Pseudomonas. The isolated bacteria hydrolyzed oxamyl to oxamyl oxime, which was not further transformed, and utilized methylamine as a C and N source. This was further supported by the detection of methylamine dehydrogenase in three of the four isolates. All oxamyl-degrading strains carried a gene highly homologous to a carbamate-hydrolase gene cehA previously identified in carbaryl- and carbofuran-degrading strains. Transcription analysis verified its direct involvement in the hydrolysis of oxamyl. Selected isolates exhibited relaxed degrading specificity and transformed all carbamates tested including the oximino carbamates aldicarb and methomyl  and the aryl-methyl carbamates carbofuran and carbaryl which share with oxamyl only the carbamate moiety.Microbial degradation is the main process controlling the environmental dissipation of the nematicide oxamyl. Despite that, little is known regarding the microorganisms involved in its biotransformation. We report the isolation of four oxamyl-degrading bacterial strains from an agricultural soil exhibiting enhanced biodegradation of oxamyl. Multilocus sequence analysis (MLSA) assigned the isolated bacteria to different subgroups of the genus  N,N-dimethyl-2-methylcarbamoyloxyimino-2-(methylthio)acetamide] is an oximino carbamate nematicide used for the control of nematodes in protected crops and potato cultivation. The recent withdrawal or restricted use of several nematicides like aldicarb, cadusafos, and fenamiphos, made oxamyl one of the leading synthetic non-fumigant nematicides in the global market. Oxamyl is a rather water soluble molecule (280 g L-1), which is only weakly sorbed by soil (Kom 21.9 ml g-1) and so is readily mobile for leaching to groundwater  and aquatic organisms  to isolate and characterize oxamyl-degrading soil bacteria, (ii) to explore the microbial metabolic pathway of oxamyl, and (iii) to identify the genes involved in the hydrolysis of oxamyl.-1 in sterile ddH2O) was used for the preparation of oxamyl-containing media. Bacteria were isolated using a selective mineral salts medium  was prepared in a similar way to the MSM liquid media except that Bacto Agar  was added. Nutrient Broth (NB) and Luria-Bertani broth (LB) were purchased from LAB M (UK) and they were prepared according to manufacturers’ instructions. Nutrient agar (NA) was prepared in a similar way to NB except that Bacto Agar (15 g L-1) was added.Analytical grade oxamyl , oxamyl oxime  carbofuran, carbaryl , methomyl, aldicarb, aldicarb sulfoxide, and sulfone , were used throughout this study. An aqueous solution of oxamyl , aiming at a soil dose rate of 10 μg g-1. Water was then added to the treated soil samples to adjust their moisture content to 40% of their water holding capacity. The two soil samples were then separated into sub-samples of 20 g, which were placed in aerated plastic bags and incubated in the dark at 25°C. Immediately after pesticide application and at daily intervals thereafter, triplicates from each soil were analyzed for oxamyl and its metabolites via HPLC.The degradation of oxamyl was investigated in a soil from a commercial banana plantation located in the area of Sitia, northeast Crete, Greece. The plantation had been treated with oxamyl twice a year for the last 5 years. Despite that, the farmer was facing severe infestation by the lesion nematode -1 of oxamyl oxime and the samples were incubated in the dark at 25°C. Immediately after the addition of oxamyl oxime in soil and 10 days later, the levels of oxamyl oxime were determined by HPLC.In a follow up experiment we investigated the involvement of microorganisms in the transformation of oxamyl oxime (hydrolysis product of oxamyl) in the same soil. Briefly, 120 g of sterilized and non-sterilized soil were treated with 8 mg kgOxamyl-degrading bacteria were isolated from soil via enrichment cultures . At the -1 of oxamyl oxime  were inoculated with 0.2 g of soil from the banana plantation which had been previously treated with oxamyl oxime. Duplicate non-inoculated controls were also included. The degradation of oxamyl oxime was followed by HPLC.A follow up enrichment was established to isolate bacteria able to degrade the main hydrolysis product of oxamyl, oxamyl oxime. Triplicate flasks containing MSMN and MSM supplemented with 20 mg L2O and DNA was extracted using the Nucleospin Tissue kit . Multilocus sequence analysis (MLSA) of the housekeeping genes 16S rRNA, rpoD, and gyrB was used for the phylogenetic classification of the isolated bacteria. Details on the primers used for the amplification of the target genes are given in Supplementary Table TM (Finnzymes), 0.2 μM of each primer, 1X buffer (DyNAzymeTM EXT buffer), 1.5 mM of MgCl2, and 200 μM of each dNTPs. The PCR thermal cycling conditions for the amplification of the 16S rRNA gene were 95°C for 5 min, followed by 25 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 2 min, with a final extension of 72°C for 10 min. PCR amplification of the gyrB and rpoD genes was performed as described by Escherichia coli  following standard procedures (Oxamyl-degrading isolates were grown in MSM + oxamyl (50 μM) agar plates for 4 days at 25°C. Bacterial cells were collected in 0.5 ml of sterile ddHocedures . Plasmid1. Subsequently, the individual alignments  were merged in Mesquite ver. 2.75  database under the accession numbers FN600408 – FN600411 (16S rRNA gene), KT808454-KT808457 (gyrB), and KT808458-KT808461 (rpoD).For the phylogenetic analysis of the isolates, three alignments, one per housekeeping gene sequenced, were prepared by using clustaloer. 2.75  forming er. 2.75 . TREECONer. 2.75  was usedudomonas . Sequenc6 colony forming units (cfu) ml-1] and the subsequent growth of the oxamyl-degrading bacteria during degradation of oxamyl were determined by serial dilution plating in NA.In all studies bacterial inocula were prepared as described by -1), methomyl (500 mg L-1), and aldicarb (500 mg L-1) were prepared in sterile ddH2O and they were used for the preparation of pesticide-supplemented MSM (10 mg L-1). In contrast, MSM supplemented with the more lipophilic carbaryl was prepared as described by Karpouzas et al., (2000). Triplicates containing MSM plus one of the compounds (10 ml) were inoculated with the degrading isolate. Triplicate non-inoculated controls were also prepared for each compound. Viable cell counts by serial dilution plating in NA gave an initial inoculum density of 2 × 106 cfu ml-1. Degradation of pesticides was determined by regularly removing and analyzing samples by HPLC.The ability of one of the isolated bacteria, strain OXA20, to degrade other carbamate pesticides was investigated. Aqueous solutions of carbofuran  or 53°C (cehA), 1 min extension at 72°C, followed by a final extension for 5 min at 72°C. The concentrations of the PCR reagents were: 200 μM of each dNTP , 1X polymerase buffer, 1.5 mM MgCl2, 1 U DNA polymerase , 0.2 μM of each primer and sterile ddH2O.Total DNA from the oxamyl-degrading bacterial isolates was screened for the presence of the known carbamate – hydrolase genes cehA gene in the DNA of the oxamyl-degrading isolates, the full length cehA gene (2385 bp) was obtained by PCR using primers cehAFLf – cehAFLr, which were designed based on the full length cehA gene sequence of Rhizobium sp. AC100 . Thermocycling conditions were as follows: initial denaturation at 95°C, followed by 35 cycles of 30 s denaturation at 95°C, 30 s annealing at 55°C, 2.5 min extension at 72°C, followed by a final extension step for 10 min at 72°C. The concentrations of the reagents in the PCR reaction were as above with the only exception that 0.4 μM of each primer were utilized. The PCR products obtained were purified, cloned and transformed. Clones identified as positive were further processed for plasmid extraction and sequencing. The sequences of the cehA gene obtained were deposited in the EMBL database under the accession numbers FR751310 – FR751313.Upon initial detection of the cehA gene in the hydrolysis of oxamyl was verified by following its expression during degradation of oxamyl. Thus, bacteria were inoculated in triplicate MSM + oxamyl (230 μM) and MSMN + succinate (0.1%). Triplicate non-inoculated controls were also included to determine the abiotic hydrolysis of oxamyl. Immediately prior to inoculation and at regular intervals thereafter, samples (1–5 ml) were removed from the flasks and used for the determination of oxamyl degradation and for RNA extraction. RNA was extracted with the Nucleospin RNA II kit  according to the manufacturers’ instructions. A DNAse treatment step  was essential to remove DNA residues from extracted RNA. The absence of DNA contamination was further confirmed by PCR of the 16S rRNA gene. DNA-free RNA was then reverse-transcribed to obtain cDNA  using random hexamers .The involvement of cehA gene was measured by RT-q-PCR using gyrB as a reference gene. New primers sets for the RT-q-PCR analysis of the cehA and gyrB genes were designed using the program Primer3 (Table 1). A single primer pair was designed for the amplification of the cehA gene from all oxamyl-degrading isolates, while two primer pairs were prepared to successfully amplify the gyrB gene of the different isolates. The specificity of the primers designed was verified by q-PCR using DNA from the targeted bacterial strains. Real-time PCR reaction mixtures contained 10 μl 2x SYBR Green PCR MasterMix , 20 pmoles of each primer,1x ROX Low, 1 μl template cDNA and sterile distilled water to a total volume of 20 μl. Thermal conditions were 95°C for 3 min followed by 45 cycles of 95°C for 15 s and 63°C for 1 min. For detection of primer dimerisation or other artifacts of amplification, a melting-curve analysis was performed immediately after completion of the real-time PCR . All reactions were performed in triplicate. Three non-template controls were included for each primer pair. Quantification of cehA gene expression was performed according to The expression of the mauA gene, which encodes methylamine dehydrogenase. This enzyme is commonly found in methylotrophic bacteria, which utilize methylamine as a C and N source (2O and used for the inoculation of duplicate MSM + oxamyl and MSM + methylamine (100 μg mL-1) agar plates. Growth on MSM without any C or N source was also tested to avoid false positives. The bacteria were sub-cultured at least three times in the corresponding media to ensure that their growth was purely on methylamine. The presence of the mauA gene in the oxamyl-degrading isolates was investigated via PCR using primers mauAII-232f (AAGTCTTGCGATTACTGGCG) and mauAII-526r (GACCGTGCAATGGTAGG TCA)  . The mau-1. Under these chromatographic conditions oxamyl and oxamyl oxime showed retention times of 3.1 and 2.3 min, respectively.Extraction of oxamyl and its hydrolysis products from soil was performed as described by For the extraction of the other pesticides studied, aliquots of 0.5 ml of the liquid cultures were mixed with 0.5 ml of methanol. The mixture was vortexed briefly and it was directly analyzed by HPLC. For the elution of carbofuran and carbaryl, a mobile phase of acetonitrile:water (40:60 v:v) was used with UV detection at 215 nm. The retention times for carbofuran and carbaryl were 5.3 and 6.1 min, respectively. Methomyl was eluted using a mobile phase of acetonitrile:water (20:80 v:v) with UV detection at 235 nm and a retention time of 3.7 min. Aldicarb and its oxidation products (sulfoxide and sulfone) were analyzed as described by Figure 1A). The degradation of oxamyl in soil followed first order kinetics with a DT50 of 1.6 days. Oxamyl degradation and oxamyl oxime formation was negligible in the sterilized soil .Degradation of oxamyl in the soil from the banana plantation proceeded rapidly with the concurrent formation of oxamyl oxime. The latter did not accumulate and it was fully degraded by day 5 . Negligible degradation of oxamyl was evident in the non-inoculated enrichment cultures.Subsequent enrichment cultures inoculated with the soil from the banana plantation showed a rapid degradation of oxamyl, which coincided with the formation of oxamyl oxime. In contrast to soil studies, oxamyl oxime was not further transformed and accumulated in the medium at the end of each enrichment cycle . In particular, strain OXA17 clustered within the Pseudomonas fluorescens subgroup and showed closest similarity to P. extremaustralis type strain. In contrast, strains OXA18 and OXA25 grouped within