in ethanol series, treated with xylene and mounted in paraffin.2O) for 5 min; (j) rinsing with ddH2O; (k) 0.5% CuSO4 treatment for 5 min to enhance the signal; (l) rinsing with wash buffer; (m) counterstaining with hematoxylin solution for 2 min; and (n) rinsing with ddH2O. All procedures except for the step (b) were carried out at room temperature. The sections were mounted in Entellan Neu  and finally examined and photographed with a Zeiss Axioskop 40 microscope .Automated immunostaining for S100P was performed using Power Vision+™ Poly-HRP IHC Kit reagents . The immunostaining method included the following steps: (a) deparaffinization of the sections using xylene and ethanol series; (b) treatment with Tris-EDTA buffer, pH 9.0, at 105°C for 15 min; (c) rinsing in wash buffer ; (d) incubation with 18-9 monoclonal anti-S100P antibodies diluted 1:20 in Universal IHC Blocking/Diluent for 30 min; (e) rinsing in wash buffer; (f) blocking with Universal IHC Blocking/Diluent for 20 min; (g) rinsing in wash buffer; (h) incubation in Poly-HRP-conjugated anti-rabbit/mouse IgG for 30 min and rinsing in wash buffer; (i) incubation in DAB  solution . Figure In addition to the mRNA analysis, we wanted to elucidate S100P protein expression by immunohistochemistry in various human tissues. For this purpose, we used a newly generated anti-S100P monoclonal antibody 18-9 produced against a recombinant fusion GST-S100P antigen. First, we verified the specificity of the MAb Fig. . In WestThe specificity of the 18-9 MAb was also evaluated by immunohistochemistry in comparison with the control MAb in parallel sections of the human stomach antrum Fig. . Both anVarious segments of the intestine were also dissected for a more detailed immunohistochemical analysis. The results indicated that all segments of the large intestine including cecum, colon and rectum (data not shown) were positive Fig. . The higin vitro and in vivo as well as of the data from various gene array studies [S100P protein has been recently considered a potential biomarker of cancer due to its frequent expression in different types of tumor tissues ,19. More studies ,20-22.In the present study, we used a newly generated anti-S100P monoclonal antibody 18-9 for immunohistochemical analysis of S100P expression in a series of normal and tumor human tissues. The comparative analysis of the new 18-9 MAb and the commercial control MAb confirmed that these two MAbs are both specific for S100P protein, but bind to different epitopes (data not shown). The immunoreactions obtained with two different S100P antibodies in parallel sections showed that the MAb 18-9 and the commercially available antibody recognize the same cell types in most cases and the subcellular distribution of the immunoreaction is similar. Both antibodies showed the highest immunoreaction in the cell nuclei. In addition, positive intracellular reactions were observed, which most probably reflects either the newly produced protein in the cytosolic compartment of the cell or the fraction of S100P molecules that interact with F-actin-binding protein ezrin . The maiBased on the immunostaining and quantitative RT-PCR results, the placenta clearly showed the most prominent expression among all normal tissues. The second most prominent immunostaining was observed in the gastric mucosa. For an unknown reason the mRNA levels in the stomach were comparable to several other segments of the gastrointestinal canal and distinctly lower than in the esophagus which repeatedly showed high mRNA signals and relatively weak or moderate immunohistochemical staining. These inconsistencies between the mRNA and protein levels might reflect tissue type-related differences in secretion of S100P protein to extracellular space, where it appears to function as a signalling molecule via activation of RAGE receptor . AlternaStructural studies have shown that S100P protein exists as a dimer and the S100P homodimer is probably more stable than those of other S100 proteins . High stThe present immunohistochemical and quantitative RT-PCR results demonstrate that S100P protein is widely expressed in both normal and neoplastic tissues. It clearly shows ectopic expression in some cancers. Based on the high expression in certain tumors, S100P could represent a potential target for novel diagnostic and therapeutic applications.The author(s) declare that they have no competing interests.SPar participated in the design of the study, tissue processing, immunohistochemical staining and light microscopy and drafted the first version of the manuscript. PP participated in real-time PCR. AW participated in real-time PCR and immunohistochemistry. AG, IO, SPas and JP participated in the design of the study and produced and characterized the monoclonal S100P antibody. ARM participated in real-time PCR. HOH participated in immunohistochemical staining. JI participated in the design of the study, tissue processing, immunohistochemical staining and light microscopy. All authors helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


We describe a vaccinialike virus, Araçatuba virus, associated with a cowpoxlike outbreak in a dairy herd and a related case of human infection. Diagnosis was based on virus growth characteristics, electron microscopy, and molecular biology techniques. Molecular characterization of the virus was done by using polymerase chain reaction amplification, cloning, and DNA sequencing of conserved orthopoxvirus genes such as the vaccinia growth factor (VGF), thymidine kinase (TK), and hemagglutinin. We used VGF-homologous and TK gene nucleotide sequences to construct a phylogenetic tree for comparison with other poxviruses. Gene sequences showed 99% homology with vaccinia virus genes and were clustered together with the isolated virus in the phylogenetic tree. Araçatuba virus is very similar to Cantagalo virus, showing the same signature deletion in the gene. Araçatuba virus could be a novel vaccinialike virus or could represent the spread of Cantagalo virus. The poxviruses comprise a family of large DNA viruses capable of infecting vertebrate and invertebrate hosts  is the causative agent of localized and painful vesicular lesions. The virus is believed to persist in wild host reservoirs , cattle, zoo animals, and domestic animals, including cats in parts of Europe and Asia. Contact of these reservoirs with susceptible animals and people can trigger the onset of disease  was considered to be the obvious etiologic agent causing this human and cattle infection. CPXV  is believed to be a result of contact with people recently vaccinated against smallpox. In fact, during mass smallpox vaccination campaigns, VACV infections were occasionally transmitted from the vesicular lesion on the vaccinee to domestic animals, usually cattle. In turn, infected animals transmitted VACV to susceptible people  with 1% antibiotic to isolate the virus by inoculations in bovine fetal kidney cell monolayers at the Instituto Biológico, São Paulo. Samples that showed cytopathic effects were analyzed by transmission electronic microscopy. Material isolated from bovine fetal kidney cell monolayers was spread on the chorioallantoic membrane of embryonated chicken eggs and incubated at 37°C for 72 h  and CPXV, Brighton strain, was provided by Dr. C. Jungwirth, Würzburg, Germany. Viruses were propagated in Vero cells and purified in a sucrose gradient as described . Samples were also added to embryonated chicken eggs so pock formations could be visualized on chorioallantoic membranes. White, nonhemorrhagic pocks were found (data not shown).PCR amplification of TK, VGF, and HA genes generated 528-, 381-, and 960-bp fragments, respectively. Amplicons were cloned into pGEMT vector and sequenced in both orientations. When compared to nucleotide sequences available in the GeneBank databases using the BLASTN program, the TK and VGF genes from Araçatuba virus were highly similar to homologous genes of VACV–WR. Optimal alignment showed similarity rates of up to 99.5% between Araçatuba virus and VACV–WR genes and minimal differences from nucleic acid substitutions. The coding region of HA gene was analyzed by alignment with similar sequences of VACV–WR and Cantagalo virus deposited in GenBank (accession nos. AF229247 and AF482758.1). The Araçatuba virus HA nucleotide sequence contained a signature deletion identical to a deletion detected in the sequence of Cantagalo virus . This feXbaI or BglII, allowing the detection of mutations at the restriction sites for these enzymes. We amplified the ATI gene from Araçatuba virus, VACV–WR, and CPXV for comparison. As described, the VACV–WR ATI amplicon generated 3 fragments after digestion with XbaI , VACV-IOC . How these viruses managed to persist in nature so long after the end of smallpox vaccination is a matter of speculation, but we think that they established circulation in some unknown wild hosts and were eventually transmitted to cattle and humans when they came in contact with populations of wild animals because of agricultural expansion.


