Avian H5N1 influenza viruses present a challenge in the laboratory environment, as they are difficult to collect from the air due to their small size and relatively low concentration. In an effort to generate effective methods of H5N1 air removal and ensure the safety of laboratory personnel, this study was designed to investigate the characteristics of aerosolized H5N1 produced by laboratory manipulations during research studies.Normal laboratory procedures used to process the influenza virus were carried out independently and the amount of virus polluting the on-site atmosphere was measured. In particular, zootomy, grinding, centrifugation, pipetting, magnetic stirring, egg inoculation, and experimental zoogenetic infection were performed. In addition, common accidents associated with each process were simulated, including breaking glass containers, syringe injection of influenza virus solution, and rupturing of centrifuge tubes. A micro-cluster sampling ambient air pollution collection device was used to collect air samples. The collected viruses were tested for activity by measuring their ability to induce hemagglutination with chicken red blood cells and to propagate in chicken embryos after direct inoculation, the latter being detected by reverse-transcription PCR and HA test. The results showed that the air samples from the normal centrifugal group and the negative-control group were negative, while all other groups were positive for H5N1.Our findings suggest that there are numerous sources of aerosols in laboratory operations involving H5N1. Thus, laboratory personnel should be aware of the exposure risk that accompanies routine procedures involved in H5N1 processing and take proactive measures to prevent accidental infection and decrease the risk of virus aerosol leakage beyond the laboratory. The pandemic H1N1 outbreak of 2009 and global threat of H5N1 in recent years have been accompanied by a large amount of laboratory-based experimental research activity using purified viruses and infected tissues and animals. The majority of these studies have focused on either natural infections from the community or induced infections in the laboratory, with very few studies considering the infection risk or outcome of laboratory personnel handling the samples. The potential of an accidental laboratory-acquired infection is well-recognized among laboratory staff and researchers. In 1941, Meyer and Eddie published the first report of laboratory infections due to the Gram-negative bacteria Brucella -9.The total number and relative frequency of bacterial laboratory-acquired infections has, in fact, declined dramatically over time -12. In cResearch into this theory has indicated that influenza virus transmission and infection can be achieved through aerosols . Many ofThe A type influenza viruses are commonly spread by the airborne route in normal circumstances. Accordingly, more research on the potential and character of aerosol spread of influenza virus has been carried out -27, and The Australian researcher, Adrian Gibbs, suggested that the A/H1N1 flu virus currently circulating around the world was created in a laboratory. Although the World Health Organization (WHO) eventually dismissed this theory, suspicion and panic were aroused in the general public about laboratory safety. There are many situations that may facilitate the spread of a pathogen from the laboratory, ranging from aerosols produced by routine procedures or misuse of laboratory equipment to uncontrollable natural disasters that impact the structural integrity of the laboratory, such as earthquake or fire. In order to regulate the potential of pathogen transmission from the laboratory, we must first gain a detailed understanding of the experimental operations that produce aerosols. To this end, this study was designed to monitor the presence of aerosolized H5N1 virus produced by normal procedures used to process the virus for experimental research and by the most frequently associated “accidents” for each, such as container breakage and accidental subcutaneous injection. This information will help to guide future experimental practice standards to ensure the safety of laboratories and laboratory personnel, thereby increasing community confidence in laboratory bio-safety.The prototype sampling device, shown in Figure At the start of each group experiment, the sampler was operated for 30 minutes to obtain baseline readings of the laboratory atmosphere. Collection of aerosols from the seven groups of experimental procedures and controls followed the steps detailed in the Materials and Methods section. The processing time was 30 min for each group, except for group I (time was 1 h), and the air sampling distance was 38 cm from the working materials. Statistics of the group temperature and relative humidity for each of the data collection processes, including operations and control of the collection, were measured automatically by the aerosol collector.None of the aerosol samples collected from any group at the time directly prior to processing of the experiment had detectable levels of H5N1, as evidenced by negative reverse transcription (Rt)-PCR and HA text results. All liquid aerosols collected from the experimental procedure of group III were also negative for H5N1 Table . The aerOur experiment was carried out in a negative pressure isolation unit with mechanical arm Figure .The three groups of procedures with simulated accidents  all produced aerosols. The controls for each were all negative. All of the aerosols that were produced contained H5N1 that was detectable by Rt-PCR and HA text Table .The field of laboratory-acquired viral infections has progressed since Sulkin and Pike’s seminal report in 1949 , but theIn fact, all of the studies performed to date by others to determine whether laboratory studies of pathogens, including viruses, bacteria and fungi, could generate infective aerosols have been based on statistical analysis of actual infections. The influenza viruses present a particular challenge to monitoring in the lab by traditional methods, since their small size and low concentration make them difficult to collect and detect from the air. Most of the bioaerosol samplers currently on the market are not suitable for collection of viruses . Furtherin vivo, indicating the infectivity of the contaminating pathogens. Future studies will aim to determine the feasibility of this approach for quantifying virus in the aerosol.Measuring infectious virus from air samples is logistically difficult. Few researchers have reported airborne influenza virus from the laboratory, and even fewer have detected the infectious capacity of influenza viruses sampled from air. Traditional methods of determining the presence of virus in an aerosol include directly applying the concentrated aerosol to infect a host cell, or as template in Rt-PCR to detect virus-specific genes. One of the most commonly used methods is Rt-PCR because of its remarkable sensitivity and specificity; however, Rt-PCR is unable to determine whether the detected virus is infectious. In our study, we combined experimental approaches that would determine both the concentration of virus in aerosols and the activity of those viruses. By exploiting the functional character of the hemagglutinin of influenza virus, we were able to estimate the concentration of influenza virus in aerosols according to the amount of adsorption that occurred with red blood cells (RBCs). By also using the aerosols to directly inoculate chick embryos, we were able to determine the ability of the aerosolized virus to proliferate This study, to our knowledge, represents the first successful attempt to directly detect influenza virus-containing aerosols generated by routine laboratory procedures. Our results provide evidence that many of the laboratory techniques used to process influenza virus for experimental analysis produce aerosols and, thereby, represent significant risks of infection to laboratory personnel and potential spread beyond the laboratory. Working in the laboratory is inevitably dangerous, but nearly all risks can be sufficiently minimized by GLP and careful monitoring of risk factors, such as presence of pathogen-containing aerosols. According to the results of this study, we plan to extend our investigations to emergency operating procedures that follow laboratory accidents with virus samples and to generate more effective strategies to prevent laboratory-generated aerosols and laboratory-acquired infections or spread.In summary, our findings demonstrated that many of the laboratory techniques used to process influenza virus produce aerosols, either through normal operations or common accidents associated with each process. Our results have great value and implications towards bio-safety and future strategies to evaluate risks of experimental virology to laboratory workers and the general public.