The five families of the LRR-RLKs except for the RPK2 family show 1N = 8 − 18 and 2N = 3 − 5. The nine families of the LRR-RLPs show 1N = 12 − 33 and 2N = 4; while 1N = 6 and 2N = 4 for the rice Os10g0479700 family and the 1N = 4 − 28 and 2N = 4 for the soybean protein family. The rule of 1N » 2N might play a common, significant role in ligand interaction, dimerization, and/or signal transduction of the LRR-RLKs and the LRR-RLPs. The structure and evolution of the LRR domains with non-LRR IRs and their proteins are also discussed. LRRs (leucine rich repeats) are present in over 14,000 proteins. Non-LRR, island regions (IRs) interrupting LRRs are widely distributed. The present article reviews 19 families of LRR proteins having non-LRR IRs (LRR@IR proteins) from various plant species. The LRR@IR proteins are LRR-containing receptor-like kinases (LRR-RLKs), LRR-containing receptor-like proteins (LRR-RLPs), TONSOKU/BRUSHY1, and MJK13.7; the LRR-RLKs are homologs of TMK1/Rhg4, BRI1, PSKR, PSYR1, Arabidopsis At1g74360, and RPK2, while the LRR-RLPs are those of Cf-9/Cf-4, Cf-2/Cf-5, Ve, HcrVf, RPP27, EIX1, clavata 2, fascinated ear2, RLP2, rice Os10g0479700, and putative soybean disease resistance protein. The LRRs are intersected by single, non-LRR IRs; only the RPK2 homologs have two IRs. In most of the LRR-RLKs and LRR-RLPs, the number of repeat units in the preceding LRR block ( Arabidopsis thaliana and Oryza sativa subsp. japonica (rice) contain over 700 and 1,400 LRR proteins, respectively  and Ricinus communis LRR-RLK [B9T4K2] have LRRNTs with 26−27CCx. The LRRCT regions are rich in His, Arg, and Lys residues, and thus are basic.BRI1/SR160 is a receptor complex for brassinosteroids that are necessary for plant development, including expression of light- and stress-regulated genes, promotion of cell elongation, normal leaf and chloroplast senescence, and flowering ,90,91,92 N1 » N2 . A. thalsteroids . The non1N = 17 − 18 and 2N= 4 (−125Yx5F. Most LRRCT regions are basic. Daucus carota PSKR contains 22 LRRs intersected by a 36-residue IR between LRR17 and LRR18. An LRRNT (with 33CCx6CCx) that is similar to that in PGIP . LR. LR20]. m fulvum ,103,104.ectively ,106,107.m fulvum ,109. Thebidopsis ,111,112 aponicus ,114. Zeaelopment . A. thalntenance . The LRRN. tabacum  is an LRR@IR protein . The homA. thaliana TONSOKU(TSK)/MGOUN3(MGO3)/BRUSHY1(BRU1), which is localized in the nucleus and is preferentially expressed in the shoot apex than in the leaves and stems, is required for cell arrangement in root and shoot apical meristems and involved in structural and functional stabilization of chromatin  and with 623 residues [B8A383_MAIZE] are paralogs of Z. mays TMK1 with 958 residues (1N= 3 and2N = 3. There are other examples; a hypothetical protein from Z. mays with 247 residues [C0PL86] and fasciated ear2 with 613 residues, O. sativa Os02g0782800 with 441 residues [Q6K7E5] and BRUSHY1 with 1,332 residues [Q6K7D3]. The occurrence of these proteins is attributed to gene duplication and deletions. Two putative uncharacterized proteins from residues . The 7171N » 2N in which 1N is variable in their individual homologs, while 2N is highly conserved. For all known LRR-RLPs, 1N = 4. The rule of 1N » 2N plays a common, significant role in ligand-interaction, dimerization, and/or signal transduction of the LRR-RLKs and the LRR-RLPs. All of the LRR domains consisting of PS- LRRs are predicted to form a superhelix and non-LRR IRs in plant LRR@IR proteins fold back into the interior or exterior of the superhelix. The present analyses suggest that some LRR-RLKs and LRR-RLPs evolved from gene duplications and recombination. The present review will stimulate various experimental studies to understand the structure and evolution of the LRR domains with non-LRR IRs and their proteins.Most plant LRR@IR proteins have LRRs intersected a single IR with


The Ugandan Ministry of Health has endorsed voluntary medical male circumcision as an HIV prevention strategy and has set ambitious goals . Innovative strategies to improve access for hard to reach, high risk, and poor populations are essential for reaching such goals. In 2009, the Makerere University Walter Reed Project began the first facility-based VMMC program in Uganda in a non-research setting. In addition, a mobile clinic began providing VMMC services to more remote, rural locations in 2011. The primary objective of this study was to estimate the average cost of performing VMMCs in the mobile clinic compared to those performed in health facilities (fixed sites). The difference between such costs is the cost of improving access to VMMC.A micro-costing approach was used to estimate costs from the service provider’s perspective of a circumcision. Supply chain and higher-level program support costs are not included.The average cost (US$2012) of resources used per circumcision was $61 in the mobile program ($72 for more remote locations) compared to $34 at the fixed site. Costs for community mobilization, HIV testing, the initial medical exam, and staff for performing VMMC operations were similar for both programs. The cost of disposable surgical kits, the additional upfront cost for the mobile clinic, and additional costs for staff drive the differences in costs between the two programs. Cost estimates are relatively insensitive to patient flow over time.The MUWRP VMMC program improves access for hard to reach, relatively poor, and high-risk rural populations for a cost of $27-$38 per VMMC. Costs to patients to access services are almost certainly less in the mobile program, by reducing out-of-pocket travel expenses and lost time and associated income, all of which have been shown to be barriers for accessing treatment. Voluntary medical male circumcision (VMMC) significantly reduces the risks of acquiring HIV in men.