the subgroup of P. putida and showed closest similarity to P. monteilii type strain. Finally strain OXA20 clustered within the P. aeruginosa subgroup, and showed closest similarity to P. jinjuensis type strain. Based on these results the oxamyl-degrading bacteria were named as P. extremaustralis strain OXA17, P. monteilii strains OXA18 and OXA25, and P. jinjuensis strain OXA20.Enrichment cultures resulted in the isolation of four pure bacterial cultures which showed complete degradation of oxamyl within 7 days. MLSA based on partial sequences of the 16S rRNA, 7 cells ml-1 at 24 h in the cultures of all the oxamyl-degrading bacteria . Degradation of oxamyl by the strains OXA17, OXA20, and OXA25 was completed within 96 h , while 7 days were needed for the degradation oxamyl by the strain OXA18 . Oxamyl degradation coincided with the formation of oxamyl oxime which was accumulated in the liquid cultures. Extending the incubation to 21 days did not result in appreciable degradation of oxamyl oxime. Negligible hydrolysis of oxamyl to oxamyl oxime was observed in the non-inoculated control cultures .Degradation of oxamyl was rapid and coincided with a build-up of bacterial growth, which reached to levels higher than 10Table 2). Negligible degradation of all tested pesticides (<10%) was observed in the non-inoculated controls (Table 2).Strain OXA20 was able to completely degrade the oximino carbamates oxamyl and aldicarb, and the aryl-methyl carbamate carbaryl within 4 days, while a period of 7 days was required for the complete degradation of methomyl and carbofuran . In particular, the cehA gene of the strain OXA18 was identical to the cehA gene sequence of the Rhizobium strain AC100, while the cehA genes carried by the other three isolates were identical and differed only in two nucleotide positions (1430 and 1494) with the cehA gene of the Rhizobium strain AC100. However, only the nucleotide polymorphism at position 1430 resulted in a change in the amino acid sequence of the resulting protein (threonine instead of asparagine). In contrast, the cehA gene sequence of Novosphingobium sp. KN65.2 was more diverse and showed four nucleotide difference with the cehA sequence of the strains OXA17, OXA20, and OXA25, and five nucleotide difference with the cehA sequences of the strain OXA18 and Rhizobium AC100 .PCR screening of total DNA from the oxamyl-degrading isolates for carbamate hydrolase genes gave positive amplification only for the cehA gene in the degradation of oxamyl, its transcription during degradation of oxamyl was followed via RT-q-PCR. The relative expression of the cehA gene (ratio of its expression in MSM + oxamyl to its expression in MSMN + succinate) followed the same temporal pattern in all oxamyl-degrading strains . The relative expression of cehA increased concurrently with the hydrolysis of oxamyl and reached to maximum levels at 71 h (OXA25), 88 h , or 111 h (OXA 17). For instance, the expression of the cehA gene in the strains OXA20 and OXA25 was 140- and 120-times higher in the oxamyl-amended cultures compared to the corresponding succinate-amended cultures .In order to verify the functional role of the mauA gene in three of the four oxamyl-degrading strains . Further attempts to amplify the mauA gene from the DNA of the strain OXA17 using alternatively primers . Sterilization of soil halted the degradation of oxamyl oxime suggesting that its transformation was biologically driven. Attempts to isolate oxamyl oxime – degrading bacteria following the same enrichment culture method failed, suggesting that its transformation in soil is probably a co-metabolic process performed by non-specialized soil bacteria or fungi. Either way, the hydrolysis of oxamyl is considered a detoxification step since oxamyl oxime is more than an order of magnitude less toxic to mammals, aquatic organisms and earthworms compared to the parent compound .The accumulation of oxamyl oxime in the bacterial cultures indicates that the oxamyl-degrading isolates were not able to exploit this metabolic product as an energy source. Thus it seems probable that the oxamyl-degrading isolates could utilize the methyl-carbamoyl moiety that is released during hydrolysis of oxamyl as a C and N source. This pathway is common among carbamate-hydrolyzing bacteria  and lead2 . The for2 . This is2 . Trabue nt paths . The nonbacteria . Based ocehA, which was first detected in a plasmid of the carbaryl-degrading Rhizobium strain AC100  or nearly identical  to the cehA gene sequence of the Rhizobium strain AC100  compared to the CEHA of the Rhizobium strain AC100 might be responsible for the different substrate specificity observed. This remains to be verified by isolation and heterologous expression of the hydrolase encoded by the cehA gene.Strain OXA20, selected as a representative of the oxamyl-degrading isolates available, rapidly degraded both the oximino carbamates aldicarb and methomyl, structurally similar to oxamyl, and the aryl-methyl carbamates carbofuran and carbaryl, which resemble oxamyl only at the methyl-carbamoyl moiety. These results further support our initial suggestion that these bacteria utilize the methyl-carbamoyl moiety produced by the hydrolysis of oxamyl as C and N source. The relaxed substrate specificity exhibited by the strain OXA20 is not common among carbamate-degrading bacteria. aldicarb . AlthougPseudomonas strains. The isolated bacteria were capable of hydrolyzing oxamyl to oxamyl oxime, which was not further transformed, and methylamine which was utilized as a C and N source. All the strains isolated carried the cehA gene, a carbaryl hydrolase gene, which was shown by transcription analysis to be responsible for the hydrolysis of oxamyl.We report the isolation and identification of four oxamyl-degrading KR and EC performed most of the laboratory work and KR contributed on the writing of the paper. DG, ES, DK, and PK contributed parts of the laboratory work. SN and MT performed the phylogenetic analysis of the isolated bacteria and the isolated hydrolase genes. ET identified and collected the soils suffering from enhanced biodegradation from where the bacteria where isolated. DK formed the idea and supervised the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In my career, I have found that obtaining fundamental insights into how one virus “works” enables the study of other viruses, which ultimately has allowed my group to investigate basic principles of viral pathogenesis and host immune restriction. A running joke among members of my laboratory is that I have never met a virus I did not like, and the more exotic the name, the more excited I am. I confess this may be true. This practice has allowed me to collect and begin study of esoteric viruses that might or might not be the next plague . Why do this?Our scientific training and experiences shape us. As an MD-PhD student in the late 1980s and early 1990s in Timothy Springer’s laboratory, I investigated the functions of two leukocyte adhesion receptors called Mac-1 (CD11b/CD18) and ICAM-1 (CD54). I recall the excitement of defining new adhesion molecule–receptor interactions and their roles in regulating immune cell function. The impact of these receptors was highlighted by the infections acquired by the rare people who genetically lacked functional proteins. At the time, I was struck by how quickly basic biology discoveries could be translated into therapeutic strategies. It seemed like every immunology faculty member had his or her own biotech company on the side, based on fundamental research from their laboratories! Since then, targeted antagonists of adhesion molecules indeed have become successful drugs for multiple sclerosis, coronary artery disease, and vascular disease.Because of this relationship between immunology and human disease and a long-standing interest in global health, I chose infectious diseases for subspecialty training. In the late 1990s, as a postdoctoral fellow in Eva Harris’ laboratory, I began working at the bench in virology by studying virus–host cell interactions of Dengue virus (DENV), a mosquito-transmitted flavivirus responsible for considerable (and growing!) morbidity on a global scale. Beyond learning molecular virology and fundamental principles of the viral life cycle in the host, I became interested in defining mechanisms of host immune restriction, viral immune evasion, and viral pathogenesis.+ T cells, CD8+ T cells, complement, and type I and II interferons can restrict West Nile virus pathogenesis.As I began searching for a faculty position, a career-changing event occurred: West Nile virus (WNV), a related flavivirus, entered into the United States and caused disease in humans and other animals. In 2001, I decided to work on WNV and put on hold my studies of DENV innate immune evasion mechanisms, even though I had secured NIH support for them (I used the funding instead to study WNV pathogenesis—I hope the NIH has forgiven me for this). I made this career decision for several reasons: (1) WNV had public health implications, and there were few scientists studying it in 2001. I assumed (which fortunately came true) that the virus would not go away within a year; (2) I reasoned that if I knew enough biology about one virus, I could apply this knowledge to study related viruses; (3) I wanted to distinguish myself from my PhD mentor, who was an emerging leader in the Dengue field; (4) I was encouraged strongly by my faculty colleagues at Washington University to study something important even though I lacked experience in critical experimental areas . This is how I began developing a mouse model of WNV infection and investigating immune mechanisms of control. Using a series of genetically engineered knock-out mice that I received from many colleagues and other approaches, my laboratory defined, over a period of ten years, how B cells, CD4As my laboratory become productive, something else happened. I learned how exciting it was to collaborate on a project with your friends in the field. Collaborations allowed the pace of our discoveries to accelerate and the use of more inter-disciplinary structural, biochemical, genetic, and immunological approaches to address unanswered questions. With my colleagues in academia and industry, my laboratory has studied basic questions, including how antibodies engage and neutralize viruses, how innate immune genes control viral infection in cells, and how viruses can evade host immune responses to promote virulence. This study of basic flavivirus biology has informed the development (by us and others) of diagnostic tests, novel vaccine candidates, antibody-based therapeutics, and analyses of immune correlates of protection.Which gets us back to the rationale for my collecting viruses. After seeing how the basic knowledge of one virus (DENV) enabled rapid progress on a related emerging virus (WNV) with implications for translational discovery, I became interested in studying many viruses. I did this for several reasons: (i) By having different viruses in the laboratory, we can test the broad significance of any given viral phenotype. This has been relevant for discerning the mechanism by which innate immune genes  restrict viral infection and interrupt pathogenesis. (ii) We can develop expertise with viruses from other families that are emerging. This has allowed us to pursue basic questions on the pathogenesis of positive-stranded alphaviruses  and negative-stranded bunyaviruses (La Crosse and Oropouche viruses). And (iii) should a new virus from these families emerge , we have the facility to begin studies quickly to address gaps in basic knowledge.I believe most of my colleagues would agree that although we cannot always predict the next major viral epidemic, if we know enough fundamental virology (and biology), we can respond quickly to ask the right questions and perform the key experiments that allow for the expedited development of disease models, diagnostics, therapeutics, and vaccines. In my opinion, transformative and translational science happens on a foundation of basic research, and we need a profound knowledge base using model systems to gain an adequate breadth and depth of understanding. Preparedness will come not from spending millions of dollars on funding translational initiatives for what we think might be the next pandemic, but rather in investing more substantively in fundamental studies that make discoveries that can be applied to all pathogens.