-1 in phosphate buffered saline  supplemented with free calcium and magnesium. Aliquots of this stock solution were stored at −80°C until use. Prior to experimentation, virus was propagated by infection in chicken eggs, and yield was determined to be 6.7 log10 50% egg infection doses per milliliter (EID50/mL). All the experiments on animals were approved by the Ethics Committee of Animal Science and Veterinary Medicine of Jilin University, First Hospital of Jilin University and Academy of Military Medical Sciences, in accordance with guidelines of the Nation Health and Medical Research Council of China.Purified avian influenza virus (A/chicken/Jilin/9/2004(H5N1), GenBank: AY653193 ~ AY653200) was obtained from our laboratory stock, and diluted 10-1, when the pressure drop across the orifice was 41 cm Hg. The tip of the capillary stem was situated 30 mm from the flask bottom; therefore, when filled with 20 mL of liquid, the nozzle outlet was 10 mm above the resting liquid surface. Airborne biological particles drawn into the 1 mm diameter nozzle and down the capillary stem, then impacted and penetrated the liquid surface [Aerosol collection was carried out with the micro-cluster sampling device that was designed by the Academy of Military Medical Sciences . Airborne biological particles were gathered using liquid impingers  that rely on inertial collection mechanisms. The flow rate was 12.5 L min surface . The adh surface .All assays were conducted in a Biosafety Level 3 setting. The containment laboratory – Biosafety Level 3 – was designed for work with Risk Group 3 microorganisms in large volumes and Risk Group 2 microorganisms at high concentrations. In addition, the Biosafety Level 3 containment laboratory was equipped with a negative pressure isolation unit, which was used in our study for simulating accidents during laboratory procedures.Normal laboratory procedures were carried out under controlled conditions for the purpose of monitoring the amount and character of aerosol produced. In group I (zootomy), chickens diagnosed with AIV upon autopsy were sectioned and the excised tissues frozen at −20°C. The total collection time was 1 h. Disinfection of the anatomical units after collection and air sampling took an additional 1 h. Control air samples were taken in the same units one day later. This process was repeated a total of six times, and six chickens were necropsied. In group II (grinding), the frozen lung tissues were thawed, ground in a mortar, and transferred to a centrifuge tube for storage at 4°C. Aerosol collections were carried out during grinding (at 20 min into the procedure) and 10 min after the homogenate was collected, for a total of 30 min. After disinfection of the area, air samples were immediately collected. This entire process was repeated six times. In group III (centrifugation) the tissue homogenates were centrifuged at 5000 rpm for 20 min at 4°C. Air samples were collected from the point of initial handling of the centrifuge tube to the centrifuge to the end of the spin when the centrifuge instrument was opened and the tube removed. To balance the centrifuge for each spin, the six homogenates were processed in groups of two, so that the experiment was repeated three times. Air sampling was performed after disinfection for use as the control. Collection times were 30 min. In group IV (pipetting), the supernatant resulting from the centrifugation step was transferred by a pipettor to a new tube, using blow-out and pull-in processes a total of 10 times to mix each sample. To ensure continuity of the experimental integrity, the pipetting experiment was carried out in another similar laboratory room that did not communicate with the centrifuge room. The same air sampling procedure was carried out for 30 min during the experiment and after disinfection. Similar to group III, the procedures of group IV were repeated three times. In group V (magnetic stirring), the virus-containing supernatant samples were diluted with PBS (pH 7.2) in a 10-fold series. This occurred by taking 1 mL of supernatant and adding to PBS in a 9 mL beaker with a stir bar that was placed on an active magnetic stirrer. Air sampling occurred throughout the entire process and after disinfection of the space, three times at 30 min each. In group VI (egg inoculation), 10 chick embryos (about 9-days-old) were inoculated with 0.2 mL diluted virus sample (10-fold diluted virus stock solution) per embryo. Air samples were collected throughout the process, including the time when the virus was drawn into the syringe, air bubbles were removed from the syringe, chick embryos were injected, and sealing wax was applied. A total of 10 chicken embryos were inoculated in each air monitoring experiment. Air samples were collected throughout and after disinfection, taking 30 min. The procedure was repeated three times, requiring a total of 30 chick embryos to be inoculated. In group VII (zoogenetic infection), five chickens (7-days-old) was intranasally administered virus at a dose of 0.2 mL per chicken. The zoogenetic infection experiments were repeated three times. Air sampling was carried out at intervals of 30 min each, and after disinfection.Laboratory simulation of accidents that most frequently occur during the routine processing of influenza viruses were carried out inside a negative pressure isolation unit equipped with a simple robotic arm and operating gloves. In accident group I (broken glass containers), glass containers holding influenza virus suspensions were dropped in a free-fall and the broken pieces collected as in a routine clean-up procedure. In accident group II (syringe-ejected influenza virus suspension), 3 mL of the virus dilution (10-fold diluted virus stock solution) was drawn into a disposable syringe, and the plunger was depressed to spray out a small amount of the virus suspension into the air. In accident group III (centrifuge tube rupture), empty microcentrifuge tubes were centrifuged at inappropriate speeds to produce cracked tubes. Then, 1 mL of virus solution was added to the tube and the tube was closed and manually squeezed until the tube ruptured and the liquid splashed out, at which point air samples were collected. All of the processes for the three groups were repeated five times. For each, air samples were collected before the procedure for use as the control. The collection time was 30 min.After virus propagation in chicken eggs, the allantoic fluid was collected and processed according to standard procedures for detections of AIV by Rt-PCR and HA text, both detections were repeated twice. The sensitivity of the Rt-PCR procedure to detect AIV was evaluated by using a 10-fold diluted virus series; AIV was detectable by this method up to 10  dilutionThe processing of samples collected by the air monitor was based on well-known hemagglutination properties of H5N1 . BrieflyTreatment of erythrocytes. Chicken blood was divided into several tubes and centrifuged at 3000 rpm for 10 min; the resultant supernatant was carefully removed by pipetting and mixed with sterile PBS (pH 7.2). Three PBS washing steps by centrifugation were carried out. Finally, the erythrocyte sediment was diluted with PBS at a 1:20 (vol/vol) ratio and 2.5% glutaraldehyde was added at a 4:1 (vol/vol) ratio and the solution was oscillated at room temperature for 45 min. The treated erythrocytes were washed four times with PBS by centrifuging (3000 rpm for 5 min each), and finally resuspended in PBS at 2% (treated erythrocytes/ volume of PBS) and stored at 4°C until use.Treatment of sample. Samples collected from experimental groups and their respective controls were incubated on ice to chill. Then, treated erythrocytes were added at a 1:2.5 (vol/vol) ratio. Adsorption was allowed to proceed for 1 h at 4°C, during which time the sample was intermittently mixed by inverting the tubes several times. After the incubation, samples were centrifuged at 5000 rpm for 10 min at 4°C, and the supernatant was discarded. The sediment was resuspended with 1 mL PBS on ice and recentrifuged at 5000 rpm for 10 min at 4°C, after which the supernatant was discarded. The sediment was then resuspended in a mixture of 0.1 mL amb-antibacterial media  and 0.1 mL PBS. The mixture was incubated for 1 h at 37°C with intermittent mixing by inverting the tubes several times. The entire volume (0.2 mL) of each sample was then used to inoculate a 9-day-old embryonated chicken egg. The inoculated eggs were incubated for 72 h at 37°C and then chilled overnight at 4°C. The allantoic fluid from each egg was collected, except in the cases where the embryo had died within 24 h after inoculation.A standard Rt-PCR test was performed to confirm the presence of the virus in allantoic fluid. A total of 0.2 mL of the allantoic fluid was used for RNA extraction by the Qiagen RNeasy Mini Kit (Cat No. 75182) and then applied to a reaction mix using reagents from the one-step Rt-PCR amplification Mini Kit (Qiagen Cat No. 210212), as follows: initial heating step, 95°C for 15 min; reverse transcription, 55°C for 30 min; initial PCR activation step, 95°C for 15 min; 35 cycles of amplification ; final extension, 72°C. Gene-specific primers for the NP gene of H5N1 were: NP-Forward, 5′-GCATTGTCTCCGAAGAAATAAG-3′ and NP-Reverse, 5′-CAGATACT GGGCHATAAGRAC-3′. The expected length of the amplicon was 320 bp.The allantoic fluid (positive by Rt-PCR) was detectable by hemagglutination test that performed to confirm the presence of the virus. Hemagglutination test was performed in 96-wells microtiter plates with 1% CRBCs.Rt-PCR, Reverse-transcript polymerase chain reaction; HA, Hemagglutination; GLP, Good laboratory practice; WHO, World Health Organization; AGI30, All Glass Impinger 30; CRBC, Chicken red blood cell.The authors declare that they have no competing interests.ZL carried out most of the experiments and wrote the manuscript. JL, ZX and ZL designed the equipment. ZD, DL, LM and LL participated in the planning of the project. DL, LM and LL collected the samples. ZL and ZD performed the Rt-PCR, analysis and interpretation of data. FG and ZX conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.