–6 In UgaIn 2009, the Makerere University Walter Reed Project (MUWRP), which is a non-profit partnership between Makerere University in Uganda and the U.S. Military HIV Research Program , began the first VMMC program in Uganda in a non-research setting. Between February and September 2009, MUWRP conducted a pilot program in two sub-counties in Kayunga district. Following the successful implementation of the pilot program, the MUWRP expanded VMMC services to men living in or near Kayunga and Mukono Districts . In addition to providing VMMC operations at health facilities, a mobile VMMC clinic began providing VMMC services to more remote, rural locations  in 2011.Although several analyses have generally evaluated the cost-effectiveness of male circumcision in different settings, 5, 9–14The MUWRP VMMC program supports activities in Kayunga, Buvuma and Mukono Districts of Uganda . Facility-based VMMC is currently provided at Kayunga District Hospital and at Kojja Health Center IV and Mukono Health Center IV, with VMMC operations performed in newly renovated “minor” theaters with 2 surgical beds in each facility dedicated to VMMC operations. The mobile clinic also provides VMMC services in Kayunga and Mukono Districts, focusing on remote villages where men do not have local access to VMMC services, including remote fishing villages near Lake Kyoga in Kayunga District with high rates of HIV. Dorsal slit and forceps-guided are the primary techniques of circumcision used in the program.Adverse events requiring extra medical care following VMMC performed by this program are rare. For example, the MUWRP conducted a pilot program in two sub-counties in Kayunga district. Clinical outcome data on 316 de-identified clients were reviewed retrospectively at one, four and fifty two weeks post-surgery (Protocol RV 277). By 4 weeThis VMMC program, both at three fixed sites and the mobile unit, utilizes staff  trained by the Ugandan Ministry of Health (MOH) for performing operations and monitoring clients. The same staff members work in the fixed sites and in the mobile program, and receive the same training. The MUWRP receives funding from U.S. President’s Emergency Plan for AIDS Relief (PEPFAR) for staffing, supplies and related activities .The mobile VMMC clinic is a truck with a self-contained surgical unit and two surgical beds mounted on the frame. A generator provides electricity for the unit (including air conditioning). The mobile program began providing services in March of 2011. The mobile clinic is “based” at a fixed site , and the MUWRP program manages the overall VMMC program (the fixed sites and mobile clinic).For both fixed and mobile sites, the overall VMMC process begins with community mobilization and recruitment activities to generate demand for the service . Once a recruit presents at a mobile or fixed site, the patient then proceeds through three main steps: (1) VMMC counseling and HIV testing; (2) completing a medical exam to confirm eligibility for the procedure that day; and (3) completing the surgical procedure (with post-operation follow up). Step 1 also includes obtaining consent from the patient for the procedure. At each of these stages, various supplies, equipment, and staff are used. The costing analysis presented here organizes the costs for each stage of the VMMC process.While called the “mobile” program, the mobile unit is driven to a location where it remains for several days , and then the vehicle returns to the home base near Kayunga District Hospital at the end of the period. For locations that are not too far away from the home base, the staff travel each day to the site (in a separate vehicle) for work and then return home in the evening. This type of location is called “staff returning”. For locations that are more distant and/or with more difficult roads, staff members remain with the mobile unit throughout the period and find accommodations and meals in the local community. The program calls this a “camping” location in the sense that staff members are lodged in nearby towns rather than returning to their homes each evening. Costs for each type of mobile program, staff returning and staff camping, along with costs for a fixed site, are included in this analysis.At the fixed site, waste is managed according to standard procedures at the site. A sanitary officer is included in staff costs for managing this waste, and other services such as incineration are included as part of the facility management and administration costs. For the mobile program, waste is collected during the day and the returned with the staff (the staff returning