We report here a case of acute lymphoblastic leukemia in remission presenting a late-onset bilateral hydronephrosis probably due to polyoma BK virus-induced proliferation of bladder endothelium on both ostii. The diagnosis was made virologically by BK virus Polymerase Chain Reaction (PCR) detection in the absence of any other bladder disease. Awareness of this late complication is necessary not only in patients after renal transplantation but also in patients after hematopoietic stem cell transplantation from matched unrelated donor. Polyomavirus BK was first isolated 1971 from the urine of an asymptomatic renal transplant patient who was hospitalized with acute renal injury and with initials BK . PrimaryAbout 50% of patients after hematopoietic bone marrow transplantation presented with BK viruria, usually within 2 months of transplantation , 11, wit2 intravenously (IV) over 3 hours every 12 hours for six doses on days 1 through 3, with mesna given by continuous infusion, vincristine 2 mg IV days 4 and 11, doxorubicin 50 mg/m2 IV day 4, and dexamethasone 40 mg daily on days 1 through 4 and 11 through 14) and was refractory when he presented in our department of hematology/oncology [2 prednisolone PO daily (days 1 through 28) plus 45 mg/m2 daunorubicin and 2 mg vincristine IV weekly . L-asparaginase (5 000 U/m2) was administered IV daily (days 15 through 28). In the second 4 weeks (phase II), the patient received two doses of 650 mg/m2 cyclophosphamide IV  together with 60 mg/m2 6-mercaptopurine PO daily (days 29 through 57) and four courses of 75 mg/m2 cytarabine IV  [A 18-year-old patient with a history of Matched-Unrelated-Donor Peripheral Blood Stem Cell Transplantation (MUD-PBSCT) was admitted to the hospital because of renal failure. In November 2007 the patient was first diagnosed with acute lymphoblastic leukemia of T cells (T-ALL) at another institution, where he was treated with induction therapy according to Hyper-CVAD Protocol ) and 10 picoM of each primer , 3 picoM of each hybridization probe (5′-GGCTGCTGCTGCCACAGGATTTTC-FL (probe 1) and 5′LC Red640-GTAGCTGAAATTGCTGCTGGAGAG-ph (probe 2), 0.25 mM dNTP's (Roche)), and 1U Platinum Taq Polymerase . Target DNA was added in a volume of 5 microliters. Two negative and 4 positive controls in different concentrations were included into each run performed on a LightCycler 1.0 instrument  under the following conditions: initial denaturation and activation of hot-start enzyme at 95°C for 30 s, followed by 45 cycles of denaturation at 95°C for 0 s, annealing of primers at 58°C for 10 s, and primer extension at 72°C for 20 s. Fluorescence was monitored by single acquisition during the annealing phase of each PCR cycle and channel setting F2/F1 (640 nm/530 nm). Differentiation of BK virus amplicons from JC virus depends on the binding of the probe 2, which perfectly matches JC virus but differs at the two underlined positions from BK virus. Thus binding to BK virus results in a shifted melting curve (61.5°C for BK versus 63.8°C for JC).Nucleic acids were extracted from 200 microliter blood or urine using magnetic beads  according to the manufacturer's instructions while from formalin-fixed, paraffin-embedded material nucleic acids were extracted using the QIAamp DNA FFPE Tissue Kit . VP2 primers lead to amplification of a 131 bp fragment . PCR reaFor detection of the viral large T-antigen, a 128 bp long fragment was amplified using modified primers from  that matPCR conditions were identical to the one described above except TaqMan probe that was used at 4 pico molar, primer annealing was 56°C for 10 s, and fluorescence acquisition was at the end of each primer extension phase at 530 nm.We described a young patient with precursor T-ALL who had received an unrelated matched donor PBSCT and one year later presented with increase creatinine levels and bilateral hydronephrosis. Nephrological and urological examination showed an epithelial proliferation of bladder on both ostii being responsible for bilateral hydronephrosis. Further pathological and molecular analysis revealed widespread endothelial infection by a BK virus. We speculate that the endothelial cell injury led to capillary injury, oedema, and tumorous proliferation on both ostii. Other organs were not involved. This case demonstrated a very late (one year after MUD-HSCT) proliferative endothelial infection by a virus related to severe viral load and hemorrhagic cystitis 4 weeks after unrelated PBSCT. Although the tumour mass could not be unambiguously attributed to BKV since the staining of large T antigen was negative in immunohistochemical analysis, large T-cell antigen and BK virus were positive by PCR, thus supporting the view that BK virus may indeed play a role in the development of this tumour. The oncogenic potential of BK virus has been well recognized , 3; GeetBK virus is an important pathogen and cause of nephropathy in renal transplant recipients, but its significance following PBSCT is less well described. Recently, O'Donnell et al. have shown in 124 allogeneic PBSCT patients that BK viruria was manifest in 65% of the patients; 17% of the patients developed viremia. The median time from transplantation to BK viremia development was 128 days and was longer for viruria (24 days) . Among cThe chronic renal failure was a multifactorial process. The transforming potential of BK virus infection has been noted early both in vitro culture models and in vivo. One of the patients with BK virus-associated nephropathy after kidney transplantation reported by Vats et al.  presenteIn summary, we could show an unusual case of late onset of bilateral hydronephrosis probably due to BK virus-induced tumorous bladder proliferation on both ostii in a young patient after MUD-PBSCT, successfully treated with operative incision and DJ catheter.


We evaluated the susceptibility, duration and intensity of viremia, and serologic responses of chickens to West Nile (WN) virus (WNV-NY99) infection by needle, mosquito, or oral inoculation. None of 21 infected chickens developed clinical disease, and all these developed neutralizing antibodies. Although viremias were detectable in all but one chicken, the magnitude  was deemed insufficient to infect vector mosquitoes. WNV-NY99 was detected in cloacal and/or throat swabs from 13 of these chickens, and direct transmission of WNV-NY99 between chickens occurred once , from a needle-inoculated bird. Nine chickens that ingested WNV-NY99 failed to become infected. The domestic chickens in this study were susceptible to WN virus infection, developed detectable antibodies, survived infection, and with one exception failed to infect cage mates. These are all considered positive attributes of a sentinel species for WN virus surveillance programs.


The author discusses the implications of a new study that used data from The Netherlands to simulate the annual and lifetime medical costs attributable to obesity. PLoS Medicine, Pieter van Baal and colleagues used data from The Netherlands to simulate the annual and lifetime medical costs attributable to obesity . Governments need to understand that public-health policies affect more than merely the sum of individual health, and sadly require greater courage to implement than does treating the sick.


Fulica atra) was conducted in Doñana, Spain. Antibody prevalence was highest in 2003, intermediate in 2004, and lowest in 2005. Some birds seroreverted <1 year after first capture. Seroconversion of birds suggests local circulation of the virus.A serosurvey for neutralizing antibodies against West Nile virus (WNV) in common coots ( In western Mediterranean countries, the frequency of outbreaks of West Nile virus (WNV) infection has increased in recent decades. Evidence for WNV circulation in Spain has remained elusive, although WNV foci have recently been identified in 3 neighboring countries  (Fulica atra) because of its high seroprevalence for WNV detected during a preliminary screening of 72 bird species . Reasons for this high seroprevalence remain unclear, although preference of this bird for mosquito-rich habitats and its relative size (weight ≈800 g) might be involved in this pattern. Birds were captured in Doñana  in a walk-in trap in October 2003 (3 capture sessions) and from September through February in 2004–2005 (12 sessions) and 2005–2006 (14 sessions). Overall, 853 captures of 515 different birds were conducted (1–7 captures/bird).We focused on the partially migratory common coot  were determined by using a micro-virus neutralization test as described  was 13.67%. This variation is similar to that observed in individual samples and repeated in different assays. In a series of 27 samples tested twice, the mean fluctuation observed was 0.29 log10 units (≈2-fold). To obtain accurate measurements of titers, particularly when assessing seroconversion/seroreversion, we analyzed samples at least twice, and when results differed, they were assayed again until a consistent result was obtained. Specificity of the test was assessed by parallel neutralization against Usutu virus (strain SAAR 1776), a flavivirus found in wild birds that belongs to the same serogroup as WNV, with a panel of sera positive for WNV by micro-virus neutralization test. All titers were higher for WNV than for Usutu virus; 93.6% were >4× higher  (<1 year of age) birds had lower antibody prevalences than adults in October . Antibody prevalence increased throughout the 2004–2005 season , but not during the 2005–2006 season   . Juvenil = 0.58) .Of 95 birds captured in 2 consecutive years, 59% had no detectable antibodies in either year, 21% seroreverted, 6.3% seroconverted, and 13.7% had antibodies in both years. Seroconversion confirms that WNV circulation is present in the study area, and seroreversion indicates that antibody titers decreased. Antibodies persisted for >1 year in some birds, although whether this was caused by reinfection, which would stimulate the antibody response, is uncertain.Of 54 birds captured at least twice in 2004–2005, 16.7% seroconverted , 3.7% seWe provide evidence for local circulation of WNV in our study area during 2004–2005. This evidence was obtained just a few months after a reported outbreak of WNV that affected humans in Algarve, Portugal, ≈100 km west of our study area. However, no increase in clinical signs or mortality rates was observed in the common coot population during the study period. The high prevalence of antibodies in juvenile birds in September–October 2003 (37.5%) and 2004 (28.8%) also suggests that WNV may have been circulating during summer and autumn of 2003 and 2004.WNV circulation decreased to low levels or was absent during the 2005–2006 winter season. There are several nonexclusive explanations for this pattern. First, the virus may not easily overwinter in Spain and thus needs to be reseeded each spring by migratory birds arriving from Africa. Nevertheless, climatic conditions probably enable the virus to survive winter because mosquitoes are present year round in the area , and seroconversion in common coots occurred by midwinter. Second, in 2005, a severe drought reduced habitat for both mosquitoes and waterbirds. Third, high seroprevalence at the end of the winter of 2005 would have resulted in effective herd immunity, which may have reduced the number of available hosts in 2006 and transmission intensity. Although this negative loop is only valid if the rate of host population turnover is low (Additional studies are needed to evaluate the role of these 3 mechanisms in the dynamics of WNV in Spain. Combining serologic results for common coots and vector sampling for virus detection may provide information needed to address these issues.