Herpesviridae, Flaviviridae, Circoviridae, Anelloviridae, Asfarviridae, and Parvoviridae families. In some cases, the putative viral sequences were virtually identical to known viruses, and in others they diverged, suggesting that they may derive from novel viruses. These results demonstrate the utility of unbiased metagenomic approaches in the detection of known and divergent viruses in the study of tropical febrile illness.Dengue virus is an emerging infectious agent that infects an estimated 50–100 million people annually worldwide, yet current diagnostic practices cannot detect an etiologic pathogen in ∼40% of dengue-like illnesses. Metagenomic approaches to pathogen detection, such as viral microarrays and deep sequencing, are promising tools to address emerging and non-diagnosable disease challenges. In this study, we used the Virochip microarray and deep sequencing to characterize the spectrum of viruses present in human sera from 123 Nicaraguan patients presenting with dengue-like symptoms but testing negative for dengue virus. We utilized a barcoding strategy to simultaneously deep sequence multiple serum specimens, generating on average over 1 million reads per sample. We then implemented a stepwise bioinformatic filtering pipeline to remove the majority of human and low-quality sequences to improve the speed and accuracy of subsequent unbiased database searches. By deep sequencing, we were able to detect virus sequence in 37% (45/123) of previously negative cases. These included 13 cases with Human Herpesvirus 6 sequences. Other samples contained sequences with similarity to sequences from viruses in the  a priori sequence, serological, or cellular tropism information is not known. In our study, we use two approaches, microarrays and deep sequencing, to virus identification that are less susceptible to such shortcomings. We used these unbiased tools to search for viruses in blood collected from Nicaraguan children with clinical symptoms indicating dengue virus infection, but for whom current dengue virus detection assays yielded negative results. We were able to identify both known and divergent viruses in about one third of previously negative samples, demonstrating the utility of these approaches to detect viruses in cases of unknown dengue-like illness.Dengue virus infection is a global health concern, affecting as many as 100 million people annually worldwide. A critical first step to proper treatment and control of any virus infection is a correct diagnosis. Traditional diagnostic tests for viruses depend on amplification of conserved portions of the viral genome, detection of the binding of antibodies to viral proteins, or replication of the virus in cell cultures. These methods have a major shortcoming: they are unable to detect divergent or novel viruses for which  Viral infections pose a significant global health burden, especially in the developing world where most infectious disease deaths occur in children and are commonly due to preventable or treatable agents. Effective diagnostic and surveillance tools are crucial for reducing disability-adjusted-life-years (DALYs) due to infectious agents and for bolstering elimination and treatment programs Dengue virus (DENV) infection is the most common arthropod-borne viral disease of humans, with an estimated 50–100 million clinical infections occurring annually worldwide a priori sequence, antigen, or cell tropism knowledge. PCR-based assays that employ degenerate primers may successfully target conserved regions within related virus groups, but unlike bacteria, viruses lack universally conserved genetic regions, such as ribosomal RNA, that can be exploited to amplify all viruses Traditional viral detection methods, such as serology, virus isolation, and PCR, are optimized for the detection of known agents Metagenomic analysis enables more systemic detection of both known and novel viral pathogens This study describes the use of the Virochip microarray and deep sequencing for the direct viral diagnosis of serum from cases of acute pediatric febrile illness in a tropical urban setting. Patient clinical data and serum samples were collected between 2005 and 2009 as part of an ongoing pediatric dengue study in Managua, Nicaragua e.g. pneumonia, were excluded. Suspected dengue cases were tested for dengue virus (DENV) infection at the Centro Nacional de Diagnóstico y Referencia (CNDR) of the Nicaraguan Ministry of Health and were considered laboratory-confirmed if: 1) DENV was isolated, 2) DENV RNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR), 3) seroconversion was observed by IgM capture enzyme-linked immunosorbent assay (ELISA) of paired acute and convalescent sera, or 4) a ≥4-fold increase in DENV-specific antibodies was demonstrated by inhibition ELISA in paired acute and convalescent sera Acute serum samples were collected from suspected dengue cases at the Hospital Infantil Manuel de Jesús Rivera (HIMJR), the National Pediatric Reference Hospital in Managua, Nicaragua, after undergoing informed consent or the informed consent procedure. Patients were enrolled in the study if they presented with fever or history of fever less than 7 days and one or more of the following signs and symptoms: headache, arthralgia, myalgia, retro-orbital pain, positive tourniquet test, petechiae, or signs of bleeding. Patients with a defined diagnosis other than dengue, Approximately one half of the suspected dengue cases testing negative by all four dengue diagnostic assays were included in the metagenomics analysis described here. 34 cases  corresponded to the subset of patients who presented within 4 days of symptom onset and who reported both fever or history of fever and rash. 89 of the samples (pool 5) were selected randomly from among the remaining samples. As positive controls, seven samples (pool 5) that had been clinically diagnosed as virus positive were included. The study protocol was reviewed and approved by the Institutional Review Boards (IRB) of the University of California, Berkeley, and of the Nicaraguan Ministry of Health.5′-CGC TCT TCC GAT CTN NNN NN-3′), 100 U Superscript III (Invitrogen), and ∼20 ng template. Following reverse transcription, Sequenase reaction buffer and 2 U of Sequenase DNA polymerase (Affymetrix) were added to samples for second strand synthesis. The Sequenase reactions were performed twice so that starting DNA templates would be converted into end-tagged library molecules. The resulting libraries were amplified by PCR using primer 5′-CGC TCT TCC GAT CT-3′. PCRs contained 1× reaction buffer, 2 µM primer, 0.25 mM dNTPs, 2 U Taq DNA polymerase, and 2 µl library template. Thermocycling conditions were 95°C for 2 min; 25 cycles of 95°C for 30 sec, 40°C for 30 sec, and 72°C for 1 minute, with a final extension of 5 minutes. These libraries were further processed for microarray hybridization and deep sequencing as described below.Total nucleic acid from 140 µl of serum was extracted using the QIAamp Viral RNA Isolation Kit (Qiagen), which co-purifies RNA and DNA. End-tagged dsDNA libraries were created essentially as previously described For microarray hybridization, a fraction of each library was amplified by PCR as above but with a modified dNTP mixture including 5--dUTP (Ambion) in lieu of 75% of the dTTP normally in the mixture. The resulting amino-allyl-containing DNA was purified using a DNA Clean and Concentrator-5 column (Zymo Research). The eluate was heat denatured at 95°C for 2 min, cooled briefly on ice, then fluorescently labeled in reactions containing 100 mM sodium bicarbonate pH 9, 10% DMSO, and 667 µM Cy3 mono NHS ester  for 1 hour at 25°C. Labeled DNA was purified using DNA-CC-5 columns and added to hybridization reactions containing 3×SSC, 25 mM HEPES pH 7.4, and 0.25% SDS. Hybridization mixtures were heated at 95°C for 2 minutes, applied to microarrays, and hybridized overnight at 65°C. Following hybridization, arrays were washed twice in 0.57× SSC and 0.028% SDS and twice in 0.057× SSC, then scanned on an Axon GenePix 4000B microarray scanner. Three analysis tools were used to analyze Virochip data: E-predict For deep sequencing, the Illumina paired-end adapter sequences were appended to library molecules using PCR, essentially as previously described In some cases, PCR and Sanger sequencing was used to confirm Virochip and deep sequencing calls and to recover additional sequence. Primer sequences are listed in −2 to 10−6 poliovirus gRNA per HeLa RNA. Randomly-primed dsDNA libraries were prepared, hybridized to the Virochip, and analyzed as described above.Full-length poliovirus genomic RNA was transcribed from MluI-linearized plasmid prib(+)XpA using T7 RNA polymerase as previously described −2). Aligning sequences were retrieved and consolidated using CD-HIT into a set of representative sequences Predicted circovirus-like replicase sequences were searched against the NCBI non-redundant protein database . Remaining reads were next aligned to the human genome using BLASTn , filtered, and again aligned to the human genome by BLASTn . After all human filtering, we reanalyzed the distribution of the complexity of reads and observed a relative enrichment of reads with LZW ratios lower than 0.54 (pools1–4) or 0.48 , and those reads were removed from further analysis. To look for reads with viral homology, we searched the non-redundant nucleotide database (nt) using BLASTn . Reads that did not map to nt were aligned to the non-redundant protein database (nr) using translated BLAST .The initial FASTQ data from each pool's lane were binned by barcode. The barcode-split reads were trimmed of non-template deriving and potentially error-prone sequence: a randomly incorporated nucleotide (N), the barcode bases, and the sequence corresponding to the random hexamer, leaving 55 , 54 (pool 3), or 90 (pool 5) bases per read. The lowest complexity fraction was identified by sequences with LZW ratios (compressed size/uncompressed size) less than 0.45 In order to make specific virus-positive calls, we implemented a set of rules to minimize false positives while maintaining sensitivity. In order to reduce the number of false positive sequences that may share identity equally with both viral and non-viral genomes, we restricted our analysis to those queries whose best alignments were only to animal viral sequences. In a number of datasets, we detected human klassevirus 1, a virus identified and studied in our lab We initially screened the serum samples with the Virochip pan viral detection microarray. This was done as a complement to the deep sequencing analysis and in order to compare the sensitivity of the two approaches. We included 7 blinded positive control samples that had been previously diagnosed in the clinic as being positive for DENV-2 (n = 4), DENV-1 (n = 1), or hepatitis A virus . The Virochip successfully identified the correct virus in all of these positive controls, and in the case of the dengue virus positive samples, the correct serotype as well . We alsoin vitro transcribed poliovirus RNA diluted into HeLa cell total RNA to the Virochip as an additional positive control to quantify Virochip sensitivity. Using the E-predict analysis tool, the lowest detectable concentration of poliovirus was 1 viral RNA per 105 HeLa RNA molecules . We sequenced 96 serum samples (pool five) on a HiSeq 2000 instrument, which provides more, longer sequences per run. The HiSeq run generated 196.4 million 97-nt sequences .E. coli and S. cerevisiae.The raw reads were first separated by barcode and analyzed as individual data sets as described in the The reads remaining after filtering were then compared to sequences in the NCBI non-redundant nucleotide and protein databases using BLASTn and BLASTx respectively. Virus-derived sequences were detected in all 7 positive control samples and in 45/123 (37%) of previously negative serum samples . In 78/1We recovered virus sequences matching the expected viral genomes in all of the positive control samples. The fraction of viral sequences in the controls spanned 4 orders of magnitude, from 0.002% to 2.8% of total reads. The two HAV positive control samples (#401) were aliquots of the same serum sample and were processed and analyzed independently. The fraction of viral reads in the duplicates was within 4-fold (0.4% and 1.2%). This demonstrates that our library preparation, sequencing, and bioinformatics pipeline is capable