model). With a camping location, staff members are housed in locations with hotels, which are also in communities with government health facilities with incinerators. In this case, the daily waste  is delivered to such facilities. The program pays no fee for this service, and no additional costs for this service is included because of the small quantities involved.This program evaluation focuses on the costs of VMMC performed in the mobile program and comparing to costs of procedures performed in a typical fixed site. All costing information is based on routine program implementation records. No human subjects data were used in this analysis.All costs above the service-delivery level, such as MUWRP program management, costs of training staff, supply chain, and so on, are excluded from this analysis. This approach is similar to previous analyses focusing on direct costs of VMMC. These hiwww.bou.or.ug/bou/rates_statistics/statistics.html). Items purchased in other currencies (USD) were converted to UGX based on the annual average exchange rate for the purchase year (Oanda.com interbank rate). Equipment costs were converted to a monthly equivalent cost based on expected working life and a 3% real annual discount rate. All costs are expressed as cost per circumcision.A micro-costing approach, from the perspective of the program implementer, was used to document and account for resources used for completing VMMC procedures through both service delivery models. All unitAlthough the VMMC procedure is provided free-of-charge in both the fixed and mobile clinics, patients incur other costs, such as the opportunity cost of their time to travel to a clinic for a procedure and recovery as well as any out-of-pocket expenses. While these costs are excluded from this analysis, such costs will influence whether a patient presents to a facility for a procedure as well as at which location.All information, assumptions, and calculations developed and used for this analysis are presented in two Excel files provided as supplemental digital content  Fixed. EFor the mobile program, the worksheet labeled “preliminary information” contains a The worksheet “1. VMMC Ed and HCT” organizes costs associated with VMMC education, counseling and consenting and HIV counseling and testing. These activities take place in a tent set up near the mobile unit, and the VMMC ED and HCT sheet first organizes costs for equipment used directly for this stage . Once the costs per month for such equipment are calculated, the costs per patient for this step  are estimated . Step 2 is the medical exam, which is completed by a clinical officer who provides the medical exam and then also performs the operation on the same patient . The exam is also completed in a tent set up near the mobile unit, and some additional basic equipment is used during the exam .Step 3 is the actual procedure, and three separate worksheets are used to organize costs for this step and for any other costs not included in the previous steps. The worksheet “S3. Equipment and Other” covers equipment costs, including the costs for purchasing and importing the mobile unit from South Africa, and various other supplies and services used in the mobile program . . Because locations with staff returning are closer than camping locations (an estimated 45 kilometers compared to 120 kilometers on average for 2012), the same worksheet “S3. Equipment and Other” estimates costs for a location with staff returning daily and for camping locations separately. The sheet labeled “S3. Staff salaries and costs” first shows standard salaries for various types of program staff, from which standard daily wages are estimated.The table labeled “S3. Staff Costs per Procedure”, which exclude staff costs from Step 1, then estimates salary costs based on the number of staff used in various capacities . Patients are counseled to return after 7 days and one month for follow up visits. At the fixed sites, these services are provided on top of performing circumcisions by the same staff, so the analysis includes such services. For the mobile program, a clinical officer remains at the site for 7 days to complete the initial 7 day follow up visit . These costs are included in the analysis. At the 7-day follow up visit, patients receiving services in the mobile program are then referred to their local health facility for the 4-week follow up visit. The cost of providing the 4-week follow up visit is not included in this analysis for the mobile program. The sheet labeled ‘S3. Supplies for VMMC procedure (not included elsewhere)’ includes a list of supplies used during the procedure itself, the most important of which is the disposable surgical sets used in the mobile program.The final sheet in the Excel file is labeled “Total cost per VMMC”, which reports costs separately for VMMC performed at sites with staff camping and staff returning (in UGX and USD). A separate Excel file shows the analysis for the fixed site and is organized in a similar fashion. The analysis for Steps 0, 1, and 2 are essentially the same for the fixed and mobile programs. For Step 3, the fixed site has only one model of service delivery (as compared to the staff camping or staff returning models