Flaviviruses tested . MSVC was equal to or one log more sensitive for virus detection than SCC for the stock Bunyaviruses (GC and ORO). For the stock Alphavirus MAY, MSVC was equally or one log more sensitive for virus detection than SCC, while for VEE SCC was equally or one log more sensitive for virus detection than MSVC. MSVC was consistently one to two sera dilutions more sensitive than SCC for the detection of Flaviviruses from patients' sera. Both methods were approximately equally sensitive for the detection of Bunyaviruses from patients' sera and equal or one dilution less sensitive for the detection of Alphaviruses from patients' sera. Additionally, MSVC detected DEN virus in five of seven DEN-3 RT-PCR positive, SCC negative patients' sera.The isolation of arboviruses from patient's low titer sera can be difficult. Here we compared the detection efficiency of Dengue (DEN), Yellow Fever (YF), Saint Louis Encephalitis (SLE), West Nile (WN), Ilheus (ILH), Group C (GC), Oropouche (ORO), Mayaro (MAY) and Venezuela Encephalitis Equine (VEE) viruses using a Modified Shell Vial Culture (MSVC) protocol to a Standard Cell Culture (SCC) protocol. First the MSVC and SCC protocols were compared using five dilutions for each of the following stock viruses: DEN-1, DEN-2, DEN-3, DEN-4, YF, SLE, WN, ILH, GC, ORO, MAY and VEE. Next, patients' original sera from which viruses  had been previously isolated were compare by the two methods using five sera dilutions. In addition, seven sera that were positive for DEN-3 by RT-PCR and negative by SCC were processed by MSVC. The MSVC protocol was consistently 1-2 logs higher virus dilution more sensitive for virus detection than the SCC protocol for all stock  The isolation of arboviruses from patient's low titer sera can be difficult.ParamyxoviradaeHerpesviridaeOrthomyxoviridaeFlaviviridaeFlaviviridae viruses for which the Shell Vial method has been tested Shell vial culture centrifugation methods for virus isolation have been shown to be more sensitive than standard isolation methods for members of the Flaviviruses: DEN-1, DEN-2, DEN-3, DEN-4, YF, SLE, ILH and WN; the Bunyaviruses: GC, ORO and the Alphaviruses: MAY and VEE. The comparison was made using two cell lines, C636 and Vero, two incubation times, four and 10 days, with stock viruses and original patients' sera from which viruses had previously been isolated.We have compared the MSVC and standard cell culture (SCC) methods of virus detection for the Flaviviruses and Bunyaviruses were titered in BHK-21 cells; the Alphaviruses were titered in Vero cells.DEN-1, DEN-2, DEN-3, DEN-4, YF, SLE, WN, ILH, GC, ORO, MAY and VEE viruses were propagated in Vero cells with Earle's Modified Essential Medium with 100 units/ml Penicillin, 100 ug/ml Streptomycin, 1 mM Sodium Pyruvate and 2% (V/V) Fetal Bovine Serum at 37°C. Upon observation of cytopathic effects, medias were collected, clarified by centrifugation at 40,000 g for five minutes and stored at −80°C until use. The Sera were collected from febrile subjects and processed for virus isolation following the SCC method  and stored at −80°C until use.2 flasks. The cells were incubated at 28°C and 37°C for C636 and Vero cells, respectively, for 4 and 10 days. The cells were scraped off the flasks, transferred to 5 ml test tubes and collected by centrifugation at 450 g for 10 minutes. The cell pellets were reconstituted with PBS and spotted onto slides and tested by indirect immunofluorescent assay (IFA) using DEN, YF, SLE, WN, ILH, GC, ORO, MAY and VEE polyclonal antibodies.100 ul of each stock virus dilution  or serum dilution  were inoculated in C636 and Vero cells propagated in T-25 cm100 ul of each stock virus dilution  or serum dilution  were inoculated in C636 and Vero cells propagated in 24 wells plates. The plates were centrifuged at 680 g for 30 minutes and incubated at 33°C and 37°C for C636 and Vero cells, respectively, for 4 and 10 days. The cells were scraped off the plates, transferred to 5 ml test tubes and collected by centrifugation at 450 g for 10 minutes. The cell pellets were reconstituted with PBS and spotted onto slides and tested by IFA using DEN, YF, SLE, WN, ILH, GC, ORO, MAY and VEE polyclonal antibodies.Flaviviruses tested by the two methods, the MSVC method consistently required one to two logs less virus for virus detection in C636 cells than the SCC method , Bunyaviruses (ORO and GC) and the Alphavirus MAY. The SCC method could be more suitable for the isolation of the Alphavirus VEE. For studies that involve the isolation of arboviruses the utilization of both C636 MSVC and Vero SCC may maximize virus isolation.MSVC is a rapid and efficient method for the isolation of


Cervical carcinoma and cervical intra-epithelial neoplasia (CIN) are likely to be associated with all sexually transmitted diseases (STDs). To help discover which (if any) of the recognised STDs might actually cause these conditions, a key question is whether one particular such association is much stronger than the others. The present study is therefore only of women newly attending an STD clinic, and compares the prevalences of cytological abnormalities of the cervix among 415 women attending with genital warts, 135 with genital herpes, and 458 with trichomoniasis or gonorrhoea. Significantly more genital wart patients (8.1%) than trichomoniasis or gonorrhoea patients (1.9%) showed dyskaryotic changes (adjusted relative risk (RR) = 5.8 with 95% limits 2.5-13.5) at, or a few months before, first attendance, while no excess whatever was seen in women with genital herpes. Moreover, half the women had a subsequent smear (at an average of 3-4 years after first attendance) and, although the diagnosis at first attendance was not related to the onset rate of dyskaryotic changes observed in these subsequent smears, it was related to the onset rate of grade III cervical intra-epithelial neoplasia (CIN III), which was found in 7 previous genital wart patients, in 2 previous trichomonas patients, but in 0 previous genital herpes patients. Thus, our findings suggest that herpes is not directly relevant to dyskaryotic change, but that one or more of the human papilloma viruses that cause genital warts may be.