of reproducibly detecting evidence of clinically relevant infections.In addition to the controls, two non-control samples contained evidence of RNA virus sequence. Both samples had reads deriving from GB Virus C  and were essentially identical to GBV-C database sequences. We detected no sequences that best aligned to dsRNA viruses or to retroviruses  sequence was detected in 13/123 previously negative samples (10.6%). The HHV-6 positive samples had an average normalized read count of 145 HHV-6 reads per sample (range: 24–411), representing 0.002% to 0.02% of the datasets , and allIn addition to HHV-6, we detected Human Herpesvirus 4  sequences in one sample. As with HHV-6, The HHV-4 sequences were virtually identical to previously reported sequences. One sample also contained reads similar to another dsDNA virus, African Swine Fever Virus (ASFV), which has been previously detected in human serum Anelloviridae virus family (TTVs) were detected in 21 (17%) samples. This frequency of detection is within the range reported previously for human serum We also identified sequences derived from single-stranded DNA viruses in some samples. In one sample we detected Parvovirus B19-derived reads with high identity to database sequences. Sequences related to various members of the Circoviridae family of ssDNA viruses were detected in 13/123 samples (10.6%). All of the sequences aligned to circovirus or circovirus-like replicase protein sequences. The alignments ranged from 36–84% amino acid identity, and appeared to derive from the replicase genes from multiple related species (Sequences similar to members of the  species . Circovi species . The low species .We termed the extended replicase-like sequences Circovirus-like NI/2007 1–3 (Cvl-NI 1–3), and compared them to a representative set of other replicase sequences . The CvlA subset of the positive samples  containeIn this study, we examined the virus diversity in serum samples from Nicaraguan children with unknown acute febrile illness. We performed Virochip microarray and deep sequencing analyses on 7 positive control and 123 undiagnosed samples. Both of these methods succeeded in detecting the expected virus in the positive control samples. Virochip analysis produced putative viral hits in 10/123 (8%) of the previously negative samples, whereas deep sequencing revealed virus or virus-like sequences in 45/123 (37%). This study demonstrates the utility of these metagenomic strategies to detect virus sequence in multiple human serum samples and is the first to utilize second-generation sequencing to simultaneously investigate many cases of acute unknown tropical illness.Monitoring the emergence and spread of novel human pathogens in tropical regions is a central public health concern. Metagenomic analysis enables more systemic viral detection of both known and novel viral pathogens 6 reads. Virus sequence detected in a clinical sample at vanishingly low copy numbers may reflect several possible host-microbe scenarios. The sequence detected may be that of a pathogenic virus capable of causing illness at low copy number or through indirect effects, a ubiquitous non-disease causing microbe, a virus outside of its primary replication site, low-level contamination, an artifact of sample collection timing/processing, or remains of incomplete immune clearance. Additional evidence must be considered in each case to define the host-microbe relationship.We detected virus sequence at concentrations as low as ∼2 in 105 for the poliovirus controls, for which there are microarray probes with perfect sequence complementarity , an acute illness with high fever and rash that typically resolves in three to seven days Exanthem subitum is a common disease of infants worldwide, and HHV-6 infection most frequently occurs between 6 and 12 months of age Determining the etiology of human diseases with symptoms that overlap with dengue-like illness is important for understanding the full spectrum of emerging or previously uncharacterized pathogens in tropical populations. In this study, 10% of acute serum samples negative for dengue virus from cases of pediatric dengue-like illness were positive for HHV-6. Primary HHV-6 infection causes undifferentiated febrile illness and After acute infection, HHV-6 can latently persist in the host quiescently, with no production of infectious virions or with low levels of viral replication. Latency is believed to endure in several cell types, including monocytes and bone marrow progenitor cells exanthem subitum, a ubiquitous self-limiting childhood illness, and dengue fever, which carries a greater risk of severe clinical complications and death.Primary HHV-6 infection is a major cause (∼20%) of infant hospitalizations in the United States erythema infectiosum (fifth disease), a condition associated with characteristic “slapped cheek” rash Similarly, the one sample positive for Parvovirus B19 sequence may be a case of acute infection with a commonly acquired childhood virus. Parvovirus B19 can manifest as Epstein Barr Virus (HHV-4) sequences were found in the serum of one patient who presented with relatively severe symptoms, and died during hospitalization . HHV-4 iIn addition to the viruses for which a plausible disease association exists, many samples contained sequences from viruses with no well-established link to human disease. These included the two samples positive for GBV-C and those containing ASFV-like, TTV-like, and circovirus-like sequences.Circoviridae family is an extraordinarily diverse group of small, single-stranded circular DNA viruses that includes cycloviruses (genus Cyclovirus) and circoviruses (genus Circovirus), which are commonly detected in human stool and blood, and also in environmental samples Giardia intestinalis or Entamoeba dispar, whose genomes encode proteins that share amino acid similarity with circovirus replicase proteins Click here for additional data file.Table S1This table lists the sequences of the oligonucleotides used in this study.(PDF)Click here for additional data file.Table S2This table lists the accession numbers of protein sequences used to construct the phylogenetic tree in .(PDF)Click here for additional data file.Table S3This table displays the clinical signs recorded during hospitalization.(PDF)Click here for additional data file.


Chitosan is a naturally occurring biopolymer which has been widely used in a variety of biomedical applications including local antibiotic delivery due to its excellent mechanical properties, biodegradability and biocompatibility. Beads are spherical, porous carriers which are prepared from various materials including chitosan.The current study aimed to fabricate a new controlled delivery system for local anti-infective treatment and to study its release behavior.Twenty beads were prepared from 1% or 2% chitosan solutions and immersed in vancomycin (VM) or teicoplanin (TN) solutions. The antibiotic release kinetics was determined by linear regression analysis supposing first order kinetics.Immersion for 3 h resulted in significant increase in the total TN release that differed from 0.5 h of immersion, except for the 1% beads immersed in VM. Increasing the chitosan concentration significantly increased the total release and antibiotic load of beads. The release of TN was more delayed compared to that of VM, which allowed a gradual release beyond 3 days. The half-life (mean ± SEM) of both types of TN-containing beads was significantly extended for 3 h immersion in comparison to 0.5 h immersion . However, neither increasing the chitosan concentration, nor immersion time did result in any significant increase in the release of VM.The current study demonstrated an improved control of TN release impregnated in beads. It can be concluded that chitosan beads might be considered as a novel carrier for TN delivery to infected bone for local anti-infective therapy. In spite of various viewpoints and concepts about the efficacy of biodegradable antibiotic beads, there is an increasing acceptance for sustained antibiotic release systems in surgical practice. After development of polymethylmethacrylate (PMMA) bone cement containing gentamicin by Bucholz and Engelbrecht, Klemm developed PMMA gentamicin chains, which are still in use together with the surgical treatment of bone and soft tissue infections today , 2. ChitTN is two- to four-fold more active than VM against both methicillin-susceptible and methicillin-resistant isolates of S. aureus with minimum inhibitory concentration MIC90) typically in the range of 0.51 and 1-2 mg/L, respectively . Chitosa0 typicalThe present study aimed to prepare chitosan beads loaded with VM and TN and investigate the release profiles of the loaded drugs from the beads intended to be used in the treatment of osteomyelitis.Vancomycin hydrochloride  and teicoplanin as Targocid®  were purchased from pharmacies. Chitosan with medium molecular weight and 85% degree of deacetylation (DD) (Aldrich 448877) Glutaraldehyde 50% , Mueller-Hinton Broth, Mueller-Hinton agar and nutrient agar were purchased from HiMedia Laboratories . All other chemicals were of reagent grade unless otherwise stated and used as received.Chitosan (1 and 2% w/v) was dissolved in an aqueous solution of 1% (w/v) acetic acid under gentle stirring (45 rpm) for 14-16 hours at room temperature. Ten grams of polymer solution was cross-linked with 0.1 mL glutaraldehyde diluted in PBS  and dropped into a liquid sodium hydroxide 1.5 M through injector . Stock s0) was plotted against time. Giving a straight line function with slope -k would show zero order release kinetic, the equation of first-order release will become as: To investigate whether drug release profiles follow zero-order kinetic,  declines exponentially as a function of time. The rate constant k in a first order reaction which can also be determined from the t 1/2. To calculate the half-life t1/2, the equation of first-order release can also be written as:Where [D] The release rate constant k was computed for each test by calculating the slope of the line for LN [D] plotted against time.To monitor any potential, chemical, or physical interference of the drug molecules with the chitosan beads, 30 random elution samples were again tested by agar diffusion method according to NCCLS guidelines , 17. TheAll data were analyzed using the SPSS software (Version 20.00). Results were expressed as mean values ± standard error of the mean (SEM). Values of P < 0.05 were considered significant. Data of all groups were proven to adhere to a standard normal distribution. Differences between groups were tested by an analysis of variences (ANOVA), followed by a post hoc comparison using the Newman-Keuls method. Correlation between the agar diffusion microbiological assay and the spectrophotometrical assay was assessed using the correlation coefficient according to Spearman.The mean weight of the twenty beads with the same dimensions prepared from 1% and 2% chitosan was 150.3 mg ± 57.1 and 200.1 mg ± 63.3, respectively. The surface and morphology of a single bead is illustrated in   . As shoIncreasing the chitosan concentration for the beads had negative effect upon total TN and VM release (P < 0.05). This was shown by a significant decrease in antibiotic release in both series of 2%-beads compared to corresponding series of 1%-beads. After each experiment, tests were conducted to determine whether the hypothesis of first-order release rate was true, and the half-life was calculated according to the above procedure. Antibiotic release profiles and t1/2 of all different series are illustrated in For the series of VM 0.5 h 1%, the differences in half-life between both antibiotics did not show significant difference . In comparison to all other investigated series, drug release was fast in 1%-chitosan beads immersed in VM solution for 3h . Here, more than 60% of total antibiotic load was released within 3 h. Elution was nearly complete (95%) after 36 h Antibiotic delivery systems for local anti-infective treatment are frequently used in nearly all cases of surgery, especially in cases of chronic osteomyelitis and deep tissue infection. The major advantage of local application of antibiotics is producing high concentration in the infected sites without toxic effects due to systemic administration. Biodegradable antibiotic delivery systems are proven to have advantages over inert polymers , 21, 22.