for the mobile program). A main difference between the mobile and fixed sites is that the mobile program uses disposal surgical kits, while the fixed sites use reusable surgical supplies (e.g. that are washed and sterilized after each use). Thus, the list of supplies used in Step 3 is substantially longer than the list for the mobile program. Three fixed sites are providing VMMC services as part of the MUWRP. Staffing and resources used are consistent across sites. For this analysis, information on the costs of facility renovation, utilities, and patient flow are based on the Mokono Health Center.Based on program information in 2012, we estimate that the cost to complete one VMMC in the mobile program was $60.79 for locations with the staff returning and $72.21 for locations with staff camping see . The difWe estimate that the cost to complete one VMMC was $34 at the fixed site. As with the mobile program, costs for community mobilization, VMMC education and HIV testing, and the exam contributed minor amounts to costs per VMMC. The cost categories for Step 3 differ somewhat between the mobile and fixed sites, and the categories are lined up in Improving access to VMMC through a mobile VMMC program that brings the service to remote, rural, low-income, and high-risk communities is estimated to cost between $27–$38 per VMMC.The cost of the disposable surgical kit, $23 in 2012, was the single largest component of cost in the mobile program, larger than total equipment costs per procedure or total salary costs of staff. Reductions in the price of these kits  will translate into direct reductions in the costs per procedure. For comparison, supplies used during the VMMC operation for the fixed sites are estimated at only $12.90, mainly because the fixed sites do not use the disposable surgical kits.Another key difference is patient flow. In the mobile program, on the days when procedures were performed, 30 procedures were completed daily (roughly one every 30 minutes during an 8 hour day with two surgeons). In contrast, 13 operations were performed on average per day. This difference in average procedures per day reflects the fact that fixed sites provides services almost every working day during the year, while days per year are fairly limited for any one location visited by the mobile unit.These cost estimates are fairly robust to wide ranges of alternative assumptions regarding equipment and patient flow. Raising the discount rate to 10% increases cost per VMMC in the mobile program by about $3. Extending the mobile unit’s working life from 10 years to 15 years reduces cost by about $3. Increasing patient flow in the mobile program from 30 to 40 procedures per day (from 15 to 20 patients per surgeon per day) reduces costs by a minor amount, while cutting patients to only 15 per day total increases costs by a minor amount ($2). In the fixed program, increasing patient flow to 30 patients per day reduces costs by about $7 . If the mobile unit operated 240 days during the year, compared to 120 actual days during 2012, costs per procedure would fall by $6–7.Even if a mobile unit and all related costs  could be procured for 50% less than the actual costs, costs would only fall by $7/6 for staff returning/camping locations. When demand for VMMCs is expected to be high at a site, the MUWRP VMMC program is able to bring along extra capacity  to perform circumcisions. This approach essentially allows the program to spread out the upfront costs for the mobile unit across more procedures, recognizing some additional costs associated with the additional test and related surgical equipment .Rather than reducing the cost of access by bringing the service closer to the targeted communities, a program could also consider alternative approaches such as paying the patient to travel to a fixed clinic. For example, in a recent study from Kenya, which randomized men to 4 payment levels if they completed a VMMC at one of the study sites , a statistically significant but absolutely small improvement in VMMC uptake after 2 months was observed in the two higher-payment groups compared to the lower two groups (e.g. from about 2% up to about 7–9%).Evaluating health service delivery requires more information than just costs to the provider. The question that cannot be addressed in this paper is if the estimated difference in average costs between the fixed and mobile settings is reasonable based on additional information that should logically be considered by policy makers. By targeting rural locations with relatively high HIV prevalence, fewer VMMCs would need to be performed to avert one HIV infection. For patients in remote locations, their costs to access services are certainly less in the mobile program compared to fixed sites. If communities in more remote locations also are poorer than communities near fixed sites, the mobile program also improve equity of access to health. Targeting communities that are relatively remote, poor, and with high HIV risks are logical strategies to improve the cost effectiveness of mobile VMMC programs based on cost per infected averted.S1 Costs Mobile(XLSX)Click here for additional data file.S1 Costs Fixed(XLSX)Click here for additional data file.