Flaviviridae family is composed of fast evolving RNA viruses that infects vertebrate  and/or invertebrate (ticks and mosquitoes) organisms. These host groups are very distinct life forms separated by a long evolutionary time, so lineage-specific anti-viral mechanisms are likely to have evolved. Flaviviridae viruses which infect a single host lineage would be subjected to specific host-induced pressures and, therefore, selected by them. In this work we compare the genomic evolutionary patterns of Flaviviridae viruses and their hosts in an attempt to uncover coevolutionary processes inducing common features in such disparate groups. Especially, we have analyzed dinucleotide and codon usage patterns in the coding regions of vertebrate and invertebrate organisms as well as in Flaviviridae viruses which specifically infect one or both host types. The two host groups possess very distinctive dinucleotide and codon usage patterns. A pronounced CpG under-representation was found in the vertebrate group, possibly induced by the methylation-deamination process, as well as a prominent TpA decrease. The invertebrate group displayed only a TpA frequency reduction bias. Flaviviridae viruses mimicked host nucleotide motif usage in a host-specific manner. Vertebrate-infecting viruses possessed under-representation of CpG and TpA, and insect-only viruses displayed only a TpA under-representation bias. Single-host Flaviviridae members which persistently infect mammals or insect hosts  were found to posses a codon usage profile more similar to that of their hosts than to related Flaviviridae. We demonstrated that vertebrates and mosquitoes genomes are under very distinct lineage-specific constraints, and Flaviviridae viruses which specifically infect these lineages appear to be subject to the same evolutionary pressures that shaped their host coding regions, evidencing the lineage-specific coevolutionary processes between the viral and host groups.Virus-host biological interaction is a continuous coevolutionary process involving both host immune system and viral escape mechanisms.  Virus-host pair is a unique situation to study coevolutionary processes, as viruses are obligate intracellular parasites and their evolution is inexorably coupled to host biology. The biological interaction of viruses and their hosts is a delicate balance of actions and counteractions between host immune system and virus escape mechanisms. Viruses are unable to replicate without infecting host cells, a process which commonly leads to host fitness decrease Flaviviridae virus family comprises small, enveloped, positive-sense, single-stranded RNA viruses which infect vertebrate  and/or invertebrate (mosquitoes and ticks) hosts. Due to their relatively small genome size and medical and veterinarian importance, several Flaviviridae viruses have already had their genomes sequenced Flavivirus, Hepacivirus and Pestivirus) which vary in their host range, specificity and outcome of infection. Flavivirus genus comprises approximately 70 viruses, and is unique among Flaviviridae genera since it contains the only arthropod-borne viruses (arboviruses) known in this family, which are transmitted to vertebrate hosts by mosquitoes or ticks via blood feeding. Some of these arboviruses are associated with severe human diseases, such as dengue virus , a very divergent Flavivirus member isolated from bats Flavivirus groups are arboviruses - a mosquito-borne and a tick-borne. The mosquito-borne group is also of paraphyletic origin and contains two main clades (the YFV and DENV lineages); the tick-borne lineage is apparently a monophyletic group Hepacivirus comprises a genus of mammal-exclusive viruses mainly transmitted by blood contact. Its type species is Hepatitis C virus (HCV), which has been found to cause hepatitis in more than 1% of the world's human population, and several other liver-related diseases such as cirrhosis and liver cancer Flaviviridae species, GB virus-B (GBV-B) Hepacivirus genus candidates by the International Committee on Taxonomy of Viruses (ICTV) Pestivirus genus is composed of several species which infect non-human mammals, mainly via oral-fecal or respiratory routes. Currently five viral species are classified as belonging to Pestivirus by ICTV rus DENV  and yellrus DENV . Flaviviavivirus . Anothermilieu. One should expect, under this assumption, a rapid and specific adaptation of arboviruses to individual hosts types when replicating in a single-host system. This was previously observed in several in vitro and in vivo experiments, where single host infection led to host-specific viral fitness increase and higher mutation rates Arbovirus is a non-taxonomic designation for RNA viruses which, in nature, replicate cyclically in vertebrate and arthropod hosts, and are transmitted to vertebrate hosts by haematophagous vectors Flaviviridae viruses which specifically infect one or both host types. As shown below, we found several viral nucleotide usage patterns which are correlated with host-specific patterns, suggesting that host-induced pressures are actively shaping dinucleotide and codon usage patterns in both Flaviviridae and hosts coding regions.Vertebrate and arthropod lineages diverged between 573 and 656 million years ago Flaviviridae Phylogenetic Analysis” section below). Hepacivirus group and Pestivirus genus were considered distinct vertebrate-exclusive groups, and Flavivirus genus was further divided into four groups according to phylogenetic relatedness and/or host type, generating the NKV, insect-only, mosquito-borne and tick-borne Flavivirus groups. Individual group composition is listed in The hosts group was divided into vertebrates and invertebrates, and viruses were divided into six groups to reflect their evolutionary history as well as their host specificity and range , so we decided to proceed with the resulting Neighbor-Joining tree .Despite the lack of phylogenetic analyses in In Among all dinucleotide usage variation found to occur in distinct genomes, perhaps a partial agreement regarding its causes was achieved only for CpG under-representation in vertebrate nuclear genomes. The current working hypothesis to explain it relies on cytosine methylation-deamination model Flavivirus groups among themselves to minimize phylogenetic distance as a possible noise source, allowing us to check for genomic features putatively induced by host range/type. All vertebrate-infecting Flavivirus groups  displayed a dinucleotide usage that mimics the vertebrate genomic signature. CpG and TpA were found to be under-represented and TpG and CpA were over-represented, probably as a compensatory mechanism for CpG avoidance. This genomic signature pattern was already observed in several vertebrate viruses with small genomes, regardless of their genomic material In order to analyze viral dinucleotide usage patterns and to look for possible correlations between them and the host-specific genomic signatures found to occur, two complementary approaches were adopted. Our first strategy consisted in the comparison of all four Flaviviridae. This proposition is further supported by the dinucleotide usage pattern found in insect-only Flavivirus group. This cluster also displayed a dinucleotide usage pattern very similar to their host group, having as only biases a TpA decrease and a TpG increase, which can be seen as a putative compensatory mechanism Flaviviridae family, West Nile Virus (WNV) HepacivirusDrosophila melanogaster, apparently does not rely on motif recognition strategies to detect foreign nucleic acids. RNA interference (RNAi)-mediated immune response appears to be a main factor to deal with a broad range of virus infection Ae. aegypti, albeit it makes no mention to what virus ligand is responsible for Toll activation Flavivirus analysis suggests that a vertebrate-specific evolutionary pressure is actively selecting both host mRNAs and viral genomes against CpG usage and possibly against TpA as well, indirectly leading to the scenario of viral mimicry of host dinucleotide usage. It is worth noting that a TpA decrease also leads to a compensatory increase in TpG, causing its higher over-representation when compared with CpA, since TpG increase is a compensatory mechanism for both CpG and TpA reduction The vertebrate immune system-mediated viral CpG depletion hypothesis can account for the CpG depletion observed in vertebrate-infecting Pestivirus and Hepacivirus) and putative (NKV Flavivirus) vertebrate-exclusive Flaviviridae groups between them and with the vertebrate genomic signature. By doing so we were able to verify how different evolutionary stories shape dinucleotide usage in viruses which share the same host taxon but are distantly related by phylogeny, thus excluding host variability as a possible source of variation. All these viral groups mimic vertebrate dinucleotide usage to some extent, with some interesting peculiarities in each group. Pestivirus genus displayed no TpA bias, a unique feature among all viral groups analyzed. This observation argues against the purifying TpA selection triggered by its presence in regulatory motifs and/or or stop codons. An RNase L-mediated selection in vertebrate-infecting viruses may be occurring if Pestivirus members are able to somehow inhibit RNase L or its activation pathway. One quite remarkable characteristic of Pestivirus biology is its ability to inhibit the type I interferon (IFN) induction by dsRNA Pestivirus IFN-β pathway inhibition Pestivirus ability to inhibit RNase L activation is possibly linked to the TpA normal levels found in this group. Nevertheless, any hypothesis to explain TpA usage among Flaviviridae must take into account TpA under-representation in insect-only Flavivirus as well, since mosquito immune system has no interferon-based antiviral mechanism and no known nucleotide motif recognition signaling pathways. Clearly more experiments must be performed in order to shed some light into this interesting, previously unknown biological phenomenon. Hepacivirus group presented the highest level of CpG and TpA frequencies and the lowest level of the putative compensatory TpG usage among all vertebrate-infecting viruses. If we consider such