Mastomys natalensis. Distribution of this rodent and relative abundance compared with M. erythroleucus correlates geographically with Lassa virus seroprevalence in humans.PCR screening of 1,482 murid rodents from 13 genera caught in 18 different localities of Guinea, West Africa, showed Lassa virus infection only in molecularly typed  Arenaviruses are emerging in the Americas and Africa and can cause hemorrhagic fevers with case fatalities of up to 15%. These viruses are mainly transmitted through contact with the excreta of their natural hosts, rodents of the family Muridae. The Old World arenavirus Lassa virus causes up to 300,000 cases of Lassa fever annually in endemic foci of 2 geographically disjunct regions of West Africa , LVL3359G-plus (5´-AGAATTAGTGAAAGGGAGAGTAACTC), LVL3754A-minus (5´-CACATCATTGGTCCCCATTTACTATGATC), and LVL3754D-minus (5´-CACATCATTGGTCCCCATTTACTGTGATC) (Note: Underlined letters represent differences in nucleotides among plus and minus primers). Because Lassa virus antigen should be frequently detectable in the natural host, and specific antibodies are known to be negatively correlated with its presence, they were not investigated in this study . A 1-step RT-PCR targeting a highly conserved region of the RNA polymerase (L) gene was performed by using primers LVL3359A-plus (5´-AGAATTAGTGAAAGGGAGAGCAATTC), LVL3359D-plus  in 6 trapping areas and high (25%–55%) in 12 trapping areas  of 1,482 murid rodents. Sequence analysis showed 96 Lassa virus strains with 96%–100% amino acid homology with the prototypic strain Josiah. Lassa virus–positive rodents were only captured in the prefectures of Faranah  and Guéckédou (Denguédou), both situated along the border with Sierra Leone . We PCR-Lassa virus was isolated in cell culture from 32 rodents, and a 631-bp fragment of the nucleoprotein gene previously used for phylogenetic analysis was sequenced (GenBank accession nos. DQ832667–DQ832699) . We dete,,Our results indicate that M. natalensis is very likely the only reservoir host of Lassa virus in Guinea. Previous studies that identified M. erythroleucus and M. huberti as reservoirs of Lassa virus in Sierra Leone may have confused the species, especially because M. natalensis and M. huberti have an identical number of chromosomes (,The relative abundance of Mastomys correlated with human Lassa virus seroprevalence in Guinea: M. natalensis was absent from the region with the lowest seroprevalence and was the only Mastomys species caught in the highest seroprevalence region. However, if both M. natalensis and M. erythroleucus were present, human Lassa virus prevalence was either low, intermediate, or high . This isThe results of this study have multiple implications for explaining the patchy occurrence of Lassa virus in Guinea and neighboring countries, as well as for Lassa fever control. First, assuming that M. natalensis is the only host of Lassa virus, natural nidality may occur in a similar fashion as that described for Bolivian hemorrhagic fever caused by Machupo virus. The Machupo virus reservoir host, Callomys callosus, has been shown by genetic analysis to be paraphyletic. The populations of rodents responsible for the maintenance and transmission of Machupo virus were monophyletic lineages different from C. callosus in non-disease–endemic regions of Bolivian hemorrhagic fever and coevolving with the virus (


Viruses occupy a unique position in biology. Although they possess some of the properties of living systems such as having a genome, they are actually nonliving infectious entities and should not be considered microorganisms. A clear distinction should be drawn between the terms virus, virion, and virus species. Species is the most fundamental taxonomic category used in all biological classification. In 1991, the International Committee on Taxonomy of Viruses (ICTV) decided that the category of virus species should be used in virus classification together with the categories of genus and family. More than 50 ICTV study groups were given the task of demarcating the 1,550 viral species that were recognized in the 7th ICTV report, which was published in 2000. We briefly describe the changes in virus classification that were introduced in that report. We also discuss recent proposals to introduce a nonlatinized binomial nomenclature for virus species. In the 7th report of the International Committee on Taxonomy of Viruses (ICTV), viruses were described as elementary biosystems that possess some of the properties of living systems such as having a genome and being able to adapt to a changing environment A virus is a general term which denotes any number of concrete objects that possess various relational properties  that arise by virtue of a relation with other objects. These relational properties, also called emergent properties, are characteristic of the viral biosystem as a whole and are not present in its constituent parts. When a virus undergoes its so-called life cycle, it takes on various forms and manifestations, for instance, as a replicating nucleic acid in the host cell or vector. One stage in this cycle is the virus particle or virion, which is characterized by intrinsic properties such as size, mass, chemical composition, nucleotide sequence of the genome, and amino acid sequence of protein subunits, among others. Virions can be fully described by their intrinsic chemical and physical properties, and that description does not entail the relational properties that belong to the virus.Confusion sometimes arises when a virion is called the virus, as, for instance, when one refers to “the picture of the virus” or to the process of “purifying the virus.” What is actually meant in such cases is a virus particle, not a virus. Confusing virus with virion is similar to confusing the entity insect, which comprises several different life stages, with a single one of these stages, such as pupa, caterpillar, or butterfly.Picornaviridae, the genus Enterovirus, or the species Poliovirus is impossible for the simple reason that abstractions, i.e. ideas, cannot be centrifuged. For the same reason, a virus species cannot cause a disease, since abstractions do not cause diseases (with the exception of psychosomatic disease). The concrete virus entity that causes a disease can, however, be a member of an abstract virus species. Concrete objects like viruses can be members of an abstract entity, that is, an entity of a different logical type, although they cannot be a part of such an abstract concept. Class inclusion or class membership is the correct relationship between a virus and the species category to which it belongs. One often reads that the species Mus musculus has been inoculated with one or other virus species. The correct statement is that a mouse (a member of the species Mus musculus) has been inoculated with a member of viral species X.Since viruses, like other biological entities, are concrete objects located in time and space, their classification is a purely conceptual construction, based on the use of abstract categories that have no spatiotemporal localization Although species is the most fundamental taxonomic category in all biological classifications, it was only in 1991 that the ICTV agreed that the concept of virus species should be uniformly applied in virus classification. For many years, plant virologists had been arguing that the concept of species was not applicable to viruses because they are not sexually reproducing organisms . In publications written in languages other than English, the use of italics for the English official species name would also indicate the alien nature of the term. In such publications, the common names of viruses will be those used in that language and not the English names. The use of italicized English instead of italicized Latin for the names of virus species reflects the emergence of English as the modern language of international scientific communication, and it also does away with the invidious task of having to coin new Latin names for all virus species The value of using italics is that it visibly reinforces the status of the corresponding species as a taxonomic entity, i.e., a formal, abstract class, distinct from the concrete viral objects that replicate and cause disease and that are written in Roman characters. Only if it is necessary to draw attention to the taxonomic position of the virus under study will it be necessary to refer to the official species name written in italics. Even then, the official name need be given only once, probably in the introduction or Materials and Methods sections . In this system, the italicized word Measles morbillivirus) and the common, nonitalicized virus name, measles virus. At present, the distinction between the species name and the virus name in most cases relies only on typography , which can lead to confusion Such a binomial system for species names would also have the advantage of clearly distinguishing between the species name written in italics (www.danforthcenter.org/ILTAB/ICTVnet/).Whether nonlatinized binomials should become the official species names of viruses has been debated within the ICTV for many years  makes sense.Although the ICTV does not have a constitutional responsibility for devising appropriate abbreviations, it has over the years published several lists of recommended abbreviations of virus names. Initially, these were abbreviations for the common names of viruses , 37.