Algae and other cobalamin auxotrophs rely on environmental cobalamin supplied from a relatively small set of cobalamin-producing prokaryotic taxa. Although several Bacteria have been implicated in cobalamin biosynthesis and associated with algal symbiosis, the involvement of Archaea in cobalamin production is poorly understood, especially with respect to the Thaumarchaeota. Based on the detection of cobalamin synthesis genes in available thaumarchaeotal genomes, we hypothesized that Thaumarchaeota, which are ubiquitous and abundant in aquatic environments, have an important role in cobalamin biosynthesis within global aquatic ecosystems. To test this hypothesis, we examined cobalamin synthesis genes across sequenced thaumarchaeotal genomes and 430 metagenomes from a diverse range of marine, freshwater and hypersaline environments. Our analysis demonstrates that all available thaumarchaeotal genomes possess cobalamin synthesis genes, predominantly from the anaerobic pathway, suggesting widespread genetic capacity for cobalamin synthesis. Furthermore, although bacterial cobalamin genes dominated most surface marine metagenomes, thaumarchaeotal cobalamin genes dominated metagenomes from polar marine environments, increased with depth in marine water columns, and displayed seasonality, with increased winter abundance observed in time-series datasets . Our results also suggest niche partitioning between thaumarchaeotal and cyanobacterial ribosomal and cobalamin synthesis genes across all metagenomic datasets analyzed. These results provide strong evidence for specific biogeographical distributions of thaumarchaeotal cobalamin genes, expanding our understanding of the global biogeochemical roles played by Thaumarchaeota in aquatic environments.Cobalamin (vitamin B Other cobalamin-dependent enzymes include ethanolamine ammonia-lyase, methylaspartate ammonia-lyase and methylaspartate mutase  or with a deoxyadenosine .Cobalamin  have been implicated in controlling bloom distributions and species successions in the ocean water column .The Integrated Microbial Genomes system  portal on 8 January 2014 , cobI_cbiL (TIGR01467), cobJ_cbiH (TIGR01466), cobM_cbiF (TIGR01465), cbiT_cobL (TIGR02469), cbiE_cobL (TIGR02467), cbiC_cobH (PF02570.10), cbiA_cobB (TIGR00379), cbiB_cobD (TIGR00380), cbiP_cobQ (TIGR00313), cobS (TIGR00317)) were selected as cobalamin pathway markers for further analysis due to their broad distribution throughout the cobalamin biosynthesis pathway. For each of these 11 genes, the corresponding HMM was used to scan for homologs in all sequence reads of each metagenome, which were initially processed into open reading frames using FragGeneScan  were used for genes of the cobalamin biosynthesis pathway that were retrieved from the TIGRfam (n<10) reduced potential statistical bias resulting from low sample sizes. To generate an overall measure of per-taxon cobalamin gene contributions for each sample, we combined data for the 11 cobalamin marker genes as follows. For a given dataset, D, the per-taxon cobalamin (cob/cbi) gene contribution (Dt) was computed as:Taxonomic frequency profiles were generated by dividing the number of cobalamin synthesis hits per taxon by the total number of hits for a given gene category. Removing samples with low overall cobalamin synthesis gene frequencies . We analyzed metadata correlations across all individual metagenome datasets and also for pooled sample datasets for larger studies, with many samples characterized by common study-specific metadata. Standard Pearson correlations were used to calculate correlation coefficients (r). Maps were generated with the maps package in R (http://cran.r-project.org/web/packages/maps/index.html), with metagenome samples plotted by their associated latitude and longitude metadata. Hierarchical clustering was performed using the Ward method with Manhattan distances in R.Correlation analyses of numerical metadata from each metagenome compared individual physical and chemical characteristics with per-taxon cobalamin gene contributions , Ribosomal_L18e (PF00828), and Ribosomal_S9 (PF00380)) that were selected from a previous publication  and possess genes encoding enzymes specific to the anaerobic biosynthesis pathway . This is supported by the presence and absence of marker genes as defined by the KEGG , ribonucleotide reductase (Nmar_1627), and methylmalonyl CoA mutase , as well as probable cobalt transporters (Nmar_0878).Genomic analysis of the ammonia-oxidizing archaeon ynthesis . Althougynthesis . N. marirchaeota  lack genthe KEGG . N. maricob/cbi gene clusters conserved across all known ammonia-oxidizing thaumarchaeotal genomes. The two relevant gene clusters are present in the genomes of Candidatus Nitrosoarchaeum limnia, Candidatus Nitrosopumilus salaria, Candidatus Nitrososphaera gargensis, and Candidatus Nitrosopumilus koreensis AR1 , Cyanobacteria (C), and Thaumarchaeota (T). At the species level, Thaumarchaeota dominated among prevalent organisms  relative to their community abundance . These results indicate that Thaumarchaeota are abundant global cobalamin producers through the anaerobic biosynthetic pathway, and may be a keystone species for cobalamin production.Our screen identified 187 223 total cobalamin biosynthetic genes across all metagenomes, 95 911 of which matched the 11 marker genes . Taxonomrganisms , comprisIn addition to evaluating all metagenome datasets together, we partitioned studies into their individual metagenome samples , each of which was associated with distinct environmental parameters. Environment-specific metagenomes varied widely in taxonomic composition of cobalamin genes, within and between studies . HierarcThaumarchaeota ranged from undetected to contributing