observations in light of the dinucleotide-depletion-by-vertebrate-immune-system hypothesis, viral escape mechanisms are likely to be occurring, as we already proposed for the TpA normal levels observed in Pestivirus group. Furthermore, Hepacivirus genus is unique among Flaviviridae family since their infection commonly evolves to persistence Retroviridae family, a constant production of viral RNA and proteins must occur in Hepacivirus members in order to maintain infection persistence. The continuous replication process of HCV is closely associated to the endoplasmic reticulum (ER), since HCV translation and replication take place here Hepacivirus genus is known for its unique ability among Flaviviridae to modulate both innate and adaptive host immune system Hepacivirus genomic signature is a direct consequence of the viral escape mechanisms that were selected due to its persistence. This genus also possesses the highest G+C content among all viral groups surveyed (data not shown), a fact which also allows a higher CpG content according to the host-induced viral CpG depletion hypothesis Hepacivirus and insect-only Flavivirus dinucleotide odds ratio values deviate less from expectation than the other viral groups, indicating that these viral groups are using dinucleotides at a frequency closer to the expected usage values.A second strategy to study vertebrate host influence in viral genome composition involved the comparison of the genomic signature of known  were found to avoid CpG-containing codons as well, since none of these codons was observed with a RSCU value above one. This observation was further supported by the analysis of tetracodonic amino acids (threonine (Thr), proline (Pro), alanine (Ala), valine  and serine (Ser) TCN codons) where, for both vertebrates and vertebrate-infecting Flavivirus, CpG-containing codons were clearly less used than the CpA-containing codons. The invertebrate host group displayed no tendency for CpG under-representation being, instead, the only taxon where CpG-containing codons were used more than CpA-containing codons for all tetracodonic amino acids. Insect-only Flavivirus also mimicked its host group in synonymous codon usage preferences, with both groups displaying as main bias the avoidance of TpA-containing codons. This viral group also displayed a much less pronounced bias for CpG under-representation when compared to the other viral groups , having the lowest difference between CpG and CpA-containing codons in tetracodonic amino acid analysis. The analysis of tetracodonic amino acid boxes in Hepacivirus group revealed almost no difference between informative codons, which is coherent with the scarcely noticeable CpG and TpA under-representation found in the dinucleotide analysis. These observations again suggest that HCV immune system escape mechanisms could be key factors to understand its nucleotide motif usage patterns. Pestivirus group displayed a strong CpG reduction bias, having the lowest RSCU values for CpG-containing codons among all groups. TpA usage pattern in this group also displayed the same intriguing phenomenon observed in dinucleotide analysis. This viral group was the only one possessing TpA-containing informative codons with RSCU values above one  RSCU values for all groups  and under-represented . In fact, visual inspection suggests a very similar codon usage pattern between the viral groups of insect-only Flavivirus and Hepacivirus and their respective host groups, which was further evidenced by hierarchical clustering analysis based on codon usage  correspond to members of the mammalian clade Boreoeutheria. Nevertheless, discrepancies between phylogenetical and codon usage trees were observed, such as the closest codon usage similarity between M. mulatta (primate) and C. Familiaris (carnivora) when compared to H. sapiens (primate). Furthermore, C. familialirs belongs to the Carnivora order, a taxon which is more related to the Artiodactyla order than to the Euarchontoglires superorder, which is composed of rodents and primates Culex and Aedes genus members grouped together. Of special interest in the second cluster was the finding of single-host viruses from Hepacivirus and insect-only Flavivirus groups that clustered together with their corresponding host group. This finding suggests that these viral groups, distantly related when comparing their protein sequences  website in GenBank format using RefSeq Vertebrata and Culicidae taxa that, after extensive literary research, are known to be infected by Flaviviridae viruses. Vertebrata dataset was composed of species Gallus gallus (chicken), Taeniopygia guttata (zebra finch), Homo sapiens (man), Sus scrofa (pig), Bos taurus (cow), Canis familiaris (dog), Macaca mulatta (Rhesus monkey), Mus musculus (mouse) and Ovis aries (sheep). Culicidae data was composed of species Culex quinquefasciatus, C. pipiens, Aedes aegypti and Ae. albopictus. In order to study specific genomic signatures of each host taxa the eukaryotic dataset was further divided into vertebrate and invertebrate groups.The host dataset consists of the entire set of complete mRNA sequences from different species of Flaviviridae virus family used in this study were obtained by an initial query into NCBI genome database for complete genomic sequences from Flaviviridae family using Entrez Flaviviridae phylogenetic analysis” below for details). Flavivirus genus was divided into four datasets - non known arthropod vector (NKV) Flavivirus, insect-only Flavivirus, mosquito-borne Flavivirus and tick-borne Flavivirus - and the two remaining genera (Hepacivirus and Pestivirus) were considered as two distinct vertebrate-exclusive groups. Viral sample names and acronyms used through this article are as follows: Apoi virus (APOIV), Border disease virus (BORV), Bovine viral diarrhea virus 1 (BVDV 1), Bovine viral diarrhea virus 2 (BVDV 2), Bussuquara virus (BSQV), Cell fusing agent virus (CFAV), Classical swine fever virus (CSFV), Culex Flavivirus (CULV), Dengue type 1 virus (DENV 1), Dengue type 2 virus (DENV 2), Dengue type 3 virus (DENV 3), Dengue type 4 virus (DENV 4), Entebbe bat virus (ENTV), GB-B virus (GBV-B), GB-C virus (GBV-C), Giraffe Pestivirus (GIRV 1), Hepatitis C virus (HCV), Iguape virus (IGUV), Ilheus virus (ILHV), Japanese encephalitis virus (JEV), Kamiti River virus (KRV), Karshi virus (KARV), Kokobera virus (KOKV), Langat virus (LGTV), Louping ill virus (LIV), Modoc virus (MODV), Montana myotis leukoencephalitis virus (MMLV), Murray valley encephalitis virus (MVEV), Omsk hemorrhagic fever virus (OHFV), Powassan virus (POWV), Reindeer 1 Pestivirus (REIV), Rio bravo virus (RBV), Sepik virus (SEPV), Tamana bat virus (TABV), Tick-borne encephalitis virus (TBEV), Usutu virus (USUV), West nile virus (WNV), Yellow fever virus (YFV), Yokose virus (YOKV)All complete genome sequences from Perl scripts were used to extract the open reading frames (ORFs) from the GenBank files downloaded from NCBI, as described in “Data Acquisition” Section. All ORFs were submitted to the following cleaning pipeline to remove possible error sources. In order to be considered as a valid gene , a given ORF should not be classified as a hypothetical product, start with a valid start codon, end with a valid stop codon, have a length multiple of three, and have no ambiguous bases, here considered as a non-standard  nucleotide. Since different nucleotide bias are known to occur in nuclear and mitochondrial genomes Flaviviridae member with a valid genome as defined by “Data treatment” section was aligned using CLUSTALW software with default parameters The complete polyprotein sequences of each A common assessment of dinucleotide bias in sequences is the odds ratio, defined as the quotient of the probability of finding a dinucleotide in a given sequence divided by the product of the probabilities of finding each nucleotide that forms the pair in the same sequence, calculated as shown in Equation 1.xy in the same sequence. As a very conservative criterion, dinucleotides with odds ratio values outside the 0.78–1.25 range were considered as having a low or high relative abundance, respectively Equation 1 – Calculation of dinucleotide odds ratiox and y denote two dinucleotides, and z and w denote the two complementary nucleotides of y and x, respectively. As an example, if x and y represents the nucleotides A and C, calculation was done as follows: P°(AC)  =  P°(GT)  =  2(f(AC) + f(GT))/(f(A)+f(T))(f(C)+f(G)).Equation 2 - Calculation of symmetrical dinucleotide odds ratioFlaviviridae members were calculated using odds ratio and hosts dinucleotide odds ratio were calculated using symmetric odds ratio.Dinucleotide odds ratio for j in a sequence x divided by the frequency expected E if all synonymous codons for the amino acid coded by j were equally frequent, as shown in Equation 3.Relative Synonymous Codon Usage (RSCU) is a common method used to estimate codon bias for all codons which code for an amino acid with degeneracy greater than one. It is defined as the observed frequency of a codon j in genome x and j. Expected values are calculated by counting the total number of synonymous codons for a given amino acid in the sequence divided by the number of existing codons that codes for it.Equation 3 – Calculation of RSCUN×M dimensions, N being the number of species and M the number of degenerated codons. Hierarchical clustering of this matrix was conducted based on Spearman's rho correlation coefficients of RSCU values, conceptually similar as done by For each organism listed in To evaluate percentage of deviation in dinucleotide and codon frequencies from the mean values, we performed the calculation of mean and standard deviation values of dinucleotide odds-ratio and RSCU measurements for host and viral groups. The main underlying idea of this analysis is that organisms subject to less constraint factors for usage of a given dinucleotide/codon should deviate from the mean value less than highly biased organisms.Table S1This table contains all species used in this study.(0.03 MB XLS)Click here for additional data file.Figure S1Dinucleotide odds ratio analysis of monophyletic Flavivirus groups.(0.56 MB TIF)Click here for additional data file.