We analyzed a database of mammal–virus associations to ask whether surveillance targeting diseased animals is the best strategy to identify potentially zoonotic pathogens. Although a mixed healthy and diseased animal surveillance strategy is generally best, surveillance of apparently healthy animals would likely maximize zoonotic virus discovery potential for bats and rodents. Nearly two thirds of emerging infectious diseases that affect humans are zoonotic, and three fourths of these originate in wildlife, making surveillance of wildlife for novel pathogens part of a logical strategy to prevent the future emergence of zoonoses  by wildlife host groups to best detect novel pathogens with zoonotic potential. In answering this question, we can better determine how host and virus taxonomy might influence our decisions about applying limited surveillance resources to a growing global health problem.www.ictvdb.org) (www.ictvdb.org) as keywords in Web of Knowledge (http://wokinfo.com/), Wildlife Disease Association meeting abstracts (http://wildlifedisease.org/wda/CONFERENCES.aspx), Google Scholar (http://scholar.google.com/), and the Global Mammal Parasites Database . The resulting 605 host–pathogen relationships included 56 unique viruses classified in 17 taxonomic families and 325 unique mammals classified in 15 taxonomic orders. We excluded rabies from our analysis because the intense research effort on this virus and its high pathogenicity in almost all of its wide range of hosts , Web of Science (http://thomsonreuters.com/products_services/science/science_products/a-z/web_of_science/), BIOSIS Previews (http://thomsonreuters.com/products_services/science/science_products/a-z/biosis_previews/), and Biologic & Agricultural Index Plus ; search terms consisted of virus names and International Committee on the Taxonomy of Viruses synonyms, host genus and species names, and common names [reconciled to the 2005 version of Mammal Species of the World  clinical signs of infection were most likely to be seen in animals monitored in laboratory settings than in the wild and 2) stressful conditions in captivity would heighten the likelihood of a normally benign pathogen leading to clinical signs (http://cran.r-project.org/bin/windows/base/), of apparent host disease as a function of host taxonomy and virus taxonomy for the subset of mammal–virus pairs for which the host order or virus family had at least 3 records in the database. We then calculated odds ratios for each host taxonomic order and virus family relative to the reference categories (Artiodactyla and Flaviviridae) and the predicted probability of being symptomatic for all species order–virus family combinations.We conducted a logistic regression analysis, using Firth’s bias reduction procedure  of infected wildlife hosts were reported to have had visible disease and 72% (n = 224) were reported without evidence of visible disease , panel A2 = 88.70, p<0.001 and χ2 = 59.45, p<0.001, respectively). Species infected with paramyxoviruses, poxviruses, and reoviruses were more likely to have visible disease , and species infected with bunyaviruses were less likely to have visible disease relative to the reference category (p = 0.01) (We found that virus family and host order were significant predictors of disease status (χ = 0.01) . Hosts i = 0.01) .Relative to the reference category, species classified in the order Chiroptera were less likely to have visible disease (p<0.001), and species in the order Rodentia were marginally less likely to have visible disease (p = 0.10) . Compare–Rhabdoviridae. Therefore, particularly for the case of novel virus detection, our results point to a mixed strategy of targeted syndromic and healthy animal surveillance across host and virus taxonomies. A mixed strategy could combine apparently healthy animal surveillance (particularly in Chiroptera) with syndromic surveillance in other wildlife and domestic animal hosts. Syndromic surveillance has proven useful where secondary animal hosts are involved [e.g., surveillance for West Nile virus (Nonhuman primates –. The mecThere are limitations to our study, particularly ascertainment and reporting biases, as acknowledged in previous studies of emerging infectious diseases (Our analysis supports a holistic, probability-based approach to zoonotic virus discovery, specifically, continued analysis of passively and actively reported deaths and increased investment in broad surveillance of healthy wildlife. The latter could be targeted geographically to those regions most likely to generate novel emerging infectious diseases (The full database of host–virus pairs and disease state.


Tobacco etch potyvirus (TEV) that show a negative relationship between virulence and accumulation rate, we first explored the evolution of virulence and fitness traits during simple and mixed infections. Short-term evolution experiments initiated with each strain independently confirmed the genetic and evolutionary stability of virulence and viral load, although infectivity significantly increased for both strains. Second, competition experiments between hypo- and hypervirulent TEV strains have shown that the outcome of competition is driven by differences in replication rate. A simple mathematical model has been developed to analyze the dynamics of these two strains during coinfection. The model qualitatively reproduced the experimental results using biologically meaningful parameters. Further analyses of the model also revealed a wide parametric region in which a low-fitness but hypovirulent virus can still outcompete a high-fitness but hypervirulent one. These results provide additional support to the observation that virulence and within-host replication may not necessarily be strongly tied in plant RNA viruses.Understanding the evolution of virulence for RNA viruses is essential for developing appropriate control strategies. Although it has been usually assumed that virulence is a consequence of within-host replication of the parasite, viral strains may be highly virulent without experiencing large accumulation as a consequence of immunopathological host responses. Using two strains of  Plant viruses have been used as model systems for exploring the mechanisms of virus evolution Virulence, which can be defined as the deleterious effects of parasites on their hosts, is a selectable trait and thus, could play an important role in the evolution of pathogens Cucumber mosaic virus (CMV) demonstrated a relationship between virulence and host genotype independent of virus multiplication Barley stripe mosaic virus were not due to changes in virus accumulation Experimental support for a positive correlation between within-host multiplication rates and virulence is limited for plant-virus systems. However, it has been shown that a positive correlation between parasite multiplication and virulence may exist only in some genotypes and/or environmental conditions for a given host-parasite system i) the most virulent parasite will outcompete the others within the host and (ii) coinfecting viruses do not compete. Mosquera and Adler Coinfection assays allow for the study of competition dynamics between different viral genotypes, and can be useful to determining what are the main forces involved in their long-term fate. For instance, using two strains that differ in virulence and multiplication rate. The outcome of such competition determines the genetic structure of the viral population and, therefore, the level of competition, as well as the phenotypic properties of such population . Theoretical models of multiple infections in which selection of parasites occurs thought competition for multiplication within host and for transmission among host have been studied Tobacco etch virus (TEV), the pathogen employed in this study, induces symptoms that range from chlorotic vein banding, mosaic mottling, necrosis and/or distortion of leaves in susceptible dicotyledonous species. Flowers, seeds and fruits are also affected. TEV has a positive sense, single strand RNA genome and taxonomically has been classified in the genus Potyvirus within the Potyviridae family. The effects on TEV fitness and virulence of random single-nucleotide substitutions have been recently characterized Nicotiana tabacum. To better interpret the experimental results, we developed a mathematical model that describes the temporal dynamics of two coinfecting viruses differing in their accumulation rate and virulence in the same way that our experimental subjects do. We first used the model to explain qualitatively the experimental results using biologically meaningful parameters, also characterizing the equilibrium values for the scenarios of coexistence, outcompetition and coextinction. The model also showed that under certain combinations of parameters a slow replicating hypovirulent strain can outcompete a fast replicating hypervirulent one.The existence of a hypervirulent strain TEV-PC2 with a low accumulation rate and of a hypovirulent strain TEV-PC76 with a high accumulation rate F1,36 = 0.178, P = 0.676), and the differences in virulence between TEV-PC2 and TEV-PC76 were maintained along the experiment , with the former strains being, on average, 9.84% more virulent than the later. Evolutionary stability was also observed for viral accumulation. While the TEV-PC2 strain viral accumulation was, overall, significantly lower than for TEV-PC76 , the differences in accumulation among the two strains remained significant along the evolution experiment : the TEV-PC76 accumulates 74% more than TEV-PC2 per gram of infected tissue. Interestingly, TEV-PC76 accumulation was undistinguishable from that of the wiltype strains . Virus accumulation values can be normalized by the average value observed for the wildtype TEV as 1.07 for TEV-PC76 and, similarly, down to 0.621 for TEV-PC2 , although the magnitude of the difference between them remained constant , being TEV-PC76 7.04% more infectious than TEV-PC2 along the evolution experiment.Contrarily to these observations, overall infectivity significantly increased with passages for both strains , suggesting a dominance of this strain in determining overall virus accumulation. These results are consistent with those previously reported by Carrasco et al.In the case of mixed infections, virus accumulation was intermediate between values characteristic of each strains but significantly grouped to the TEV-PC76 values (i) one virus was completely outcompeted by its counterpart and is not present in the plant anymore or (ii) it may still be present but at a concentration that is under the method detection level. To determine what of these two options was correct, we made virus preparations from all plants, regardless their infectious status, and used them to continue the evolution experiment. At passage three, however, all population switched radically into a strict TEV-PC76 hypovirulent population, suggesting that the second possibility was, indeed, the case. Furthermore, this dominance of the TEV-PC76 strain is consistent with previous results from head-to-head competition assays against the wildtype virus showing that the hypervirulent TEV-PC2 had lower competitive fitness than the hypovirulent TEV-PC76 Next, we determined the composition of viral populations on each coinfected plant 7 dpi by means of the RT-PCR followed by diagnostic restriction analyses. Only ∼43% of the coinoculated plants were diagnosed as coinfected . In somex1 and x2. The model is similar to the one analyzed by Solé et al.The previous results indicate that a hypervirulent virus that reaches low viral accumulation is outcompeted by a hypovirulent one but that it reaches high accumulation. In order to disentangle the effects between accumulation and virulence we developed a dynamical mathematical model for virus competition together with differential virulences. The model was formulated by means of a two-species time-continuous dynamical system considering as state variables two populations of viruses, named P, to complete its reproductive cycle  and that the utilization of such factor by the virus translates into a defects in cell cycle and thus