as much as ∼70–80% of the cobalamin genes in various samples from the Microbial Initiative in Low Oxygen areas off Concepcion and Oregon , Western Channel Observatory Microbial Metagenomic Study  studies  with sample depth , sample depth (r=0.73), phosphate (r=0.61) and dissolved organic nitrogen (r=0.60). The strongest negative correlations with thaumarchaeotal cob/cbi genes included bacterial cell number (r=−0.53) and ammonium concentration (r=−0.40). Low ammonium mixing ratios are consistent with a role for marine Thaumarchaeota as high affinity ammonia oxidizers  are grown in medium prepared without vitamin supplementation (12-independent methionine synthesis pathway (metE) is absent in the genome of N. maritimus SCM1, growth in the absence of vitamin supplementation provides strong supporting evidence for thaumarchaeotal cobalamin synthesis. Direct measurements of gene expression and cobalamin production by pure cultures of N. maritimus SCM1 and additional Thaumarchaeota would confirm thaumarchaeotal cobalamin synthesis and provide additional information on the structure, regulation and fate of this vitamin when produced by marine Thaumarchaeota. Although all known ammonia-oxidizing archaea possess the genetic capacity for cobalamin synthesis, the absence of homologous genes in Caldiarchaeaum subterranean , a possibility is that high cyanobacterial photosynthetic production of molecular oxygen interferes with the anaerobic thaumarchaeotal cobalamin biosynthetic pathway, which possesses an oxygen-sensitive intermediate  in deep marine water columns (The metagenomic data are also consistent with observed geographical and temporal patterns of thaumarchaeotal abundances. In particular, the urea-fueled polar and halocline abundances of 12 maxima in ocean waters below the marine photic zone are consistent with an important role for Thaumarchaeota in marine cobalamin production, given their high proportional abundance with depth. Previous observations of increased cobalamin concentrations in ocean mesopelagic zones (12 concentrations did not correlate with upwelling index off the coast of Baja California, Mexico (12 production (Thaumarchaeota were responsible for vitamin B12 production in this experiment, and implies that thaumarchaeotal productivity may be limited by cobalt concentrations. Importantly, vitamin B12 was shown to influence phytoplankton growth and community composition in samples taken from the subarctic Gulf of Alaska (12 in freshwater environments as well (Thaumarchaeota may directly influence phytoplanktonic communities in polar surface waters, freshwater and possibly in marine surface waters via mixing with ocean water column vitamin pools.Observations of vitamin B as well . TogetheThaumarchaeota are among the most abundant global cobalamin vitamin producers. All known ammonia-oxidizing members of the Thaumarchaeota possess the genetic capacity to produce cobalamin, and data from multiple metagenomic studies reveal niche partitioning of this keystone community function. Thaumarchaeotal cobalamin synthesis genes demonstrate spatial and temporal biogeographical distributions. Combining pure culture experimental work and field studies will help assess the proportion of marine cobalamin pools derived from archaeal synthesis, identify the role of archaeal cobalamin in marine biogeochemistry, and characterize links between global thaumarchaeotal cobalamin production and microbial community composition and succession.Thaumarchaeotal vitamin production in most of the global ocean would have enormous potential impacts on microbial communities and food web dynamics. Here we provide evidence that


Apodemus agraricus). The main manifestation of HFRS, including acute kidney injury, increases vascular permeability, and coagulation abnormalities. In this paper, we review the current knowledge of the pathogenesis of HFRS including virus factor, immunity factor and host genetic factors. Furthermore, the treatment and prevention will be discussed.Hantaan virus (HTNV) causes hemorrhagic fever with renal syndrome (HFRS), which is a zoonosis endemic in eastern Asia, especially in China. The reservoir host of HTNV is field mouse ( Hemorrhagic fever with renal syndrome (HFRS) has been a major epidemic mainly in Asia and Europe. About 100,000 cases of HFRS are documented annually , Amur virus(AMV), Seoul virus (SEOV), Dobrava virus (DOBV), or Puumala virus (PUUV), each of which causes diseases with differing severity. HTNV or DOBV may cause the most severe form of HFRS and have the highest morbidity rates ranging from 5 to 10% . The disease typically progresses through five phases: febrile, hypotensive shock, oliguric, polyuric, and convalescent Table . AdditioKidney injury frequently occurs in HFRS and the most prominent pathological presentation is acute tubulointerstitial nephritis following the infiltration of inflammatory cells , is likely to happen, and, thus, results in a high fatality rate. It was demonstrated that the agitation, conjunctival hemorrhage, coma, were also negatively correlated with survival outcome  and colloidal gold method. A safe, rapid and specific serotyping method for diagnosis was developed by using the recombinant hantavirus nucleocapsid protein (NP) as antigen  by various Pattern-recognition receptors (PRRs). Among the different receptors that participate in the recognition of microbial invaders, Toll-like receptors (TLRs) play important roles in mediating the innate response  are cytokines which are produced in response to viral infection and play an important role in regulating viral replication. The IFN-inducible MxA is considered to be important for control of infections with hantaviruses. MxA inhibits representative members of