Costs associated with HPV-related diseases such as cervical dysplasia, cervical cancer, and genital warts have not been evaluated in Sweden. These costs must be estimated in order to determine the potential savings if these diseases were eradicated and to assess the combined cost-effectiveness of HPV vaccination and cervical cancer screening. The present study aimed to estimate prevention, management, and treatment costs associated with cervical dysplasia, cervical cancer, and genital warts from a societal perspective in Sweden in 2009, 1 year before the quadrivalent HPV vaccination program was implemented.Data from the Swedish cervical cancer screening program was used to calculate the costs associated with prevention , management , and treatment of CIN. Swedish official statistics were used to estimate treatment costs associated with cervical cancer. Published epidemiological data were used to estimate the number of incident, recurrent, and persistent cases of genital warts; a clinical expert panel assessed management and treatment procedures. Estimated visits, procedures, and use of medications were used to calculate the annual cost associated with genital warts.From a societal perspective, total estimated costs associated with cervical cancer and genital warts in 2009 were €106.6 million, of which €81.4 million (76%) were direct medical costs. Costs associated with prevention, management, and treatment of CIN were €74 million; screening and management costs for women with normal and inadequate cytology alone accounted for 76% of this sum. The treatment costs associated with incident and prevalent cervical cancer and palliative care were €23 million. Estimated costs for incident, recurrent and persistent cases of genital warts were €9.8 million.Prevention, management, and treatment costs associated with cervical dysplasia, cervical cancer, and genital warts are substantial. Defining these costs is important for future cost-effectiveness analyses of the quadrivalent HPV vaccination program in Sweden. Each year 34 300 women in Europe are diagnosed with cervical cancer, and 16 300 die from this disease , and a rThe Swedish cervical cancer screening program has been in place since the early 1960s, and has effectively reduced the prevalence of cervical cancer in the country by 50% . HoweverHigh-risk HPV testing, i.e. detection of high-risk HPV DNA, is an effective screening method to prevent cervical cancer. When used in primary cervical cancer screening, samples taken by clinicians show the highest sensitivity to detect CIN , though Genital warts are benign mucosal or skin lesions that are common in sexually active men and women. They are usually located at sites that are exposed to epithelial contact during sexual intercourse, most commonly at external genital sites, but they can also be found at internal sites such as the vagina and the uterine ectocervix in women, and the anus in both men and women. Genital warts are often asymptomatic, but they can be psychologically troublesome, and sometimes cause itching, burning, and dyspareunia. Treatment of genital warts depends on several clinical aspects, such as patient preference, planned or known pregnancy, and location and extent of the lesions. Since genital warts are benign and sometimes regress spontaneously, a wait-and-see approach can sometimes be applied. In Sweden, the topical treatments podophyllotoxin and imiquimod are most commonly used for external genital warts, and in some cases destructive treatment and/or surgical removal is also necessary. Internal genital warts on the other hand usually require destructive treatment and/or surgical removal . All of Cervical cancer screening does not prevent HPV infection, but two prophylactic HPV vaccines were licensed in Europe in 2006 and 2007 respectively, with the hope of eliminating the HPV infections that cause cancerous lesions. In 2010, the Swedish National Board of Health and Welfare launched the quadrivalent HPV vaccination program, in which girls 10–12 years of age are vaccinated for free as part of the school vaccination program. The quadrivalent HPV vaccination program aimed to protect against cervical cancer, cervical intraepithelial neoplasia (CIN), vulvar dysplasia, and genital warts caused by the HPV types 16, 18, 6 and 11. In order to estimate the benefits of the quadrivalent HPV vaccination program, decision-makers need to know the prevention, management, and treatment costs that were associated with these HPV-related diseases before the program was introduced. In this way, costs obtained before and after the introduction of HPV vaccination can be compared. As of yet, no study has done this in Sweden. Therefore, this study aimed to estimate the prevention costs , management costs , and treatment costs of CIN, cervical cancer, and genital warts from a societal perspective in Sweden in 2009, 1 year before the introduction of the quadrivalent HPV vaccination program.http://www.sfog.se). As per the guidelines, it was assumed that women with inadequate cytological results were referred back to the outpatient clinic for repeat cytology performed by a midwife, and that women with abnormal cytological results were referred to a gynecologist for immediate colposcopy and biopsy. Also as per the guidelines, it was assumed that women with histologically confirmed CIN2+ had one follow-up visit to undergo loop electrode excision procedure (LEEP), which is the most common treatment currently utilized in Swedish health care. The number of treatment procedures performed among women with CIN2+ was taken from statistics presented by the National Board of Health and Welfare [Data on the number of adequate cytological results  and inadequate cytological results (i.e. results that could not be determined) were obtained from a 2009 report on the Swedish cervical cancer screening program . The rep Welfare . As per  Welfare .The number of incident and prevalent cases of cervical cancer and cervical cancer mortality in Sweden were retrieved from the NORDCAN Database . To estiWomen with histologically confirmed cervical cancer were assumed to be referred for clinical staging according to FIGO procedures, and treated as previously reported in detail . For casrates sic) in the number of new and recurrent cases of genital warts among 20–24-year-olds that were similar to case-reported data from Sweden [There is a lack of register data on genital warts, as they are not included in Sweden’s mandatory surveillance of infectious diseases. Therefore, for individuals under 45 years of age we used epidemiological data from a recent study based on prescription drug registers and patient registers to estimate the incidence of genital warts in Sweden in 2009 . In addim Sweden ,29. Althm Sweden ,30. For www.scb.se). Assuming 20 workdays per month and the activity rate in Sweden, the cost per work day (€138.4) and work hour (€22.9) was used to calculate indirect costs. Based on the cost per work day, total indirect costs were calculated for the prevention, management, and treatment of CIN, as well as the treatment of cervical cancer and genital warts. No indirect costs were estimated for women and men aged 65 years and older, or aged 16 years or younger.A societal perspective, including all direct medical costs and indirect productivity-related costs, was used to estimate costs, which are presented in Euro (€) using the 2009 average exchange rate (€ 1 = Swedish Kroner 10.62) . Direct Direct and indirect costs  were maiThe resources used to calculate the treatment costs associated with incident cervical cancer were based on our previous study . These cFor terminal cervical cancer, the number of patients with who died in 2009 was multiplied by all palliative care costs , includihttp://www.tlv.se). Indirect costs included costs related to travel time, applied to both genders, and treatment time [Direct costs of visits for non-pharmacological or pharmacological reasons, or for treatment including diathermy, cryotherapy, laser, or surgical excision, were extracted from the Patient Level Clinical Costing database. Costs for topical cream treatments were obtained from The Dental and Pharmaceutical Benefits Agency  revealed normal results, 6 502 (1%) inadequate results, and 32 509 (5%) abnormal results . Of all In 2009 there were 441 women with incident cervical cancer in Sweden, the majority (68%) of whom were under 65 years of age, and 158 women died from cervical cancer, the majority (63%) of whom were older than 65 years of age . ApplyinWe applied the figures obtained by extrapolating data from the Swedish study  and dataThe estimated total annual cost associated with the prevention, management, and treatment of CIN, cervical cancer, and genital warts in 2009 was €106.6 million, of which €81.4 million (76%) were direct medical costs. Depending on the sensitivity analysis, total annual cost ranged from €66 million to €121 million.Overall, the results showed that in 2009 the total annual costs associated with the prevention, management, and treatment of CIN, cervical cancer, and genital warts were considerable, at €107 million. Of these costs, €74 million (70%) were associated with the prevention, management, and treatment of CIN, and an additional €23 million (20%) with the treatment of incident and prevalent cervical cancer, which occurs despite the existence of the Swedish cervical cancer screening program, and palliative care. The estimated annual cost of the treatment of incident and prevalent cervical cancer cases is close to the estimated cost of prophylactic HPV vaccination in Sweden . The cosTo our knowledge, this is the first attempt to estimate the annual costs of the HPV-related diseases cervical dysplasia, invasive cervical cancer, and genital warts in Sweden. The estimates are based on the best available data, i.e. the Swedish cervical cancer screening program, epidemiological data on the incidence and prevalence of cervical cancer, and mean estimates of managing and treating genital warts obtained from clinical experts via self-administered questionnaires and reviewed responses.However, this study has several limitations that need to be acknowledged. For example, our overall treatment costs for genital warts were similar to those from other studies ,35, but There are also limitations concerning screening and the number of treatment procedures for women with histologically confirmed CIN. Women who were diagnosed with a CIN1 lesion before 2009 that later developed into CIN2+ would have been referred to immediate treatment, which was not accounted for. Also, in 2009 some county councils used liquid-based cytology and HPV testing to follow-up abnormal cytological results, which may have led to an overestimation of costs. Moreover, women who are HPV-negative at this follow-up are not referred for colposcopy or biopsy, thus the total number of referrals in our study would be somewhat lower.Travel time to and from care sites was based on estimates from Stockholm, which may not be representative of all of Sweden, and costs may therefore be underestimated for geographical areas where distances are greater.We believe that the assumptions made to calculate costs are valid, given the existence of national guidelines for the HPV-related diseases considered in this report, and surveillance data for screening. However, there is a possible underestimation of costs associated with incident and prevalent cervical cancer as potential overlaps in the use of health care resources for incident and prevalent cases diagnosed during the previous year cannot be determined. No costs for recurrent cases were accounted for, which would likely underestimate the total annual costs for the treatment of prevalent cervical cancer cases. Our cost estimates for the treatment of cervical cancer by FIGO stage were similar to those used in a study from the United Kingdom .Our study highlights the significant economic burden (€107 million) of the HPV-related diseases cervical dysplasia, invasive cervical cancer, and genital warts for the 9.34 million affected inhabitants of Sweden in 2009, which is somewhat higher than other studies performed in European countries. A study from the United Kingdom reported annual direct costs for cervical cancer screening, management, and treatment of genital warts of £208 million (for 60 million inhabitants) . In FranWe used the human capital approach to estimate the value of productivity losses due to