C. gattii may be spread through soil disturbances, wind, water, distribution of tree and soil byproducts, and human movement. Cryptococcus gattii infections in humans and animals without travel history to Vancouver Island, as well as environmental isolations of the organism in other areas of the Pacific Northwest, led to an investigation of potential dispersal mechanisms. Longitudinal analysis of C. gattii presence in trees and soil showed patterns of permanent, intermittent, and transient colonization, reflecting C. gattii population dynamics once the pathogen is introduced to a new site. Systematic sampling showed C. gattii was associated with high-traffic locations. In addition, C. gattii was isolated from the wheel wells of vehicles on Vancouver Island and the mainland and on footwear, consistent with anthropogenic dispersal of the organism. Increased levels of airborne C. gattii were detected during forestry and municipal activities such as wood chipping, the byproducts of which are frequently used in park landscaping. C. gattii dispersal by these mechanisms may be a useful model for other emerging pathogens.Recent  Cryptococcus gattii can infect the pulmonary and central nervous systems of humans and animals and was until recently regarded as a predominantly tropical organism , in 1999 . With the exception of 1 Gulf Island location with extremely high C. gattii concentrations in soil, these “hotspot” areas were all on Vancouver Island, which suggests that this is the primary area of C. gattii colonization. Approximately 10% of trees were positive for C. gattii, including >10 species. The highest airborne C. gattii concentrations were detected during the Northern Hemisphere summer but with propagules sufficiently small to cause infection present throughout the year. C. gattii was detected in fresh water and seawater in several locations.A large-scale study of the environmental distribution of C. gattii and exported tree species, particularly eucalypts, has been speculatively linked to its dispersal . Sampling was conducted in the environments surrounding the residences of those with reported infections, including homes, habitats, and nearby parks and wooded areas.C. gattii. Positive sites were resampled in June and December of 2005, including the original positive tree(s) and adjacent trees.A 35-km traffic corridor connecting 2 highly visited provincial parks was sampled at ≈500-m intervals (publicly accessible areas only). This sampling was performed on 6 nonconsecutive days in October 2004 and included 92 sites, 64 located at the roadside (designated “road sites”) and 28 located ≈100 m from the road (designated “forest sites”). For this series of samples, 169 trees of 8 different species were swabbed, and soil samples were collected from the rhizospheres of 77 of these trees. Global positioning system coordinates and tags were used to identify trees. Sites were designated positive by the presence of at least 1 tree or soil sample positive for C. gattii in the environment. Except in these analyses, all data presented consider only the first swab, soil, and air samples collected at each sampling point to minimize statistical artifacts.Many sites were sampled multiple times within 2–3 years to investigate the longitudinal colonization patterns of C. gattii aerosolization, air samples were collected during 2 independent tree removal efforts in a C. gattii–endemic area of Vancouver Island. A red alder (Alnus rubra) and a Douglas fir (Pseudotsuga menziesii var. menziesii) tree were removed by arborists on the same day in August 2002. The trees were tested for C. gattii colonization by swab and air sampling immediately before felling; in addition, air samples were collected at different tree heights during felling.To investigate the effect of forestry activities on In collaboration with a municipal garden waste removal service, swabs of garden waste and nearby trees were collected from properties on 17 residential streets in October 2003. Air samples were collected around the garden waste and at the outlet of the wood chipper. Samples of wood chips were also collected from the wood chipper.C. gattii carriage on vehicles not involved in the sampling effort. Two hundred vehicles in a mainland university campus parking lot and 200 vehicles in a Vancouver Island university campus parking lot were randomly selected for testing.The wheel wells of vehicles used for Vancouver Island sampling were routinely swabbed; 63 swabs have been taken since July 2003. In addition, wheel wells of privately owned mainland- and Vancouver Island–based vehicles were swabbed to evaluate C. gattii sampling were taken at various Vancouver Island sampling sites during July 2003–June 2004. In addition, 9 swabs were taken from surfaces that came in contact with C. gattii–positive footwear . To investigate the potential for survival of C. gattii on footwear, shoes worn in a C. gattii–endemic area of Vancouver Island were stored for 333 days, with swabs taken periodically. The shoes were worn in nonendemic areas for short periods (≈4 h) on days 144, 153, and 154.Eighty swabs of footwear worn by personnel participating in C. gattii by using canavanine-glycine-bromothymol blue media  methods  Access 2002, and statistical analyses were performed with SPSS 14 . Geographic data were assembled, and sampling points were mapped on 1:50,000 scale National Topographic System of Canada (NTS) grids .C. gattii tree and soil colonization. C. gattii in these locations. Intermittently positive swab and soil results were also observed, in which the series of samples effectively oscillated between positive and negative for C. gattii over time with no discernable seasonal pattern. This intermittent positivity was probably due to fluctuations in the cryptococcal population over time, above and below limits of detection. This situation might arise following the introduction of C. gattii to a new location or substrate.We initially investigated longitudinal patterns of C. gattii introduction to new areas of the Pacific Northwest, in which the fungus must adapt to new microclimates and compete with local microbiota. Areas of recent C. gattii dispersal may go through a period of intermittent positivity before either failing to become colonized (transience) or becoming permanently colonized. Determinants of colonization resulting in high C. gattii concentrations include low soil moisture and organic carbon content .We hypothesize that permanent colonization is established once the cryptococcal population reaches a critical mass. This concept forms the basis of a model for C. gattii in BC. We assessed C. gattii positivity at sites along a largely recreational traffic corridor that traverses both the Coastal Douglas Fir and Coastal Western Hemlock xeric maritime biogeoclimatic zones. Of the 169 trees sampled by swabbing on this route, 12 (7.1%) were positive for C. gattii, representing 10 sampling sites. Two of 77 soil samples (2.6%) from different sites were positive at low concentrations (25–50 CFU/g). No significant difference was observed between the C. gattii positivity of road sites and forest sites. Interestingly, the positive sampling sites were clustered at small towns, services, or attractions such as provincial parks  with a boat launch, hiking trails, and facilities for camping and picnics; 170,900 persons visited the park in 2005 . Forty water samples were collected at 4 park sites during 11 separate sampling trips in a 32-month period, and at least 1 positive sample was obtained on each sampling trip. C. gattii concentration detected at each. The boat launch site was associated with the highest rate of sample positivity and C. gattii concentration, followed by the picnic site and a historic artifact site, all of which are high-traffic visitor areas. Within the campground, water samples were collected from a creek feeding into the lake near the picnic site. These positive creek samples probably account for some of the C. gattii isolated from lake samples at the picnic site, although the observed C. gattii concentrations from creek samples were far lower than that of the lake samples. By contrast, 6 water samples taken in areas of the lake with limited public access were all negative for C. gattii. Many swab and soil samples collected from the public sites were positive for C. gattii (data not shown). While C. gattii in the water from these locations may be in part seeded by contaminated soil or tree debris, we suggest that human activities in and around the lake contributed to the dispersal of C. gattii to these recreational areas.Water sampling at a different provincial park (NTS grid 092F/07) also yielded evidence in support of anthropogenic C. gattii in several bodies of fresh water and seawater around Vancouver Island, and viability assays indicate the organism’s potential to survive for at least 1 year in fresh water and seawater . These data provide some insight into the mechanism of transmission of cryptococcal infection reported for a considerable number of wild marine mammals in the Strait of Georgia (Sampling in other areas of BC detected C. gattii was detected in 22 (35%) of 63 samples, including samples taken several weeks after return of the vehicle to a non–C. gattii–endemic area, after the vehicle had been professionally washed. In addition, C. gattii was detected on 10 (5%) of 200 privately owned vehicles on Vancouver Island and 1 (0.5 %) of 200 on the BC mainland. The isolates obtained from the Vancouver Island–based vehicles represented molecular types VGIIa, VGIIb, and VGI (including coisolation of VGIIa and VGIIb from 1 vehicle), while the isolate from the mainland-based vehicle represented VGIIa. While we have not assessed the C. gattii positivity of cars that are actively traveling between Vancouver Island and the mainland, vehicles could certainly be involved in the mechanical dispersal of C. gattii propagules. Approximately 8 million private and commercial vehicles are transported between Vancouver Island and the BC mainland each year  of 80 swab samples from the footwear of persons participating in sampling. In addition, 5 (56%) of 9 swabs of surfaces contacted by positive footwear were positive for C. gattii, which indicates that the fungus can be transferred to contacting surfaces and may be redistributed to some extent by this mechanism. Similarly, passive transport of C. gattii by wild and domestic animals could be involved in dispersal.C. gattii carried on footwear was investigated over time. Swabs from shoes worn for environmental sampling were positive at day 0 and were consistently scored as 2+ when unworn (up to day 144). The active wearing of the shoes reduced viable C. gattii levels; C. gattii was detected following activity on day 144 but not after activity on days 153 and 154. However, viable levels rebounded slightly by day 183 (1+) and remained detectable on day 333 (1+). Genotyping of isolates from this footwear showed isolates belonging to the VGIIa, VGIIb, and VGI subtypes, reflecting the diversity observed at the sampling site where the shoes became contaminated on day 0 (data not shown). While different footwear materials and activity patterns are likely to influence viability and dispersal of C. gattii, these observations suggest that footwear could serve as mechanical vectors for C. gattii.The viability