the Bunyaviridae family by interacting with an early step of virus replication. MxA protein can inhibit PUUV and Tula virus (TULV) replication in both viral protein and RNA accumulation in virus-infected cells  directed against G1 and G2 displayed neutralizing activity, while the MAbs against N protein did not  has been used in the treatment of HCPS and HFRS  was reported to have a high activity against a panel of Bunyaviruses  by cytopathic effect and virus yield reduction  has become an important and widely used therapy for critical HFRS patients accompanied with mutiorgan injury, ARDS, pulmonary edema, fluid overload, severe electrolyte disturbances, cerebropathy, and AKI. Intermittent hemodialysis (IHD) is often chosen as the first choice by clinicians for AKI patients with stable hemodynamic status and less potentially fatal complications.HTNV is a major agent causing HFRS. The disease is obviously very complex. We think that the dysfunction of all the organs is mainly due to the capillary leakage and further studies should be emphasized on this aspect. Although the pathogenesis of HFRS is still unclear, our understanding on this disease has improved in recent years.As the initial reason of the disease, the virus itself plays an important role in the course of pathological development. With the progress of the virus nucleic acid detection technology, it has become more easy and accessible to detect hantavirus RNA and structural protein in clinical cases. The role of virus in different clinical stages and whole disease process needs to be further studied.It has been widely accepted that the immune response plays an important role in hantavirus pathogenesis. A number of studies have focused on adaptive immunity in the past. High levels of hantavirus specific antibodies and presence of IC in the blood were detected. More and more scientists have been paying attention to early activation of immune response to hantavirus antigens. Recently, lots of studies indicate that innate immunity stimulates adaptive immunity as well as innate immunity itself has antiviral functions. We believe that future efforts should be made to find the mechanism of the innate immunity in the pathogenesis of the disease.Strong antiviral treatment has improved the prognosis of many kinds of viral diseases. Therefore, a new potent antiviral drugs against hantavirus should be developed. It should be emphasized that more and more studies are now focusing on the pathogenesis of increased capillary leakage. This research direction may provide a great inspiration for the searches on effective therapies and exploring potential therapeutic targets. Further important questions mainly refer to the identification of risk factors, preventive measures and vaccination. Answers had better be found through close cooperation from scientists of various fields.Wrote the paper: HJ, HD, LW. Revised the manuscript: PW, XB.This work was supported by the National Natural Science Foundation of China (No.81373118), the National Basic Research Programme of China (973 Programme No.2012CB518905), and the Substantial Development Program of Technology Innovation and Clinical Research in Tangdu Hospital (2013-4241370U2).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.


Chikungunya virus (CHIKV) is a mosquito-borne pathogen that emerged in Brazil by late2014. In the country, two CHIKV foci characterized by the East/Central/South Africaand Asian genotypes, were established in North and Northeast regions. Wecharacterized, by phylogenetic analyses of full and partial genomes, CHIKV from Riode Janeiro state (2014-2015). These CHIKV strains belong to the Asian genotype, whichis the determinant of the current Northern Brazilian focus, even though the genomesequence presents particular single nucleotide variations. This study provides thefirst genetic characterisation of CHIKV in Rio de Janeiro and highlights thepotential impact of human mobility in the spread of an arthropod-borne virus. Alphavirus, family Togaviridae, endemic in parts ofAfrica, Southeast Asia and on the Indian subcontinent. It usually producesa non-fatal febrile illness in humans, associated with rash and severe arthralgia (Chikungunya virus (CHIKV) is a mosquito-borne pathogen that belongs to the genusthralgia , and occThe first autochthonous CHIKV case in the Americas occurred in the Caribbean (Island ofSaint Martin) in late 2013 . After tUntil November 2015, Rio de Janeiro state, located in the Southeast of Brazil, had onlyregistered imported CHIKV cases . It is3Here, we performed a phylogenetic analysis of four CHIKV identified in the Rio de Janeirostate in 2014-2015. These CHIKV strains were from individuals with recent travel history tothe Caribbean region . Their main clinical manifestations were fever, arthralgia and exanthema.Whole-genome sequencing was performed on an Illumina HiSeq 2500 system  using 2 x 100 bp paired-end readsgenerated with Nextera XT libraries. Bioinformatic analyses allowed the recovery of nearlycomplete genome of the CHIKV virus from the 2015 case (RJ/CHIKV/2015). E1 gene sequenceswere recovered by PCR and Sanger sequencing from other three 2014 cases. The sequences weresubmitted to GenBank under accession number KU355832-KU355835. Phylogenetic trees wereconstructed using Neighbor-Joining method and was evaluated by thousand bootstrapreplicates.The phylogenetic analysis of full-length genomes reveals that the RJ/CHIKV/2015 belongs tothe Asian genotype (97-99% identity) and clusters together with other Brazilian importedcases - Guadalupe to Belém, Pará and Dominican Republic to Recife, Pernambuco - and anautochthone case identified in Amapá (middle 2014), as well as with strains from theCaribbean and Mexico , panel AAedes albopictus mosquitoes  of the all four imported Rio de JaneiroCHIKV showed that the other three strains also belong to the same Asian genotype cluster, panel Bsquitoes .This study provides original genomic information of non-autochthonous CHIKV strainsidentified in travelers coming from the Caribbean region to Rio de Janeiro. This is thesecond most populous metropolitan area in Brazil and the primary national and internationaltourist attraction city of the country. Severe outbreaks caused by other arboviruses,Dengue and Zika virus, sharing the same mosquito vector as CHIKV have been oc- curring inthe country as well in Rio de Janeiro . Ourres