work absenteeism related to cervical cancer and genital warts. One critique that is often raised with this approach is that it discriminates against elderly who are no longer part of the workforce. This is of course highly relevant for the indirect costs for cervical cancer, since the majority of women diagnosed are of retirement age. However, the screening program affects women of working age, making the indirect costs higher for this particular group. In order to obtain a more accurate deliberation of all indirect costs imposed on Swedish society as a result of cervical cancer and genital warts, a comprehensive assessment is necessary and must be carried out using national registry-based data, which provide individual characteristics including age at diagnosis, disease severity, and inability to work due to sick leave, early retirement, or premature death, which would contribute to heterogeneity in our study population and might influence the total estimated indirect cost.This cost analysis gives important insight into HPV-related diseases in Sweden since it provides recent cost data on the prevention, management, and treatment of CIN, cervical cancer, and genital warts from a societal perspective. Despite the limitations of this study, these results are of interest due to the recent introduction of the quadrivalent HPV vaccination  program. In clinical trials, the quadrivalent vaccine has been shown to reduce lifetime risk of cervical cancer caused by the included HPV types by 47% to 100%, depending on age and coverage ,44, and There are still questions as to whether boys should be included in the HPV vaccination program, and how to wisely design the Swedish cervical cancer screening program to adapt to a situation with a lower incidence and prevalence of precancerous lesions. The costs reported in this study may be incorporated into future cost-effectiveness analyses comparing strategies with different available HPV vaccines alongside the cervical cancer screening program.This study only provides a partial estimate of the cost of HPV-related diseases. Other HPV-related diseases such as cancer of the vulva, vagina, anus, penis, head and neck, and recurrent respiratory papillomatosis were not included in this analysis. Future research should aim to estimate the total direct medical and indirect costs associated with all major HPV-related diseases in Sweden, and to identify cost drivers of relevance to guide decision-making regarding future investments in HPV prevention programs.S1 File(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Bean common mosaic virus (BCMV) is widespread, with Phaseolus species as the primary host plants. Numerous BCMV strains have been identified on the basis of a panel of bean varieties that distinguish the pathogenicity types with respect to the viral strains. The molecular responses in Phaseolus to BCMV infection have not yet been well characterized.Phaseolus vulgaris L., cultivar ‘Stringless green refugee’) to two BCMV strains, in a time-course experiment. We also report the genome sequence of a previously unreported BCMV strain. The interaction with the known strain NL1-Iowa causes moderate symptoms and large transcriptional responses, and the newly identified strain (Strain 2 or S2) causes severe symptoms and moderate transcriptional responses. The transcriptional profiles of host plants infected with the two isolates are distinct, and involve numerous differences in splice forms in particular genes, and pathway specific expression patterns.We report the transcriptional responses of a widely susceptible variety of common bean (Phaseolus vulgaris L.). Virus infection initiated a suite of changes in gene expression level and patterns in the host plants. Pathways related to defense, gene regulation, metabolic processes, photosynthesis were specifically altered after virus infection. Results presented in this study can increase the understanding of host-pathogen interactions and provide resources for further investigations of the biological mechanisms in BCMV infection and defense.We identified differential host transcriptome response after infection of two different strains of Bean common mosaic virus (BCMV) in common bean (The online version of this article (doi:10.1186/s12864-016-2976-8) contains supplementary material, which is available to authorized users. Bean common mosaic virus (BCMV) is a seed borne, aphid-transmitted virus with worldwide distribution [Phaseolus vulgaris L.) is the main host of this virus, although it also infects other Phaseolus species [Potyviridae, it is a single-stranded, positive-sense RNA virus approximately 10 kb in length and encodes an open reading frame for a polyprotein containing 10 genes and a eleventh gene is created by ribosomal slippage in the P3 protein and is called PIPO [ribution . Common  species , 3. This species –6. As a led PIPO –10. Eachled PIPO .R) genes [I locus, and recessive loci, bc-u, bc-1, bc-12, bc-2, bc-22, and bc-3 [I locus is associated with a cluster of R gene homologs belonging to the TIR-NB-LRR (Toll/interleukin- 1-nucleotide binding site-leucine rich repeat) plant immunity receptors [eIF4E [There are 8 pathogenicity groups in the BCMV complex, identified by the virus response on a standard set of differential bean lines –13. The R) genes , 15. Sevand bc-3 , 14, 16.s [eIF4E ). Althous [eIF4E , 19.Host plants activate a number of signaling cascades to recognize different pathogens and to develop suitable defensive strategies –24. DefePhaseolus vulgaris L.) to two BCMV strains on a widely susceptible common bean line, Stringless green refugee. Previous work has demonstrated that pathogenicity group I is present in Iowa and only shows symptoms on bean lines containing no known BCMV resistance genes [Despite the economic importance of BCMV, the molecular pathways associated with BCMV infection are not fully characterized. In order to identify host plant response upon BCMV infection, we characterized the response of common bean . Fourteen days after planting, the first true leaves were mechanically inoculated by first dusting 600-mesh carborundum and then spreading 50 mm potassium phosphate buffer, pH 7.0, containing infectious leaf sap of BCMV NL1-I  was conducted to identify the strain. P. vulgaris cultivars Dubbele witte (PI 377736), Stringless green refugee (PI 560052), Black valentine stringless (PI 549537), Pinto UI 114 (PI 549846), Sutter pink (PI 549706), Monroe (PI 599016), Top crop (PI 554129), Imuna (PI 326420), Redland’s greenleaf B (PI 599004), Great northern UI 123 (PI 549668), Red mexican UI 34 (PI 549732), Sanilac (PI 549695), Michelite 62 (PI 549693), Great northern UI-31 (PI 549671) and Puregold wax (PI 599002) were planted in the greenhouse in individual pots under ambient summer conditions . At 14 days after planting, the first true leaves were inoculated as previous. For each cultivar there was a healthy, mock inoculated plant and between 2–6 infected plants depending on seed germination. One month after inoculation, symptoms were recorded and the symptomatic leaf was selected for testing by ELISA. Leaves were ground in Indirect sample extraction buffer (provided with kit) and applied to the ELISA plate for testing with the potyvirus group test  per company instructions.To further characterize the Unknown strain (BCMV-S2), a test of Leaf tissue was harvested from healthy and virus infected plants using three biological replicates and at several developmental stages. The first trifoliolate leaf of five plants for each replicate was collected at four days post-inoculation, then the second at six days and so on every two days until 14 days post-inoculation. Harvested tissues were immediately frozen in liquid nitrogen and were stored at −80 °C. Two leaves for each time point were tested for the presence of virus by ELISA using the general potyvirus antibody from Agdia Inc., leaving three leaves of each replicate for RNA extraction. Similarly, uninoculated leaves were used as healthy controls.Total RNA was extracted from the infected and control leaf tissues using Qiagen RNeasy® Plant mini kit  as per the manufacturer’s instructions. The extracted RNA was purified using the Qiagen RNA purification kit and treated with DNaseI to remove any DNA contamination. Subsequently, RNA samples were tested for their quantity and quality using a NanoDrop spectrophotometer and Agilent 2100 Bioanalyzer. RNA was extracted from three treatment groups at two developmental stages and three biological replicates  for a total of 18 RNA sequencing libraries that were prepared using 1–3 μg of total RNA from healthy and treatment groups using TruSeq RNA Sample Preparation Kits  per the manufacturer’s protocol. Each library was barcoded and multiplexed in pools of six samples per lane. A total of three lanes were used on the Illumina HiSeq 2000. The sequencing reaction produced single-end reads of 50 bps. The sequencing was conducted at the DNA sequencing core facility, Iowa State University, Ames, IA.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The sequencing adapters and low quality bases at the 5 prime and 3 prime ends were removed using quality scores 20 or below in Trimmomatic [http://www.phytozome.net/) and high quality reads were aligned against it using Tophat software [p value < 0.05 with FDR correction) and their fpkm values. Various splicing events were annotated using ASTALAVISTA bioinformatics software [The single-end raw read data obtained above was processed further, as follows. Reads were separated by sample using the barcode information. The quality of raw reads was evaluated using fastqc  pathway analysis was performed using KOBAS 2.0 [p-value of 0.05 or less was used as threshold to determine significantly enriched pathways. To identify the putative up-regulated or down-regulated transcription factors (TFs) in the expression data, Hidden Markov Model (HMM) motif sequences of different TFs were downloaded from Legume Transcription Factor (TF) Database [Lotus japonicus, and Medicago truncatula. The HMM motif sequences were blasted against the DE gene sequences. The putative TFs were detected using 90 % sequence homology and e-value ≤ 10−10.The mapping information was used to count the number of raw reads using HTSeq package . The HTSOBAS 2.0 . The traOBAS 2.0 . A p-valDatabase . This dahttp://www.ncbi.nlm.nih.gov/). The unmapped raw reads from individual time points were extracted and combined separately for the NL1-I and the unknown potyvirus treatment in a single dataset. The resulting datasets were searched against the virus database to identify the virus-specific read sequences. The matched read sequences were extracted to perform a de novo assembly of the virus genome using default settings in the Trinity de novo assembly platform [A virus BLAST database was built using a large collection of genomes and genes of different plant virus strains from NCBI , virus was not detected by ELISA in the first non-inoculated trifoliolate leaves. At six dpi although no symptoms were observed, NL1-I was detected by ELISA in the same grouping of trifoliolate leaves as day 4 in two of the three replicates, and BCMV-S2 was detected in all three replicates. Both viruses were detected in the non-inoculated leaves in all replicates at the subsequent sampling times of 8, 10, 12, and 14 dpi. Plants infected by either virus became symptomatic at between 11–12 dpi. Based this time course, we used the 4 dpi (the first time point before any positives were recorded) and the 8 dpi  samples for RNA-Seq analyses.P. vulgaris L. version1) genome [de novo assemblies for the complete genome sequences of BCMV-NL1-I (10049 bases) and BCMV-S2 (10048 bases). A search of one of the recovered and assembled viral genomes matched the NL1 genome present in NCBI (Genbank Accession AY112735), so we refer to that virus and assembly as NL1-I. A comparison of the NL1-I and the BCMV-S2 strain nucleotide sequences showed a significant number of polymorphic sites (identity = 9823/10048) between the two virus genomes  genome . Reads sTo investigate the genetic mechanisms of common bean-BCMV interactions, we analyzed the RNA-Seq data to determine how alternate splicing and transcriptome profiles were altered in response to BCMV-NL1-I and BCMV-S2 at early 4 days) and late (8 days) stages of infection. The bioinformatics analysis pipeline is presented in Additional file  days andP. vulgaris v1.0). The overall mapping