in symptoms. We defined the available quantity of such cellular factor as avP decreases as viral populations grow in size. We assumed P0 = 1 to be a mean and constant concentration value of the limiting cellular factor. iK  are the affinity constants for the limiting cellular factor, and ir the realized growth rates for the i th viral strain, being max,ir the maximum replication rate when the cellular factor is present at infinite concentration and is not limiting viral growth. We would like to highlight that the model assumes that the virus accumulation is entirely determined by replication rate. This assumption may not be entirely realistic from a virological perspective, since accumulation may also depend of other factors such as cell-to-cell and systemic movements, but it is convenient from the mathematical point of view, since it avoids considering spatial correlations, and, in addition, does not require of knowledge about viral spread, which has not been gathered in the above experiments. In our formulation the virulence of a given strain i is proportional to iK and this proportionality creates the observed tradeoff between accumulation rate and virulence shown in Eq. (3). We assumed that both viral strains compete in a finite bounded system , using a logistic-like constraint  that couples both populations and introduces a competition term associated to the growth inside the host. The parameter ijβ in the logistic term corresponds to the interspecific competition rates. As previously noted, the experimental results suggested no major interference between both viruses, therefore we considered symmetric interspecific competition i.e., ijβ = jiβ≡β, and for simplicity we hereafter used β = 1. Finally, we assumed degradation rates  to be symmetric i.e., δ1 = δ2≡δ. Following the previous empirical observations, the model does not consider changes in virulence and virus replicative fitness along time.In order to introduce virulence we assumed that viruses need a cellular factor, t = 0.1). The terms inside the Jacobian matrix for this dynamical system,iθ = iK+avP, and iη = max,ir(1−x1−x2), with i = 1, 2.We analytically and numerically studied Eqs. (1) and (2). All numerical results were performed solving the differential equations with the fourth-order Runge-Kutta method  imposed by the logistic-like constraint (with carrying capacity C0 = 1), and thus are not analyzed. The first fixed point is the trivial equilibrium, P1* = , where both strains have zero population numbers. The stability of such a point is obtained by linearizing the flow and computing the eigenvalues from det(J(0)−λI) = 0, withP2* =  and P3* = , withP2*, if stable, involves the outcompetition of the second viral strain, x2, by the first one, x1; while P3* involves the reverse scenario, that is, the virus population x1 is outcompeted by the x2 population whenever this is a stable fixed point. The stability of these two equilibria was numerically studied under the parameter ranges shown in Equations (1)–(2) have six fixed points. As we were interested in the scenarios of extinction and of outcompetition, we focused our analyses in three fixed points: one involving the extinction of both strains and the two equilibrium points involving the extinction of one viral strain and the survival of the other one. Together with these three equilibria, there is another fixed point that can involve the coexistence of both viral strains (see below), as well as two other fixed points that involve the outcompetition of one of the two strains. However, numerical investigations for these latter two equilibria indicate that, under the parameter regions we are studying , the nonP4* = , withWe note the existence of another fixed point involving the asymptotic coexistence of both viral populations, which is given by max,ir) as well the parameters related to virulence  we numerically characterized the possible scenarios of virus extinction (equilibrium P1*), outcompetition (equilibria P2* and P3*) and coexistence (equilibrium P4*). The model qualitatively reproduced the outcome of the competition experiments discussed in the previous section. To reproduce the experimental observations TEV-PC76 we set the relative accumulation rate to maxr,1 = 1.07 and virulence to K1 = 0.817. For TEV-PC2, we also set maxr,2 = 0.621 and virulence to K2 = 1.221. The iK values were fixed to the empirical virulence values determined by Carrasco et al.max,ir, in the parameter space . The results showed that for the values of iK previously mentioned, TEV-PC76 outcompetes TEV-PC2. Actually, for these values of accumulation rate, the only combination of virulence that allows the outcompetition of TEV-PC76 by TEV-PC2 would be a lower virulence for the later, thus indicating that when two viruses are competing, a slower replicator can still outcompete a faster one if there are large differences in virulence. All the time series computed under the biologically meaningful parameter values −λI) = 0, are λ±<0. For the same parameters, and extensively, to all the parametric region covered by the gridded surface of P2*, is stable.By studying the parameters denoting accumulation  has a lower fitness. The results displayed in x1 outcompetes the second virus population (with a largest fitness), for low values of K1 . In K1 = 0.2<K2 = 0.7,  where the slower replicator outcompetes the fastest one. We also analyzed numerically the effect of the initial conditions +x2(0) = 1) on the asymptotic dynamics found in the parametric regions studied in P2* in the gridded surface and P3* in the flat surface), thus indicating that scenarios of bistability are very unlikely. To illustrate the dynamics and the basins of attraction we represent in x1, x2). We specifically used the same parameter values used in The previous results showed that a hypovirulent virus could outcompete a hypervirulent one provided that the former is a faster replicator. Actually, the effect of virulence seems to be important in the outcompetition dynamics. For instance, in the parameter space displayed in In conclusion, in agreement with the experimental results, a fast replicating hypovirulent viral strain can outcompete a hypervirulent one provided it replicates slower. Our model also shows a wide region in parameter space for which slow replicating and hypovirulent strains can still outcompete fast replicating hypervirulent ones.In this work we have studied two mutants of TEV differing in virulence and multiplication rate but not in infectivity rate. The first mutant genotype (TEV-PC76) has a higher multiplication rate and is hypovirulent relative to the wildtype genotype, and the second one (TEV-PC2) has a lower fitness and is hypervirulent The results of the evolution experiments with mixed infections indicate a rapid switch in population composition where the genotype with higher multiplication rate and hypovirulent outcompetes the hypervirulent one but with lower multiplication rate. Two different phenomena can be called to explain this observation. First, to consider that virulence is not a determining factor and it is just the replication rate what drives the result of competition. The second interpretation is that virulence plays an important role (and probably in connection with replication), and a hypovirulent virus can outcompete a hypervirulent one. In order to further explore these two hypotheses, we developed and studied a two-species Lotka-Volterra mathematical model describing the competition dynamics between two viruses considering as relevant parameters the multiplication rate and virulence. By using the relative replication rate values obtained in the experiments, as well as virulence values previously estimated for these two viruses Among the model predicted asymptotic dynamics, we have characterized a small region in parameter space where the coexistence of both viral genotypes may be possible. We note that such a region allows for the existence of polymorphic viral populations containing variants that differ in virulence and accumulation rates even within the same host; or in other words, two opposed evolutionary strategies in terms of virulence and replication can coexist. Therefore, no tradeoff between virulence and replication may be at play in our pathosystem.Our results suggest that if hypervirulent but slow replicating and hypovirulent but fast replicating strains  had to evolve in nature, a rapid extinction of the hypervirulent would take place due to differences in accumulation rates. Indeed, if we assume an equal mutation rate for both viruses with a similar rate of infectivity, the quick switch to a pure hypovirulent population may not let any chances for a possible recombination event or time enough for TEV-PC2 to evolve by itself into a faster replicator while still retaining high virulence.et al.BglII (Fermentas) and transcribed into 5′-capped RNAs using SP6 mMessage mMachine® Kit (Ambion Inc.). Transcripts were precipitated , collected and resuspended in DEPC-treated water Nicotiana tabacum cv. Xanthi plants were inoculated by abrasion of the third true leaf with 4 µg of 5′-capped RNA produced by in vitro transcription using mMESSAGE mMACHINE® SP6 kit (Ambion Inc.) for the first passage , was used as our surrogated wildtype  passage . Plants 3 and CCl4 each were added per gram of sample, then mixed. After centrifugation , the upper aqueous phase was taken and filtered through Miracloth . Precipitation of viral particles was done by adding 0.11 volumes of a solution 40% PEG8000, 17.5% NaCl and incubation on ice with agitation for 30 min. After centrifugation  supernatant was removed. Finally, the pellet was resuspended in 20 µL of buffer  per gram of sample. The virus suspension is conserved at −80°C in 25% of sterile glycerol.Seven dpi inoculation infected plants were collected (except the inoculated leaf) and 2 mL of extraction buffer  per gram of tissue added. Whole plant where sampled to avoid the random effects associated with bottleneck colonization of different leafs by different viral subpopulations. After homogenization, 1 mL of CHClChenopodium quinoa leaves The evaluation of virus accumulation per gram of infected tissue was performed by inoculating serial dilutions of viral samples on N. tabacum plants were inoculated either with TEV-7DA, TEV-PC2, TEV-PC76 or a 1∶1 mix of TEV-PC2 and TEV-PC76. Four independent evolution lineages were started on each case for two independent replications. Seven dpi, total plant tissue was homogenized as described above and virus extracted. Virus accumulation per gram of infected tissue was assessed and equal numbers of 20 LFU used to initiate the next evolution passage.The following short-term evolution experiments were performed, each designed to assess the evolutionary stability of virulence and several fitness traits during single or multiple infections. For single infections, −2 to 10−6. Each dilution was used to inoculate 20 N. tabacum plants. Seven dpi all infected plants were counted. The trimmed Spearman-Karber method was used to evaluate the ID50 after the first, the third and fourth passage An equal number of LFUs was taken for each virus and diluted in the range 10Virulence was defined as the reduction in host's fitness associated with infection. Practically, this was done by quantifying the total number of germinating seeds from infected plants in relation to the number of germinating seeds produced by healthy plants Restriction enzymes Eco81I and EcoT14I were used to check which viral genotype was present at each evolution passages. Eco81I cleaves the mutant TEV-PC2 P1 sequence but not the corresponding wildtype sequence of TEV-PC76 at this locus, whereas EcoT14I cleaves the mutant TEV-PC76 NIa-Pro sequence but not the wildtype sequence found in this locus for TEV-PC2.The effect of genotype (main factor) and passage number (covariable), as well as their interaction, on virulence, virus accumulation and infectivity were assessed by a model II ANOVA. Both factors were treated as random ones. Prior to analyses, virus accumulation data were log-transformed to achieve normality and homoscedasticity of variances. Statistics were done with SPSS v16.