of C. gattii was investigated during the scheduled removal of 2 trees in an area of Vancouver Island where C. gattii had been found. Both trees tested positive for C. gattii by swab and by adjacent air sampling done immediately before they were felled. C. gattii concentrations in air samples taken during tree cutting, limb removal, and chipping activities. All air samples, collected at varying heights, tested positive during the felling of both trees. Airborne concentrations increased during felling of the red alder, but no substantial change was observed for the Douglas fir. However, for both trees, air samples taken during branch chipping indicated much greater (10- to 140-fold) airborne C. gattii concentrations than were observed during quiescence. Aerosolization of C. gattii through such activities is likely to increase the risk of exposure and the dispersal of cryptococci through wind.The concentration of airborne 3 and <5 CFU/m3C. gattii, respectively. Similarly, air samples taken at the opening of the wood-chips storage bag detected 2,256 and 1,494 CFU/m3 after 1 and 2 years, respectively, indicating long-term aerosolization of propagules from these tree byproducts. C. gattii was also isolated from 2 samples of wood chips  collected from within a wood chipping machine during a municipal cleanup of garden waste. Wood debris sampled directly from the chipper blade yielded 18 CFU/g of C. gattii, and an air sample taken at the outlet of the chipper yielded 19 CFU/m3 of C. gattii. Woodchips obtained from this service are used primarily to cover trails in the local parks . These data indicate that forestry activities and the distribution of tree byproducts may facilitate the mobility of C. gattii through both aerosolization and mechanical dispersal.A log and a sample of wood chips from 1 of the removed trees were retained in the laboratory for 2 years, stored in sealed plastic bags at room temperature. Air samples taken close to the log after 1 and 2 years detected 25 CFU/mC. gattii infection in domestic cats and dogs in BC  and could potentially be aerosolized through both large- and small-scale soil disturbances such as deforestation, landscaping and gardening, vehicles traveling on dirt roads, or rain splash . The unique opportunity for investigation of C. gattii as an emerging pathogen in BC has facilitated insight into the ecology and distribution of this pathogen. We believe the dispersal mechanisms of C. gattii could be applied as a model for other organisms.The emergence of C. gattii dispersal discussed here are similar to those described in a number of reports for other mammalian pathogens. For example, Coccidioides immitis is similar to C. gattii in that it primarily colonizes soil, and disease is acquired by inhaling aerosolized spores. Coccidioidomycosis outbreaks have occurred as a result of soil disturbances as well as windborne dispersal. Such outbreaks were documented after a California earthquake (The mechanisms of Blastomyces dermatitidis is the cause of blastomycosis outbreaks in humans and animals in Wisconsin (C. gattii from bodies of water in BC. B. dermatitidis infection has also been reported in 2 captive California sea lions in the adjacent US states of Wisconsin and Illinois (C. gattii may be important, including the detection of multiple C. gattii strains in the wheel wells of vehicles and on footwear. Footwear and farm vehicles were found to be involved in mechanical transmission of porcine reproductive and respiratory syndrome virus in Minnesota (Phytophthora ramorum, causing sudden oak death in North America and Europe, is mediated by human activity as well as natural factors (We have compiled evidence that suggests human-mediated dispersal of C. gattii introduction to the Pacific Northwest has not been established, although 1 hypothesis implicates importation of contaminated trees (C. gattii could also have been introduced by mechanical vectors such as vehicles or footwear, or by wooden pallets or crates (C. gattii dispersal through passive transport as well. Certainly, a large number of migratory birds pass through the disease-endemic area on Vancouver Island.A specific route of C. gattii, until more study is done on conditions favoring or inhibiting de novo colonization, attempts to restrict further dispersal of the organism would be impractical and unlikely to succeed. However, public health, medical, and veterinary personnel, as well as the public in the Pacific Northwest and elsewhere, must be made aware of C. gattii and symptoms of infection so the disease can be identified correctly and managed effectively.Given numerous possibilities for dispersal of


Eidolon helvum to both Nipah  and Hendra  viruses. Virus neutralization tests further confirmed seropositivity for 30% (7/23) of Luminex positive serum samples. Our results indicate that henipavirus is present within West Africa.Henipaviruses are emerging RNA viruses of fruit bat origin that can cause fatal encephalitis in man. Ghanaian fruit bats (megachiroptera) were tested for antibodies to henipaviruses. Using a Luminex multiplexed microsphere assay, antibodies were detected in sera of  Pteropus spp. fruit bats identified as their reservoir hosts Henipaviruses are emerging fatal zoonotic RNA viruses with Pteropus spp. bats was assumed to limit henipavirus distribution. In Madagascar, however, henipavirus antibodies were found in the non-pteropid bats, Eidolon dupreanum, and Rousettus madagascariensis, but both species were sympatric with seropositive pteropid bats The distribution of E. helvum, with 23 of 59  showing reactivity to henipavirus: 23 showed reactivity to NiV  and 13 showed reactivity to HeV . All the HeV-seropositive bats showed reactivity to both viruses. Of the 23 E. helvum samples seropositive using the Luminex binding assay, seven were positive using virus neutralization tests (VNTs) .The numbers of each species of bat tested and the serology results using a Luminex binding assay E. gambianus  and H. monstrosus  gave positive readings for NiV using the Luminex binding assay, but gave negative results with the VNT. No other bats gave seropositive results for henipavirus infection.One serum sample from each of E. helvum tested were positive to both HeV and NiV , but for HeV, females were marginally significantly more likely to be seropositive .Many of the  and NiV . The degE. helvum, we attempted to determine a possible case reproduction rate (R0) for henipavirus infection in this species using:As a high level of seropositivity was detected in 0 = 1.6 (95% CI: 1.3–2.0).This attempt assumes that infection with henipavirus within the bat population is endemic, stable, and randomly dispersed, that lifelong immunity is detectable serologically and all seropositive animals have lifelong immunity and that seropositivity is to a single virus (Luminex binding assay results for NiV were used). Based on these assumptions, RE. helvum fruit bats in Ghana, providing the first evidence of henipavirus infection in Africa. Mainland Africa has no Pteropus spp. fruit bats E. helvum is widely distributed across sub-Saharan Africa. It is known to inhabit the equatorial tropical forest region and is believed to migrate into savannah regions annually Pteropus species would indicate that a member of the genus Henipavirus has spread through, or is circulating in, the E. helvum population in Accra, the capital city of Ghana. The identity of the infecting virus (or viruses) that elicited the antibody response to henipavirus remains unknown.We found seropositivity to henipavirus in E. helvum antibodies. It is worth noting that most of the serum samples gave a higher reading for NiV than HeV, indicating the virus(es) circulating in the West African E. helvum populations is more NiV-like. The seropositive animals were apparently healthy. This suggests that E. helvum might be similar to pteropid bats, surviving infection and possibly acting as a reservoir host.As in studies of henipaviruses in pteropid bats H. monstrosus had an MFI just above the cut-off, and not enough individuals were caught to evaluate seroprevalence. Epomophorus gambianus, however, was caught in large numbers and the seropositive individual exhibited a high binding MFI to NiV.Two bats from other species were seropositive for NiV using the Luminex binding assay, but were negative using the VNT. The sample from E. helvum sera, henipavirus seropositivity was confirmed in around one third of the Luminex positive samples using VNT. The lower seroprevalence detected using VNT is most likely due to a low level of antibodies circulating in the bat population . The fluorescence-based Luminex assay is much more sensitive than the conventional VNT, and hence is expected to pick up more positive samples. Another possibility is the presence of more than one henipavirus species in E. helvum, antibodies to one of which are unable to neutralise either HeV or NiV, but can cross react with their G proteins in the Luminex assay. This is less likely due to the fact that the G proteins of henipaviruses are responsible for receptor binding and, as in most other paramyxoviruses, the target of neutralizing antibodies. It is therefore expected that G-reactive antisera would neutralize live virus if their G-reacting antibodies are at a sufficiently high level.Although the Luminex binding assay used in this study has not been stringently validated with bat sera due to lack of the required number of known positive and negative bat sera from different species, it has been extensively tested with known positive and negative sera of other species and shown to perform better than conventional ELISA-based binding assays in terms of both sensitivity and specificity E. gambianus and E. helvum raises the question as to whether this is a feature of species susceptibility or of species ecology. E. helvum lives in large, densely populated colonies numbering hundreds of thousands: the urban colony sampled in this study comprised at least 500,000 bats. This is in comparison to the much smaller (tens or hundreds) and less dense colonies of E. gambianus, thus populations of this species would be less likely to sustain henipavirus infection. There is, however, genetic evidence that Eidolon spp. differ from other African fruit bats Eidolon. The Eidolon genus has been found to be more closely related to other genera, including Pteropus, than to the other proposed African clades; African Rousettus, Epomophorine and Myonycterine The difference in henipavirus seroprevalence between 0 value for henipavirus in the E. helvum colony sampled in this study was higher than those estimable from other seroprevalence datasets for henipavirus in pteropid bats Although caution is needed in interpretation, the RPteropus spp. fruit bats. An additional priority for future research is the strengthening of medical surveillance for encephalitis in Africa and the investigation of henipavirus involvement in patients suffering from encephalitis, particularly where alternative diagnoses, such as rabies and cerebral malaria, have not been confirmed.Further to the serological results obtained in this study, work is now underway to confirm the presence of henipavirus infection in African bats using reverse transcriptase PCR and virus isolation. This is also necessary to characterize the virus(es) concerned and to make comparisons