percentage ranged from 87.4 % to 96.4 %, with the percentage of uniquely aligned reads varying from 80.0 % to 88.1 % ; and when NL1-I reads are mapped against the NL-I genome, there are 6 SNPs  ; and when NL1-I reads are mapped against the BCMVS2 genome, there are 192 SNPs (4 heterozygous)  has a well-characterized role in various plant growth processes and biotic and abiotic stress conditions , 47–50. To determine the statistical significance of various splicing events, we used the cuffdiff program , 37 to ep-value for multiple testing corrections less than 0.05 [We evaluated the coverage of our transcriptome data by considering a gene model as “expressed” if it contained or overlapped with at least three uniquely mapped reads. Approximately 80 % of the published gene models were expressed using this criterion . This gene is the small subunit of ribulose bisphosphate carboxylase and is involved in photosynthesis. The down regulation of this photosynthetic gene in two independent virus treatments suggests that the photosynthetic pathway is highly responsive to virus infection. Another gene, Phvul.010G120600, was induced specifically in the BCMV-S2 treatment and is a NAM  protein, involved in transcriptional regulation. Interestingly, the Phvul.005G073400 was not down-regulated at 8 dpi in either virus strain (suggesting a time point specific response), but in the S2 leaves at 8 dpi, Phvul.010G120600 continued to be induced (1.4214 at 4 dpi compared to 5.8475 at 8 dpi).Comparison of gene expression profiles of healthy and virus treated plants at 4 dpi identified only 1 and 2 DE genes in response to NL1-I and BCMV-S2 inoculation, respectively. A single gene, Phvul.004G073400, was down-regulated under both virus treatments . The Arabidopsis TAIR database (https://www.arabidopsis.org/) was searched to complete annotations of few unannotated common bean genes. Among the top upregulated genes, the receptor-like protein kinases , pathogenesis-related proteins , and oxidative stress related genes  were present  gene ontology (GO) terms associated with DE genes were analyzed at 8 dpi for each virus treatment. The analysis was performed with respect to all genes present in the common bean genome. Various overrepresented GO terms associated with biological, molecular and cellular processes were identified for both NL1-I Fig.  and BCMVp < 0.05) using unique DE genes for the NL1-I treatment. In contrast, ribosome, ribosome biogenesis, and diterpenoid pathways were enriched (p < 0.05) using uniquely expressed genes under the BCMV-S2 treatment.We also performed a pathway analysis using KEGG (Kyoto Encyclopedia of Genes and Genomes) to understand the relation between gene expression pattern and associated pathways for each virus treatment. Different datasets were created for upregulated genes, downregulated, unique, and shared DE genes for each virus treatment. The complete KEGG pathway analysis is presented in Table p < 0.05) under both virus treatments. In contrast, certain downregulated gene specific pathways were unique to each virus treatment. For example, biosynthesis of secondary metabolites, metabolic pathways, carbon fixation in photosynthetic organisms, glyoxylate and dicarboxylate metabolism, and pentose phosphate pathway were unique to NL1-I treatment, while linoleic acid metabolism, alpha-Linolenic acid metabolism, valine, leucine and isoleucine biosynthesis, diterpenoid biosynthesis, and valine, leucine and isoleucine degradation were downregulated (p < 0.05) in the BCMV-S2 treatment. Similar analysis was conducted using the upregulated genes. The results indicated that pathways related to ribosome, ribosome biogenesis, plant pathogen interaction, and glutathione metabolism were shared between two virus treatments and were significantly upregulated (p < 0.05). Certain pathways such as protein processing in endoplasmic reticulum, phenylalanine metabolism, N-glycan biosynthesis, phenylpropanoid biosynthesis, and protein export were uniquely upregulated after the NL1-I treatment, while no unique pathway was observed for the BCMV-S2 treatment.To discern the identity of various induced and repressed pathways upon virus infection, we used the upregulated and downregulated genes separately for KEGG analysis Table . Pathwayhttp://genome.tugraz.at). The normalized expression values from the DE genes in each virus treatment were combined to obtain twenty distinct clusters . This is followed by mitochondrion processes (cluster 11) having 8.2 % of total genes (n = 423). Similarly, clusters 3 and 18 have the smallest sizes  and represent enriched pathways in cellular, photosynthetic, protein modification, binding and catalytic activity. Furthermore, clear patterns are evident in up regulation  and down regulation .Similar expression patterns between different genes often illustrate functional correlation between them. To identify various expression modules related to specific pathways, the DE genes were clustered with k-means clustering using Genesis software  http://gers Fig. . Also, tn = 164), catalytic activity , mitochondrion , reproductive , iron binding  etc., were represented by a single cluster. However, several clusters represented more than one functional classification. For instance, clusters 13 (n = 113), 15 (n = 356), 18 (n = 109), and 20 (n = 138) exhibit a broader level of functional activities related to protein modification, transferase activity, catalytic activity, and metabolism. Overall, this analysis suggests that many pathways respond to virus infection in a modular fashion by co-expressed gene components. Also, identification of co-expressed genes within the same cluster indicates possible interactions between different pathways responding to virus-induced stress.The GO enrichment analysis, using genes in individual clusters, showed that a number of clusters belong to one specific functional category. For example, specific functional classes such as membrane  and identified the matched regions in DE gene sequences . This database includes sixty-one TF families from Glycine max, Lotus japonicus, and Medicago truncatula. The TFs were categorized using separate datasets for upregulated genes and downregulated genes for each virus treatment (Tables The roles of different transcription factors (TFs) under various biotic and abiotic stress conditions have been extensively studied , 57, 58.t Tables . A totalt Tables . The memt Tables . In contt Tables . OverallP. vulgaris and the associated expression changes at two different stages of BCMV infection. We further described the identification and genome sequence of a previously unknown BCMV strain used in this study. At the start of experiment, the identity of one BCMV strain was not known. We utilized the unmapped sequences to produce a de novo assembly of the genomes of two virus strains used in this study. Comparison of genome sequences from the two viruses with known virus sequences in NCBI (http://www.ncbi.nlm.nih.gov/) revealed them as most similar to the strain NL1 of BCMV. However, further sequence comparison of both strains suggests the two viruses are distinct strains of BCMV. To clearly differentiate the transcriptional response to two BCMV strains and to further characterize the severity of symptoms of BCMV-S2, we conducted RNAseq on two time points during infection on a universally susceptible line, stringless green refugee. The analysis of alternate splicing (AS) and differential gene expression (DGE) patterns clearly indicate a strain-specific host response upon infection. A larger number of significant (padj < 0.05) AS events were observed for the BCMV-S2 (342) strain than the NL1-I (172) strain. However, the trend was opposite in terms of DGE, and fewer genes showed differential expression in BCMV-S2 (2099) than for the NL1-I (4676) strain. Overall, these results indicate that the viruses represent two different strains of BCMV which activate differential host transcriptional responses upon infection and that these differences may correlate both to the expanded host range of S2 and the increased severity of the symptoms in susceptible cultivars. Moreover, there is also a high level of overlap between DE genes (upregulated and downregulated) across virus treatments – which suggests that common regulatory pathways respond to the different virus stimuli. In contrast, identification of uniquely expressed genes implies strain-specific induction in each case.This study describes the regulatory landscape of P. vulgaris L. These stages were selected to represent represented early onset of infection to full systemic spread of virus infection in common bean leaves as determined by ELISA. We determined that although we did not observe symptoms until day 12, we could identify viral reads as early as day 4. We identified a number of putative AS events, previously unannotated in the common bean genome. The distribution ratios of detected AS events were consistent across healthy and virus inoculated samples. However, the frequency of occurrence was different for various AS events. Intron retention (IR) and alternate acceptor events represent the most frequent types of AS events (31–35 %). This observation is consistent with previous studies in other monocot and dicot species [Panicum mosaic virus infection in Brachypodium distachyon [Plants respond to stress conditions in part by producing spliced isoforms , 48, 50  species , 62. An stachyon . Howeverstachyon  The decrstachyon , 64. Stastachyon , which cstachyon –65.Arabidopsis thaliana with Turnip mosaic virus (TuMV) [Similarly, host gene expression levels show drastic changes for a large number of genes after virus infection. Also, the expression changes were clearly different for NL1-I and BCMV-S2 strains, which further highlights the differential host responses upon infection with two strains of the same virus. In our study, the initial sampling stage (day 4) defines the start of host response as revealed by expression changes in only 1 and 2 genes for NL1-I and BCMV-S2 treatments, respectively. In particular, a noticeable repression in expression level of a ribulose bisphosphate carboxylase gene (Phvul.004G073400) was detected under both virus treatments. This observation suggests that photosynthetic pathways quickly respond under pathogen stress conditions. This result is consistent with previous data during infection of s (TuMV) . Analysis (TuMV) –71. PathCorresponding changes in photosynthetic pathways and various metabolic activities after pathogen infection have been reported earlier –74. ThesRice stripe virus and Rice tungro spherical virus infection [Various transcription factors (TFs) have well-characterized roles under different stress conditions , 57, 58.nfection . Nonethenfection –83. ThisPhaseolus vulgaris L.) plants. Plants undergo genome level changes in transcript expression levels and patterns during virus infection. Systemic virus infection rewires gene regulatory networks in the host plant. Our analyses increase the understanding of system level changes associated with BCMV infection, and provide a basis for future explorations of plant response to infection with BCMV.Infection of two different strains of bean common mosaic virus (BCMV) clearly showed differential transcriptome response in the common bean (