Although human torque teno viruses (TTVs) were first discovered in 1997, still many associated aspects are not clarified yet. The viruses reveal a remarkable heterogeneity and it is possible that some genotypes are more pathogenic than others. The identification of all genotypes is essential to confirm previous pathogenicity data, and an unbiased search for novel viruses is needed to identify TTVs that might be related to disease.The virus discovery technique VIDISCA-454 was used to screen serum of 55 HIV-1 positive injecting drug users, from the Amsterdam Cohort Studies, in search for novel blood-blood transmittable viruses which are undetectable via normal diagnostics or panvirus-primer PCRs.A novel torque teno mini virus (TTMV) was identified in two patients and the sequence of the full genomes were determined. The virus is significantly different from the known TTMVs (< 40% amino acid identity in ORF1), yet it contains conserved characteristics that are also present in other TTMVs. The virus is chronically present in both patients, and these patients both suffered from a pneumococcal pneumonia during follow up and had extremely low B-cells counts.We describe a novel TTMV which we tentatively named TTMV-13. Further research is needed to address the epidemiology and pathogenicity of this novel virus. Anelloviridae family which includes also torque teno mini virus (TTMV), formerly known as TTV-like Mini virus (TLMV), and TT midi virus (TTMDV), discovered in 2000 and 2007 respectively [Anelloviridae family contains 11 genera and TTMV belongs to the genus Betatorquevirus, where the TTV and TTMDV are in Alphatorquevirus and Gammatorquevirus genera respectively [Human torque teno virus (TTV) was first discovered in a Japanese patient with acute post-transfusion hepatitis in 1997 ,3. Accorectively ,7. The Aectively ,5. All tectively -9.Using alternative splicing, TTV generates three mRNA species and produces at least six proteins by alternative translation initiation . TTV ORFIn search for novel viruses which are potentially transmitted via blood-blood contact, we screened serum of 55 HIV-1 positive injecting drug users from the Amsterdam Cohort Studies via VIDISCA-454, an unbiased virus discovery method that can detect both RNA and DNA viruses . From th7HX3CXCX5H motif was found in the ORF2 of the new TTMV described here (CX7HX3CXCX5H) at the AA positions 15–34. A palindromic sequence (GGGGGCTCCGCCCCC) is repeated two times in the 3′end of genome and a GC-rich region is also present in the non-coding part, as similarly reported for TTMV, TTV and TTMDV [The genome of the virus carries the characteristic features of TTMVs . In general the two viruses were very similar, with the same genome composition and overall less than 1% difference in nt identity. The variation was mainly in the ORF1 gene (1.1%), and the ORF2 genes were 100% identical at nucleotide level.Phylogenetic analysis shows that TTMV-13 does not cluster with any of the known TTMVs Figure . The novAnelloviridae . Furthermore, HIV-1 was detected as expected .The patient (D50) in which the virus was originally detected was male, known HIV-1 positive since entry of the Amsterdam Cohort Studies, and followed for 43 months until death (April 1989 - January 1993). The patient was diagnosed with AIDS, 6 months after entering the study (pneumocystis carinii pneumonia). At the same time (6 months after study entry) zidovudine treatment was started. Seven months before death the patient developed a pneumococcal pneumonia, which was treated with sulfamethoxazole. The sample in which the TTMV was detected was collected 16 months since study entry (June 1991). Besides the novel TTMV, the VIDISCA-454 sequence reads also showed that the patient was coinfected with HCV  and several known members of the To investigate whether the virus was circulating among injecting drug users, a diagnostic-TTMV13-specific-real time PCR was developed and 548 samples from 385 HIV-1 positive injecting drug users, randomly chosen from the Amsterdam Cohort Studies, were screened for presence of the virus (collection dates 1985–1995). One additional patient carrying the virus was identified. This patient (D11) was male, HIV-1 and HCV positive since entry of the Amsterdam Cohort Studies, and followed from June 1993 until death June 1998. AIDS was diagnosed 22 months since study entry (candida oesophagitis). At that time the patient had been treated with zidovudine for 20 months, which was supplemented with lamivudine after AIDS diagnosis. The patient developed a pneumococcal pneumonia and died due to a pneumococcal sepsis 5 years after study entry. The TTMV positive sample was collected 9 months since study entry (January 1994).3 -104 copies/ml) except one peak in 1994.To investigate whether the TTMV infection was chronic, sequential serum samples form both patients collected over the study period were screened for viral DNA. In both patients the virus was detectable over a prolonged period in time. Patient D50 was TTMV-13 positive during the first 26 months of follow up, whereas patient D11 became positive 7 months after study entry and cleared the virus at month 27 : a total of 26 HIV-1 positive injecting drug users and 19 HIV-1 positive men having sex with men were examined for the presence of the virus but none of the serum or EDTA-plasma samples resulted positive. Attempts to express the viral capsid protein or parts of this protein in a prokaryotic protein expression system (pET100/TOPO in E.coli Rosetta 2 (Novagen)) were unsuccessful, therefore no serological test could be developed.In search for a disease association, all collected clinical and laboratory data were examined for specific factors which were in common for both TTMV-13 positive patients. Both patients have suffered a pneumococcal pneumonia during follow up and both patients had extremely low B-cells counts  and were at the end stage of HIV-1 disease. It is known that HIV-1 affects humoral immunity through changes in the B-cell compartment , loss ofWith the discovery of the novel TTMV-13 we are one step further with revealing the full spectrum of genotypes and sequence variation among anelloviruses.3 . N- and O-linked glycosylation sites and signal peptide cleavage sites were predicted using the NetNGly 1.0, NetOGly 3.1, and SignalP 4.0 analysis tools, from the Center for Biological Sequence Analysis (http://www.cbs.dtu.dk/services/).VIDISCA-454 was performed with 110 μl of serum/EDTA-plasma samples as previously described . Full leNucleic acids were extracted from serum samples using the Boom method . Absoluthttp://www.ncbi.nlm.nih.gov/genbank/) were used for the analysis (accession numbers are listed in Figure Available complete TTMV genome sequences in GenBank database (Two complete genome sequences of TTMV-13 obtained in this study have been deposited in the GenBank database under accession numbers KF764701 and KF764702.TTV: Torque teno virus; TTMV: Torque teno mini virus; TLMV: TTV-like mini virus; VIDISCA: Virus discovery cDNA-AFLP; ICTV: International committee on taxonomy of viruses; HIV: Human immunodeficiency virus; AIDS: Acquired immunodeficiency syndrome; ARD: Acute respiratory disease; HCV: Hepatitis C virus; RSV: Respiratory syncytial virus; PPE: Parapneumonic empyema; PBMC: Peripheral blood mononuclear cell; ORF: Open Reading Frame.The authors declare they have no competing interests.SMJF carried out the characterization of the virus infections, the prevalence of the infection, the full length sequencing and the writing of the manuscript. MFJ participated in protein expression, MD, MC and SMJF performed the VIDISCA-454 and analyzed the sequence data, KAD and NK carried out the virus culture, MB analyzed the clinical, cell biology and virology data of the patients, BPXG, MP collected the clinical and behavioral data. MC and FJH performed the phylogenetic analysis. LH designed the study and participated in drafting the manuscript. All authors read and approved the final version of the manuscript.HIV-1 Virus load and CD4 cell counts of the VIDISCA-454 analysed patients.Click here for filePrimers used for full length genome sequencing.Click here for file


Powassan virus is endemic to the United States, Canada, and the Russian Far East. We report serologic evidence of circulation of this virus in Alaska, New Mexico, and Siberia. These data support further studies of viral ecology in rapidly changing Arctic environments. Flaviviridae, genus Flavivirus) with recent and increasing prevalence. The only member of the tick-borne encephalitis (TBE) serogroup of flaviviruses endemic to North America, POWV is an emerging cause of human illness and death  (Powassan virus (POWV) is a tick-borne virus , Alaska (2004–2005) and throughout the southwestern United States (2005–2007  under UnSerum samples and supernatants were tested by using a strip immunoblot assay (SIA) with recombinant DTV envelope glycoprotein. Because POWV and DTV are serologically indistinguishable, this antigen binds antibodies specific for DTV, POWV, or other closely related viruses.In brief, antigen was adhered to a nitrocellulose membrane, and strips were produced with the following antigens and markers: Coomassie blue (orientation control), specific pathogen–free mouse serum (negative control), serum from mice inoculated with DTV envelope glycoprotein (DTV-positive control), mouse IgG (IgG-positive control), and purified DTV E-glycoprotein (test). Approximately 0.25 µg of DTV envelope glycoprotein was used per 2-mm test strip. Samples were tested at a 1:200 dilution, and antibody was detected by using an alkaline phosphatase–conjugated secondary antibody (goat antimouse IgG). Colorimetric intensity was assessed and DTV envelope glycoprotein–positive results were compared with 3+ and 1+ IgG control bands.Myodes rutilus) (6.2%) (M. gapperi) (6.7%). In the southwestern United States, DTV-reactive antibodies were found in New Mexico in 2 Peromyscus species mice: the piñon mouse (P. truei) and the deer mouse (P. maniculatus)  that were collected sympatrically. The deer mouse is of particular interest because it is the primary host of Sin Nombre virus, the etiologic agent of hantavirus cardiopulmonary syndrome in North America  . In soutIxodes angustus) from coastal southeastern Alaska  in 2009 from captured mammals  to Siberia (M. rutilus)  . The sersampling . The incMyodes spp. voles in Alaska and Siberia and from Peromyscus spp. mice in the southwestern United States.Our findings augment knowledge of distribution of TBE serogroup flavivirus in the Nearctic and will guide further studies of New World TBE serogroup flavivirus ecology. Future work will focus on acquisition of viral isolates and nucleic acid sequences from