with henipaviruses found in Australian and Asian E. helvum in Ghana poses interesting questions regarding henipavirus ecology within African bat populations and its potential for zoonotic emergence. E. helvum is widely distributed across sub-Saharan Africa where it commonly forms extremely large colonies in close proximity to both man and domestic animals. E. helvum is also a common and important source of bushmeat in West Africa, thus presenting another possible conduit for zoonotic emergence.In conclusion, serological evidence for henipavirus infection in E. helvum, using nets on poles. Up to 1% of body weight of blood was taken from the propatagial vein prior to release. A total of 206 bats of six species were caught, sampled and tested , woodland on the outskirts of Accra (savannah habitat), and in forest habitat at Pra, Kibi, Adoagyiri and Oyibi. The last sampling site was in a plantation along a woodland/forest border. All sites were within 180 km of each other. Bats were captured either using 6–18 m mist nets or, for roosting d tested .Epomophorus gambianus (n = 89) and E. helvum (n = 59), were tested in sufficient numbers for reasonable inferences to be made about seroprevalence rates: 59 being the sample size required to have 95% confidence of finding at least one seropositive in a large population given a 5% seroprevalence, assuming random sampling E. helvum samples were derived from the colony in central Accra, whereas E. gambianus was sampled across all habitats. We assumed the sampled E. gambianus and E. helvum were from single metapopulations.Two species, Sera were tested for antibodies binding to the recombinant HeV and NiV G proteins in a Luminex multiplexed binding assay


Clinicians in Asia should consider this disease when diagnosing acute febrile illnesses. –4 substitutions/site/year, which is similar to rates estimated for mosquito-borne flaviviruses. KFDVs were estimated to have shared a common ancestor in ≈1942, fifteen years before identification of the disease in India. These data are consistent with the view that KFD represented a newly emerged disease when first recognized. Recent common ancestry of KFDVs from India and Saudi Arabia, despite their large geographic separation, indicates long-range movement of virus, possibly by birds.Kyasanur Forest disease virus (KFDV) is enzootic to India and maintained in ticks, mammals, and birds. It causes severe febrile illness in humans and was first recognized in 1957 associated with a high number of deaths among monkeys in Kyasanur Forest. Genetic analysis of 48 viruses isolated in India during 1957–2006 showed low diversity (1.2%). Bayesian coalescence analysis of these sequences and those of KFDVs from Saudi Arabia and the People’s Republic of China estimated that KFDVs have evolved at a mean rate of ≈6.4 × 10 Flaviviridae and genus Flavivirus  and the red-faced bonnet monkey (Macaca radiata).Kyasanur Forest disease virus (KFDV) is a member of the mammalian tick-borne virus group  of the family >30%  of Shimoga District . By 1972More recent studies have identified KFDV in Saudi Arabia and the People’s Republic of China  per the manufacturer’s protocol. RNA was dissolved in 50 μL of nuclease-free water. cDNA was prepared separately for structural genes and NS5 by using avian Moloney virus reverse transcriptase . Briefly, 10 pM of each gene-specific reverse primer  and incubated at 42°C for 45 min and then 85°C for 5 min.2, and 10.0 pM of each primer pair as described in cDNA was amplified by using 1U of Taq DNA Polymerase, 10× PCR buffer , 0.2 mmol/L dNTP, 1.5 mmol/L MgClThe quality of each sequence was monitored by using Sequence Analysis software version 5.1 (Applied Biosystems). Sequences were assembled by using Kodon software version 2.1 . Sequences were processed to give 720 nt of the structural gene (nt positions 500–1220) and 620 nt of the NS5 gene (nt positions 9440–10080) and submitted to the National Center for Biotechnology Information  NCBI . Multiple sequence alignments were generated by using the MAFFT function  for the KFDV dataset. Final data analysis included a Markov chain Monte Carlo chain length of 50,000,000 generations with sampling every 1,000 states.The Modeltest 3.7 software program , and rodents (Rattus rattus wroghtoni and R. blanfordi)  and NS5 (620 nt) genes of 48 KFDV isolates from India collected over the past 5 decades  showed aanfordi) . The seqAll KFDV isolates from India differed from the Alkhurma variant of KFDV (–4 substitutions/site/year (95% highest probable density [HPD] 4.1–8.8 × 10–4 substitutions/site/year). This estimate is similar to rates for other flaviviruses analyzed by using similar Bayesian coalescent methods, including a rate of 2.17 × 10–4 substitutions/site/year obtained for 23 St. Louis encephalitis viruses collected during 1933–2001 , the isolates from Saudi Arabia (1995–2004) . These v2 in Sagar and Sorab taluks of Shimoga District. However, after 1972, epizootics and epidemics were recognized in several new foci, increasingly more distant from the original focus.Analysis estimated that the mean time to the MRCA for all the KFDV isolates was only 64 years (95% HPD 51–84 years) before 2006 (the year when the most contemporary virus was isolated). The analysis estimated that these viruses shared a common ancestor as recently as ≈1942. Analysis of only KFDV isolates from India provides a slightly more recent estimate of their MRCA (≈1948), just 9 years before identification of the disease in Kyasanur Forest in 1957. This finding correlates well with the perception of local villagers and healthcare providers in the Kyasanur Forest area that this was a newly emerged disease . Attempts to examine the relationship between genetic differences in a virus isolate relative to geographic location did not show any notable findings because of small differences and distances involved. However, the 1972 virus 72166 was isolated from a tick in the village of Gadgeri in Sirsi (Uttara Kannada District), which is north of Shimoga District. The 2006 virus A106 was isolated from a person south of Shimoga District, in Mangalore (Dakshina Kannada District) and further from the original virus epicenter . AlthougBayesian analysis estimates that the 1995 and 2004 KFDV isolates from Saudi Arabia shared a common ancestor in 1992. The node connecting these viruses with the 2006 KFDV isolate from India was in 1977, and a strongly supported node (1.0) shows that the 1972 and 2006 KFDV isolates from India shared a common ancestor with the viruses from Saudi Arabia in ≈1969. The simplest interpretation of these data and the epidemiologic observations would be that KFDV was introduced from India into Saudi Arabia in the late 1970s or the 1980s.Similar findings of low genetic diversity and recent common ancestry were reported for the KFDVs from Saudi Arabia in a more limited study of 11 virus isolates collected over a 5-year period (1994–1999). Only 0.4%, 0.6%, and 0.9% genetic diversity were found in the E, NS3, and NS5 gene fragments of these isolates, respectively  and amplifying (vertebrate) hosts, including rodents and shrews, and possibly monkeys and cattle. Increased human populations in the Sagar and Sorab taluks in the early 1950s may have been the primary catalyst for emergence of KFD in 1957. During 1951–1961, the population of Sagar Taluk increased 116%, bringing with it increases in deforestation, cattle grazing, and extension of paddy fields and cleared grazing areas deeper into previously forested areas (H. spinigera ticks, and an association between cattle and increases in tick larval density has been described (Haemaphysalis spp. ticks, which have been shown to be infected with KFDV. Thus, cattle would likely increase tick densities in cleared forest areas most frequented by humans. In addition, rats, shrews, and mice are highly susceptible to KFDV infection, and numerous virus isolates have been obtained from organs of infected animals (It is unclear what factors influenced the apparent emergence of KFDV in Shimoga District, India, in 1957 and in the Makkah/Jeddah region in Saudi Arabia in 1994. Also unknown is how this tick-borne virus moved over the large distance between these regions. A considerable amount of knowledge has been accumulated with regard to the ecology of KFDV in India (Haemaphysalis spp. and other tick genera, particularly larvae and nymphs (A high percentage of birds in the affected area are positive for antibodies reactive with KFDV and infested with The current known distribution of KFDV is limited to relatively restricted areas of India and Saudi Arabia. However, it is likely that the virus exists in other areas in cryptic enzootic cycles or is associated with unrecognized or undiagnosed disease. This finding, together with the distance separating the KFDV-affected areas in India and Saudi Arabia, despite their relatively recent common ancestry, suggests that KFD has the potential to flare up in other regions because of virus movement or ecologic changes in the area. Clinicians should consider KFD in a differential diagnosis when considering acute febrile cases with compatible symptoms in other regions of Asia and the Middle East.


Although rhTRIM5α blocks accumulation of reverse transcription products, proteasome inhibitors rescue reverse transcription while maintaining a block to infection, supporting the involvement of the proteasome in the mechanism of restriction.First, rhTRIM5α was examined by immunofluorescence and characterized with specific ubiquitin antibodies. Next, mutations were made in rhTRIM5α to prevent involvement in the ubiquitin-proteasome pathway, and mutants were analyzed by immunofluorescence, flow cytometry and real-time PCR. A mutant virus was constructed to prevent ubiquitination of capsid, and used to infect cells expressing rhTRIM5α to characterize restriction and reverse transcription. Finally, a fluorescently tagged subunit of the proteasome, LMP2-GFP, was co-expressed with rhTRIM5α and analyzed by fixed and live cell microscopy.Staining with specific ubiquitin antibodies revealed that rhTRIM5α cytoplasmic bodies contained polyubiquitinated proteins, but proteasome inhibitors decreased polyubiquitinated proteins within cytoplasmic bodies. rhTRIM5α mutants showed reduced restriction of HIV-1, and localized to cytoplasmic bodies with different patterns of ubiquitination. The mutant virus exhibited reduced infectivity and an intermediate phenotype with regard to the block of reverse transcription. Analysis of fluorescently tagged proteasomes in cells expressing rhTRIM5α showed that proteasomes were recruited to rhTRIM5α cytoplasmic bodies in the presence of virus, and live cell microscopy revealed interactions between virus and proteasomes.The relocalization of polyubiquitinated proteins upon proteasome inhibition may indicate an efflux of polyubiquitinated proteins, implicating cytoplasmic bodies in the process of ubiquitination. Analysis of rhTRIM5α mutants revealed that rhTRIM5α may play a partial role in conjugating and receiving ubiquitination. The mutant virus showed that ubiquitination of capsid is not required for rhTRIM5α restriction, but reverse transcription is affected. Finally, proteasomes are recruited to rhTRIM5α cytoplasmic bodies in the presence of virus, which may lead to proteasomal destruction of rhTRIM5α-virus complexes.