Bunyaviridae family have revealed a remarkable diversity in their spike organization. Despite this structural heterogeneity, in every case the spikes seem to be composed of heterodimers formed by Gn and Gc envelope glycoproteins. In this review, current knowledge of the Gn and Gc structures and their functions in virus cell entry and exit is summarized. During virus cell entry, the role of Gn and Gc in receptor binding has not yet been determined. Nevertheless, biochemical studies suggest that the subsequent virus-membrane fusion activity is accomplished by Gc. Further, a class II fusion protein conformation has been predicted for Gc of hantaviruses, and novel crystallographic data confirmed such a fold for the Rift Valley fever virus (RVFV) Gc protein. During virus cell exit, the assembly of different viral components seems to be established by interaction of Gn and Gc cytoplasmic tails (CT) with internal viral ribonucleocapsids. Moreover, recent findings show that hantavirus glycoproteins accomplish important roles during virus budding since they self-assemble into virus-like particles. Collectively, these novel insights provide essential information for gaining a more detailed understanding of Gn and Gc functions in the early and late steps of the hantavirus infection cycle.In recent years, ultrastructural studies of viral surface spikes from three different genera within the  Bunyaviridae family, in which over 300 viruses are grouped into five different genera: Nairovirus, Orthobunyavirus, Phlebovirus, Tospovirus and Hantavirus . Se. Se77]. The mature HTNV Gn protein has been shown to start at residue 18 after its first AUG codon , accordiIn order to exit from the ER, heteromultimerization of Gn and Gc glycoproteins seems to be a pre-requisite ,82,83. TAs described, hantaviruses expose spikes that likely consist of four Gn and four Gc proteins ,17. In tBunyaviridae family [in vitro and in vivo in the future.The assembly of the structural components of enveloped viruses occurs generally at specific sites, mostly at microdomains on the plasma membrane or in the internal compartments of the cell ,6. In the family ,89,90,91e family ,92. Howee family ,60,92. Ie family ,93  . A recenBunyaviridae family, the formation of VLPs has been also reported for phleboviruses [Within the oviruses ,114. AmoAs described above, the hantavirus surface spikes form locally ordered patches with a four-fold symmetry ,17. In aIn order to achieve the pinching-off reaction of budding virus particles, many enveloped viruses recruit the cellular ESCRT  machinery that mediates the formation of multivesicular bodies. These cellular complexes are recruited by ubiquitination of residues within proteins present at the membrane of internal compartments (reviewed in ). In a rStructural perspectives of virus structures during early and late events of virus replication have allowed for a discerning of the dynamic transitions of envelope proteins on virus particles; although these proteins form part of the defined spike complexes of mature particles, during cell entry and exit they undergo re-arrangements. Structural plasticity allows them to accomplish their functions during virus assembly, budding, maturation, receptor interaction and membrane fusion as well as to hide epitopes from the neutralizing antibodies of the host cell immune system ,120,121.In this sense, the different spike electron densities observed between TULV and HTNV cryo-ET maps ,17 see  could co(i)During Gn and Gc biogenesis, the glycoproteins seem to require hetero-oligomerization in order to exit the ER (see above). Before entering the secretory pathway, fusion proteins must shield their fusion peptides; this is usually achieved through intermolecular interactions . In the (ii)It has been known for a long time that Gn forms SDS-resistant oligomers, even under reducing conditions ,83,84,85As described above, the particularity of the spike density found in the TULV cryo-ET map  allows oThe notion that TULV particle spikes may correspond to Gn tetramers interconnected by Gc dimers has also been proposed by others . In the Bunyaviridae family. Despite this information, studies on the assembly and budding mechanisms of these viruses are still in their infancy. Recently, more novel insights have been gained regarding the entry processes used by these viruses to invade target cells. Hopefully, more pieces of the puzzle will be uncovered in the near future, such as the molecular structures of the hantavirus glycoprotein ectodomains and the understanding of how the glycoproteins are involved in the different coordinated and successive steps during the viral life cycle. Finally, these insights may allow for the rational design of effective countermeasures against these highly pathogenic viruses.The pieces describing the steps of the hantavirus life cycle are just starting to be characterized and brought together as a complex puzzle. An amazing diversity in the structures of viral-spike complexes has been found among three genera of the


Bemisia tabaci complex, often in a species-specific manner. Papaya leaf curl China virus (PaLCuCNV) has been recorded to infect several crops including papaya, tomato and tobacco in China. To help assess the risks of spread of this virus, we compared the acquisition, retention and transmission of PaLCuCNV among four species of whiteflies, Middle East-Asia Minor 1 (MEAM1), Mediterranean (MED), Asia 1 and Asia II 7. All four species of whiteflies are able to acquire, retain and transmit the virus, but with different levels of efficiency. Transmission tests using tomato as the host plant showed that MEAM1 transmitted PaLCuCNV with substantially higher efficiency than did MED, Asia 1 and Asia II 7. Furthermore, accumulation of PaLCuCNV in the whiteflies was positively associated with its efficiency of transmitting the virus. Altogether, these findings indicate that MEAM1 is the most efficient vector for PaLCuCNV in the four species of whiteflies, and suggest that risks of PaLCuCNV pandemics are high in regions where MEAM1 occurs.Begomoviruses are transmitted by cryptic species of the whitefly  Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a species complex of over 35 cryptic species, recently ascertained by evidence provided by molecular phylogenetic analyses and reciprocal crossing experiments between genetic groups12451The whitefly Begomovirus (family Geminiviridae), which are one of the most important groups of plant viruses in tropical and subtropical regions of the worldBegomovirus, with its more than 200 species, is exclusively transmitted by whiteflies of the B. tabaci complex in a persistent-circulative mode6791011Papaya leaf curl China virus (PaLCuCNV) was a newly isolated monopartite begomovirus without satellite DNA in Guangxi Province of China, and has been causing a severe leaf curl disease on papaya in southern China1214Most of the whitefly-transmitted viruses belong to the genus B. tabaci complex have been recorded, consisting of 14 indigenous species and the two invasive species MEAM1 and MED16B. tabaci fauna occurs in the southern, southeastern and southwestern regions of ChinaB. tabaciB. tabaci complex may transmit a given begomovirus with various levels of efficiency or specificity, and each of the whitefly species may only be able to transmit certain viruses19202122In China, so far 16 species of the B. tabaci complex. We chose for the tests four species of whiteflies, two invasive MEAM1 and MED because they are the most abundant whiteflies across China including Guangxi and in many other countries, and two indigenous Asia 1 and Asia II 7 because they are among the most abundant of the indigenous whiteflies in south and southwestern regions of China, where PaLCuCNV was firstly recorded121316While PaLCuCNV has been spreading in China, no data are yet available on the efficacy of putative vectors of PaLCuCNV, and how the virus got disseminated12B. tabaci complex were originally collected from four localities in China and were maintained in the laboratory (Gossypium hirsutum cv. Zhe-Mian 1793) as cotton is a non-host plant of PaLCuCNV and a suitable host for all the four whitefly species. The insects were reared in a climate-controlled chamber at 26 ± 1 °C, LD 14:10 h and 60 ± 10% relative humidity. Half of the cotton plants in a culture were replaced by fresh ones every 60 days in an alternate manner so as to offer the whiteflies some new plants to colonize every 30 days. The purity of each of the four cultures was examined every three to five generations by randomly sampling 30–50 adults as described in Qin et al. (2013)mtCOI ) PCR-RFLP technique and further confirmed by sequencing of 5–10 individuals. To obtain virus-infected tomato plants (Solanum lycopersicum Mill. cv. Hezuo 903), an infectious clone of PaLCuCNV was used to generate sources of inoculumFour cultures of four cryptic species of the whitefly boratory . All culFor each of the four whitefly species, approximately 300 newly emerged (0–24 h), non-viruliferous adult whiteflies were drawn from their respective culture and transferred to feed on two PaLCuCNV-infected tomato plants enclosed in an insect-proof cage. The lower two older leaves of the plants, which showed mild or no symptoms, were covered by an insect-proof mesh bag one day before the whitefly transfer. Care was taken to gently place the adults evenly onto all the exposed leaves. Following the adult transfer, we randomly collected 10 adults from leaves of the two plants at the end of 13 designated acquisition access periods  on the plants. The adults collected were stored at −20 °C and later assayed individually for detectable PaLCuCNV DNA by conventional PCR (hereafter PCR).For each of the four whitefly species, approximately 400 newly emerged (0–24 h) non-viruliferous adults were transferred to feed on two PaLCuCNV-infected tomato plants enclosed in an insect-proof cage for 48 h AAP to obtain a 100% viruliferous whitefly population as determined above. Then viruliferous adults of each species were collected and released to feed on a healthy cotton plant, a non-host of PaLCuCNV. Following the initial release, a group of 10 live adults were collected at 0, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30 and 40 d respectively. During the collection process, the adults remaining on the cotton were transferred onto a new cotton plant at 15 and 30 d respectively to avoid emergence of new adults. To examine whether the virus might survive in the cotton leaves and whiteflies might acquire the virus through feeding on cotton, we also made detection of the virus in the cotton leaves that had been fed by viruliferous whiteflies for 1, 5, and 10 d. The insects and leaf samples collected were stored at −20 °C and later assayed individually by PCR for detection of PaLCuCNV DNA.For each of the four whitefly species, approximately 500 newly emerged (0–24 h) adults drawn from their respective cultures were released to feed on two PaLCuCNV-infected tomato plants for 48 h. The viruliferous adults were then collected and inoculated in groups of 1, 5 or 10 to feed on the second leaf from the bottom of an uninfected tomato plant at the three true-leaf stage using a clip-cage, for a 48 h inoculation access period. To quantify PaLCuCNV in these whiteflies after a 48 h AAP plus a 48 h inoculation access period, we randomly collected 15 female adults from each of the treatments with 10 adults per plant and conducted quantitative PCR (qPCR) for each individual. The plants were then sprayed with imidacloprid at a concentration of 50 mg/l to kill all the adults and eggs and kept for symptom development in insect-proof cages. With four whitefly species and three inoculation densities, we had 12 treatments each with 21–37 plants (replicates). After 30 days, the tomato plants in each of the treatments were examined to determine virus infection by PaLCuCNV symptoms and further by PCR.For quantification of PaLCuCNV DNA in whiteflies after various durations of feeding on PaLCuCNV-infected tomato plants, approximately 600 newly emerged (0–24 h), non-viruliferous adults of each of the four whitefly species were transferred to feed on three PaLCuCNV-infected tomato plants enclosed in three insect-proof cages. Thereafter we randomly collected 10 female adults from leaves of each plant at the end of 0, 24, 48 and 96 h AAP respectively. The insects samples collected were stored at −20 °C and later assayed as sub-samples of 10 female adults each by qPCR for quantifying PaLCuCNV DNA.et al. (1999)mtCOI gene of the whitefly: primer C1-J-2195: 5′-TTGATTTTTTGGTCATCCAGAAGT-3′ and primer L2-N-3014: 5′-TCCAATGCACTAATCTGCCATATTA-3′et al. (2002)mtCOI and 56 °C for primers of PaLCuCNV. The PCR products were electrophoresed on a 1.0% agarose gel in a 1×TAE buffer containing GelRed  and visualized by a Gel-Doc 2000 system .The DNA from individual whitefly adults was extracted according to the methods of De Barro and Driver (1997)v2 gene was used for quantification with the primers: forward 5′-GACCCGCCGATATAGTCATT-3′, reverse 5′-GTTTGTGACGAGGACAGTGG-3′. β- actin gene forward primer 5′-TCTTCCAGCCATCCTTCTTG-3′ and the reverse primer 5′-CGGTGATTTCCTTCTGCATT-3′ were used with each sample as a normalization gene for verifying equal concentrations of whitefly genomic DNA. Amplifications were performed using the SYBR® Premix Ex Taq™  and ABI Prism 7500 fast real-time PCR system  with SYBR green detection  and the relative abundance of viral DNA was calculated using the 2−Δ Ct method.For quantifying PaLCuCNV in the whiteflies, qPCR assays were conducted. For the whiteflies collected in the transmission assay, DNA extraction method was the same as that of conventional PCR assay mentioned above. For quantifying PaLCuCNV DNA in whiteflies following 0, 24, 48 and 96 h AAP, each group was placed on parafilm and ground in 40 μl of ice-cold lysis buffer with the round end of a sterile 0.2-ml PCR tube, and then washed twice using another 100 μl lysis buffer. Extracts were incubated at 65 °C for 1 h and 100 °C for 10 min, and then were centrifuged briefly. The aqueous supernatant was used as template for qPCR amplification. PaLCuCNV A 2 × 4 test of independence was applied to compare frequencies of virus-infected versus uninfected plants between the four whitefly species at each of the three inoculation adult densities. A 2 × 3 test of independence was applied to compare frequencies of virus-infect versus uninfected plants between the three inoculation adult densities for each of the four whitefly species. The relative concentrations of PaLCuCNV in whitefly adults used in the transmission test were compared by a one-way analysis of variance (ANOVA) at a 0.05 significance level followed by LSD tests. The relative virus concentrations as affected by whitefly species (four levels) and AAPs (three levels) were compared by a two-way ANOVA at a 0.05 significance level followed by LSD tests. All the data analyses were performed using IBM Statistics SPSS 20.0.The whiteflies collected after various AAPs were scored for the presence of viral DNA by PCR. PaLCuCNV DNA was detected in 10% of the tested MED adults with a 30 min AAP and in 10% of all the four whitefly species with a 1 h AAP . The freAs detected by PCR, nearly all of the viruliferous adults of each of the four species retained PaLCuCNV DNA for up to 4–5 days after virus acquisition and transfer to feed on cotton plants . Thereaf2 = 51.473, df = 3, P < 0.001; 5 adults per plant: χ2 = 33.197, df = 3, P < 0.001; 10 adults per plant: χ2 = 13.397, df = 3, P = 0.004). In addition, as the number of adults per plant increased from 1 to 10 the levels of successful virus transmission increased significantly in MED , Asia 1 and Asia II 7 . MEAM1 also achieved higher levels of successful transmission as the number of adults per plant increased and approached 100% successful transmission when 10 adults per plant were inoculated; however, the increase was not significant statistically  apparently due to the already high level of transmission at 1 adult per plant  and AAPs  had highly significant effects on the relative virus concentrations in the whiteflies . The results show that the relative virus concentration was the highest in MEAM1, followed by Asia II 7 and MED, and finally by Asia 1  or Tobacco curly shoot virus (TbCSV)Tomato yellow leaf curl virus (TYLCV)30323334313234When offered access to virus-infected tomato plants, it took the whiteflies a minimum of 0–60 min to acquire detectable amount of PaLCuCNV DNA . BecauseAbutilon mosaic virus, following a 4 d AAP on infected abutilon plants both DNA A and DNA B of the virus were detected in the whitefly throughout a period of 15 days of an experiment while the viral CP was only detected in the first seven days30Following a 48 h AAP on PaLCuCNV infected tomato plants, Asia 1 may retain PaLCuCNV DNA for up to 20 days, while MEAM1, MED and Asia II 7 may retain the virus for 40 days or longer . Compari19Tomato Yellow Leaf Curl China Virus (TYLCCNV) can be transmitted to tomato and tobacco by MEAM1 and Asia II 3 but not by MED or Asia II 119et al. (2014)et al., (2015)Tomato yellow leaf curl Thailand virus (TYLCTHV) and Tomato leaf curl Taiwan virus (ToLCTWV) more efficiently than the MED, and can transmit TYLCTHV more efficiently than ToLCTWV; Li et al.(2010)et al. (2001)Chino del tomate virus more efficiently than the invasive MEAM1. Brown and Idris (2005)B. tabaci species complex based on mtCOI were in concordance with that of the CPs for the genus Begomovirus, suggesting the co-evolutionary histories of begomovirus CPs and their B. tabaci vectors. Their analysis suggests that viruses and vectors that originate from the same geographical region are likely to be the most efficient combinations for transmission40In this study, we demonstrate that MEAM1 can transmit PaLCuCNV more efficiently than the other three species of whiteflies , and var19et al. 14et al.,et al., unpublished data). Interestingly, from 2007 to 2010 when MEAM1 and MED co-occurred in Henan in the central-north region of China, a pandemic of PaLCuCNV in tomato crops was recorded thereThe whitefly MEAM1 most likely entered China in the mid to late 1990s, whereas MED was first detected from ornamental plants in Yunnan Province of China in 2003164216444546et al. (2010)et al. (2015)In conclusion, the current study shows that MEAM1 is an efficient vector of PaLCuCNV and risks of the virus pandemics are high in regions where MEAM1 occurs. Nevertheless, the efficiency of transmission of a virus by a whitefly species may also be affected by the host plantset al. 1020 repoet al. 2015, are yHow to cite this article: Guo, T. et al. Comparison of transmission of Papaya leaf curl China virus among four cryptic species of the whitefly Bemisia tabaci complex. Sci. Rep.5, 15432; doi: 10.1038/srep15432 (2015).


Evidence of infection was low despite frequent exposure to infected poultry and low use of personal protective equipment. The risk for influenza A(H5N1) virus infection is unclear among poultry workers in countries where the virus is endemic. To assess H5N1 seroprevalence and seroconversion among workers at live bird markets (LBMs) in Bangladesh, we followed a cohort of workers from 12 LBMs with existing avian influenza surveillance. Serum samples from workers were tested for H5N1 antibodies at the end of the study or when LBM samples first had H5N1 virus–positive test results. Of 404 workers, 9 (2%) were seropositive at baseline. Of 284 workers who completed the study and were seronegative at baseline, 6 (2%) seroconverted (7 cases/100 poultry worker–years). Workers who frequently fed poultry, cleaned feces from pens, cleaned food/water containers, and did not wash hands after touching sick poultry had a 7.6 times higher risk for infection compared with workers who infrequently performed these behaviors. Despite frequent exposure to H5N1 virus, LBM workers showed evidence of only sporadic infection. During July and August 2010, using a structured questionnaire  Figure 3All blood specimens were transported to the icddr,b laboratory in Dhaka on frozen cold packs at 2°C –8°C. Specimens collected outside Dhaka were centrifuged at the end of each day to separate serum and then transported. Specimens collected in Dhaka were transported to and centrifuged at icddr,b the same day. All serum samples were split into 3 aliquots and stored at icddr,b at −70°C. One aliquot was shipped on dry ice to the Influenza Division at the Centers for Disease Control and Prevention  for H5N1 serologic testing.,>40  and confirmation by an H5-specific Western blot  only among poultry workers with paired serum samples. Candidate risk factors were collinear, precluding the use of a regression model. Therefore, we performed the Kaiser-Meyer-Olkin test to assess the applicability of factor analysis for this dataset  in LBMs in Dhaka and 17% (15/89) in those outside Dhaka. Most refusals were due to an unwillingness to provide a serum sample. We collected data from 101 nonpoultry workers, all of whom were from Dhaka. Overall, compared with nonpoultry workers, poultry workers were younger (median age 28.0 years [interquartile range (IQR) 22.5–38.0 y] vs. 36.0 years [IQR 32–40 y]) and more likely to be male (100% vs. 78%) and to smoke (58% vs. 34%) (p<0.001) .Of 404 poultry workers, 9 (2%) were seropositive for H5N1 virus antibodies at baseline (95% CI 1%–4%). During November 2009–March 2010, routine icddr,b poultry surveillance identified H5N1 virus at 11 (92%) of the 12 LBMs and in 25 (93%) of 27 monthly samples. We obtained a second blood specimen from 278 (72%) of 387 participating poultry workers from the 11 LBMs  Table 1.Of 9 seropositive poultry workers at baseline, 5 remained seropositive and 1 was seronegative for H5N1 virus antibodies at follow-up  Figure; H5N1 virus was not detected by routine poultry surveillance in 1 subdistrict LBM during the study period. We collected follow-up data from 12 (71%) of 17 participating poultry workers at this LBM 1 year after baseline enrollment, and all 12 were seronegative for H5N1 virus at enrollment and follow-up. The overall seroprevalence of antibodies to H5N1 virus among poultry workers from all LBMs during the study period was 5% . In comparison, none of the 101 nonpoultry workers was seropositive (95% CI 0%–4%).In LBMs where H5N1 virus was detected through routine poultry surveillance, we followed 278 poultry workers, of whom 266 were H5N1 virus–seronegative at baseline. These 266 workers contributed 30,043 days (≈82 years) of observation between the collection of paired blood samples, resulting in an incidence of 7 cases/100 poultry worker–years (95% CI 3–16). Using this incidence, we estimate that the annual incidence of H5N1 virus infection after exposure to H5N1 virus at the study LBMs was 50 cases per 721 enlisted poultry workers.Seventeen (94%) of the 18 workers who were seropositive or seroconverted against H5N1 virus and 180 (66%) of the 272 seronegative workers reported exposure to poultry through >1 activity. None of the workers who were seropositive or who seroconverted reported exposure to poultry at home, at their farm, or at another place.Three sets of behaviors explained 95% of the variability among risk behaviors at baseline and follow-up. However, the risk for H5N1 virus infection (risk ratio) was not equal for each set of behaviors  Table 2.Poultry workers who frequently performed high-exposure behaviors had a 7.6 times higher risk for H5N1 virus infection compared with poultry workers who infrequently performed high-exposure behaviors when they also infrequently performed medium-exposure behaviors (p<0.001) . PoultryOur study demonstrates that, despite frequent exposure to infected poultry and low PPE use, LBM workers in Bangladesh had low serologic evidence of H5N1 virus infection. These results also suggest that cross-sectional seroprevalence studies may underestimate the risk for H5N1 virus infection if conducted outside the peak time for H5N1 virus circulation or if samples are obtained from infected workers long after exposure to the virus .,,,Two percent of poultry workers were H5N1 virus–seropositive at baseline. This finding suggests that previous infection with H5N1 virus was uncommon despite the frequent exposure of workers to poultry. One of the workers who was seropositive at baseline became seronegative at follow-up, possibly because neutralizing antibodies decreased below the threshold for laboratory detection  virus, we could not use it for confirmation of seropositivity and seroconversion in this study. Fourth, poultry workers in Bangladesh were engaged in multiple activities, making it difficult to identify which specific behavior was the predominant risk factor for H5N1 virus infection. Last, we were unlikely to have accurately ascertained clinical illness associated with H5N1 virus infections because of the lag between collection of H5N1 virus–positive poultry and environmental surveillance samples and the collection of follow-up blood samples from workers.In conclusion, our study suggests that a low but substantive proportion of LBM poultry workers in Bangladesh become infected with H5N1 virus after unprotected, ongoing sporadic exposures to H5N1 virus–infected poultry and virus-contaminated environments of LBMs. The risk behaviors identified in our study may help public health officials explore interventions to interrupt poultry-to-human transmission of H5N1 virus and other avian influenza A viruses among the poultry workers. The cost of any interventions needs to take into account the anticipated potential modest benefit of decreasing an infrequent event with uncertain pandemic potential.Questionnaires administered to the poultry workers and nonpoultry workers during a study of avian influenza A(H5N1) virus infection among workers at live bird markets.Workers’ baseline characteristics, exposure to poultry, and use of personal protective equipment; contribution of individual behaviors of poultry workers for each set of exposure behaviors; and timeline for testing.


Extensive surveillance in bat populations in response to recent emerging diseases has revealed that this group of mammals acts as a reservoir for a large range of viruses. However, the oldest known association between a zoonotic virus and a bat is that between rabies virus and the vampire bat. Vampire bats are only found in Latin America and their unique method of obtaining nutrition, blood-feeding or haematophagy, has only evolved in the New World. The adaptations that enable blood-feeding also make the vampire bat highly effective at transmitting rabies virus. Whether the virus was present in pre-Columbian America or was introduced is much disputed, however, the introduction of Old World livestock and associated landscape modification, which continues to the present day, has enabled vampire bat populations to increase. This in turn has provided the conditions for rabies re-emergence to threaten both livestock and human populations as vampire bats target large mammals. This review considers the ecology of the vampire bat that make it such an efficient vector for rabies, the current status of vampire-transmitted rabies and the future prospects for spread by this virus and its control. Lyssavirus; family Rhabdoviridae. It is now considered a re-emerging disease in different countries of the world and is associated with increased rates of reservoir contact [Rabies is a zoonotic disease caused by viruses belonging to the genus  contact .The dise contact . Through concerted action across the Americas, rabies virus transmission from dogs to humans has been controlled in virtually all countries of the region. This is reflected in the continued decline in human rabies over the past two decades . HoweverThe earliest description of human death associated with vampire bat attacks comes from the time of the Spanish conquest of the Americas in the 16th century . Rabies Vampire bats preferentially prey on livestock . LivestoBovine rabies in Latin America is commonly called derriengue, a Spanish word for a fatal paralytic disease . The infThe increase in the abundance of species such as vampire bats in the Americas has had a direct impact on human and animal health ,18. MoreDesmodus rotundus , intramuscular (IM) and sub-cutaneous (SC) routes. Disease development was dependent on inoculation route and virus dose with IC inoculation inducing rabies in all recipients, whereas only a high virus dose  caused disease in the majority of bats inoculated by other routes. Virus was detected in both salivary glands and saliva from bats that developed rabies. Later studies have provided descriptions of rabies in vampire bats. The initial sign in captive bats is a decrease in blood consumption [Rabies in vampire bats has been investigated through experimental infection. Moreno and Baer  inoculatsumption  leading sumption ,30. RABVsumption . This issumption . There aThe rabies virus genome is a negative-sense, single-stranded RNA genome approximately 12,000 base pairs in length. This contains five discrete coding sequences that encode the structural proteins, nucleoprotein, matrix and glycoprotein, and non-structural proteins, phosphoprotein and RNA-dependent polymerase. All lyssaviruses contain this simple genomic structure . The maiUntil the introduction of virus typing by monoclonal antibodies, it was impossible to distinguish between rabies viruses isolated from infected brain material. Typing, using this method, allows the differentiation of viruses based on the binding, or not, of panels of monoclonal antibodies directed against the virus nucleoprotein  or glycoComparison of genomic sequences in order to discriminate rabies viruses began in the early 1990s  and has Artibeus lituratus) were identified to be a variant associated with hematophagous bats through the use of monoclonal antibody panels. In Brazil it has been demonstrated that rabies viruses isolated from Brazilian frugivorous bats (Artibeus spp.) were phylogenetically characterized as the vampire-bat related RABV [D. rotundus, was found in 71.4% (15/21) of non-haematophagus bat species sampled. AgV-3 was also identified in 90.9% (10/11) of the frugivorous bats Artibeus lituratus and 50% of the insectivorous bats , and the nectivorous Pallas’s long-tongued bat (Glossophaga soricina). The genetic variant related to haematophagous bats was identified in 75.8% (22/29) of the studied samples. Those results were also observed in studies with samples isolated from bats from the state of São Paulo and other Brazilian regions. An intensive investigation of vampire bat rabies in Peru and neighboring countries identified four discrete lineages [The ability to associate a particular virus lineage with a particular reservoir host has greatly assisted in revealing the interrelationships between rabies virus and New World bats. Analysis of RABVs in North American insectivorous bats indicate that particular lineages are associated with particular bat species, possibly through adaptation to the host, and that transmission between species is rare or rarely detected . Howeverted RABV . Also inted RABV  found thlineages . In addiCochliomyia hominivorax) that in extreme cases can lead to death [Fossil vampire bats have been detected throughout the Americas dating from the late Pleistocene age over two million years ago. Images of what are clearly vampire bats were created by the Mayan civilization and descriptions of vampire bats were recorded by Spanish explorers in the 16th century . It has to death .Bats have been associated with a large range of zoonotic viruses . HoweverDespite this apparent picture of decline in rabies cases, in certain countries there is evidence that cases have increased over this twenty year period. For example in Mexico, numbers of rabies cases fluctuate widely from year to year with no clear trend in the prevalence of disease . Many ofD. rotundus, is currently the main cause of human rabies in Brazil [Historically, human mortality due to rabies transmitted by vampire bats has remained low because bats do not usually attack humans. However, prior to the 1970s, there had been 150 human deaths reported that were attributed to transmission by vampire bat attacks . Aggressn Brazil . A recenn Brazil  and Box The number of rabies cases associated with wild species, especially those transmitted by vampire bats, seems to be increasing and the patterns of occurrence of this disease are also changing ,64,65. T3) but glucose levels were normal. The patient developed a fever with a temperature of 38.4 °C and he became generally less responsive. On August 20th, rabies virus-specific IgM and IgG were detected in the patient’s CSF and serum. On August 21st, after discussion with the patient’s family, ventilator support was removed that the patient died shortly after. Rabies virus was confirmed by detection of viral antigen in brain tissue taken at postmortem. A link to vampire bats was suggested by antigenic typing of the virus isolated from the patient and genetic analysis of nucleic acid sequencing derived from reverse transcription polymerase chain reaction.In July, 2010, a 19 year old male migrant worker from Mexico developed fatigue with pain in his left hand and shoulder. He sought medical attention on July 30th and was transferred to New Orleans on August 3rd when his symptoms persisted. Further physical examination revealed generalized areflexia and a drooping left upper eyelid. A cerebrospinal fluid (CSF) sample contained elevated white blood cells  in the vampire bat population. Further surveillance did not detect any rabies cases in cattle within the control area but some cases were reported to the south east of this zone bringing into question the long-term efficacy of this strategy when dealing with a highly mobile host species.The observation of vampire rabies movement suggests that a landscape is covered in a network of interacting bat colonies, which include maternity roosts and satellite male roosts. Interactions are frequent, often daily, and can include behavior such as social grooming and blood-meal sharing . This enThe impact of vampire predation on livestock is such that a number of control methods have been used. The most dramatic and destructive is that of colony destruction that in extreme cases has included the dynamiting of caves  or gassiD. rotundus are treated and destroyed. A variation of this approach is to coat the walls of roosts with anticoagulant although this again can lead to indiscriminant killing of other bat species and the anticoagulant can persist in the environment for years. An alternative is to inject cattle with low levels of anticoagulant that has no effect on livestock and does not affect the suitability of meat for the food chain but is of sufficient strength to be lethal to vampire bats. A variation of this approach is to apply the anticoagulant to fresh wounds in a paste formulation, relying on the behavior of the vampire bats to return to a prior prey location. This approach is more costly although it is specifically targeted at vampire bats. If protection of rabies is the main concern then vaccination of cattle is effective but again is costly and not always adopted by farmers.More targeted methods of control include the capture of vampire bats and coating the animal with Vaseline containing an anticoagulant such as Warfarin. The bat is then released and through mutual grooming spreads the anticoagulant throughout the colony leading to the death of its members. A disadvantage of this approach is that often bat species other than Anticoagulants have been used in most Latin American countries since the seventies. In Mexico, its use is indicated in the national recommendations in response to a disease outbreak. However, despite its application for more than 40 years, rabies in vampire bat populations has increased over time. From the 1970s rabies was present in approximately 65% of the area inhabited by the common vampire bat, and currently in the 21st century, almost 100% of this area shows bovine paralytic rabies (et al. [Streicker and co-workers  reportedet al.  should bet al. .Experimental vaccination against rabies of vampire bats using a range of applications including both parenteral and oral administration has been attempted and shown to be effective ,75. HoweD. rotundus to move north and introduce another rabies variant into US wildlife [Reports of both livestock and human cases of rabies are increasing, mainly due to the continuing encroachment of human populations into areas occupied by vampire bats and there is now strong evidence that the range of vampire bats is also increasing. It is possible that vampire bats could move further north and into the United States, bringing with them the risk of rabies virus transmission to humans and livestock. Fossils of vampire bats have been found in a number of US states from warmer periods in earth history . This suwildlife . In ordewildlife  have sug