Ebolaviruses cause a severe and often fatal haemorrhagic fever in humans, with some species such as Ebola virus having case fatality rates approaching 90%. Currently, the worst Ebola virus outbreak since the disease was discovered is occurring in West Africa. Although thought to be a zoonotic infection, a concern is that with increasing numbers of humans being infected, Ebola virus variants could be selected which are better adapted for human-to-human transmission.To investigate whether genetic changes in Ebola virus become established in response to adaptation in a different host, a guinea pig model of infection was used. In this experimental system, guinea pigs were infected with Ebola virus (EBOV), which initially did not cause disease. To simulate transmission to uninfected individuals, the virus was serially passaged five times in naïve animals. As the virus was passaged, virulence increased and clinical effects were observed in the guinea pig. An RNAseq and consensus mapping approach was then used to evaluate potential nucleotide changes in the Ebola virus genome at each passage.Upon passage in the guinea pig model, EBOV become more virulent, RNA editing and also coding changes in key proteins become established. The data suggest that the initial evolutionary trajectory of EBOV in a new host can lead to a gain in virulence. Given the circumstances of the sustained transmission of EBOV in the current outbreak in West Africa, increases in virulence may be associated with prolonged and uncontrolled epidemics of EBOV. Ebola virus (EBOV) causes severe haemorrhagic fever in humans and non-human primates. Due to the high mortality rate, potential transmission from person-to-person contact and the lack of approved vaccines or anti-viral therapies, EBOV is classified as a hazard group 4 pathogen. The case fatality rate is related to the species of ebolavirus. EBOV has the highest case fatality rate (up to 90%) while Reston virus (RESTV) is not pathogenic for humans. However, RESTV can cause viral haemorrhagic fever in non-human primates and illustrates the potential zoonotic threat of ebolavirus –3. Outbr1,2), VP30, VP24 and the RNA-dependent RNA polymerase (L). GP1,2 is the primary structural component and is exposed on the surface of the virus particle. The protein mediates host cell attachment and fusion [1,2 is necessary for protection against lethal EBOV challenge [Ebola virus is an enveloped non-segmented negative-sense single-stranded RNA virus with a genome of 19 kb in length encoding several proteins including: nucleoprotein (NP), virion protein (VP) 35, VP40, the surface glycoprotein .1,2 is transcribed through two disjointed reading frames in the genome. These two reading frames are brought together by slippage of the viral polymerase complex at an editing site (a run of seven A residues) to insert an eighth A residue, generating an mRNA transcript that allows read-through translation of GP1,2 [1,2, thereby allowing sGP to bind and compete for anti-GP1,2 antibodies [in vitro and in vivo models of infection [There are several properties of the polymerase complex that contribute to virulence. The mRNA encoding GPof GP1,2 ,9. Durinof GP1,2  includintibodies . The edinfection .-4 and 7.06 × 10-4 nucleotide substitutions/site/year [-9 nucleotide substitutions/site/year [Similar to other viruses with RNA genomes and which code an RNA dependent RNA polymerase, the L protein is unlikely to have extensive error correction, if at all. The molecular evolutionary rate for EBOV has been estimated at being between 2.2 × 10ite/year , whereasite/year . TherefoEBOV is proposed to be a zoonotic infection. Fruit bats are believed to be the natural reservoir and are not thought to develop disease . A concein vivo using a guinea pig model of infection. EBOV is initially non-pathogenic in guinea pigs, but becomes more virulent and adapted to replicating in this host [In order to investigate how the EBOV genome changes with increasing pathogenicity, a forced evolution model was used in which EBOV was sequentially passaged his host ,17.4 TCID50 EBOV, with spleens harvested 7 days post infection (as a source of progeny virus). Virus titre was determined and a new inoculum prepared before administering 104 TCID50 EBOV to a new group of guinea pigs. This was repeated until there was clinical and virological evidence that the virus adapted to the guinea pig host. Animals were observed for 2 weeks post infection. Weight data indicated that guinea pigs showed a minimal response to the initial challenge, whereas with subsequent passages weight loss exceeding 10% was observed  and the virus was serially passaged to develop uniform lethality in guinea pigs  with the polyA carrier in the viral isolation kit. Sequence analysis indicated an increased proportion of sequence reads mapping to the EBOV genome with passage Table  we found1,2, suggesting that viral mRNA might be co-purified or that there is editing of the genome itself during viral replication. Previous work has shown that approximately 20% of GP mRNA analysed from Ebola virus infected cells can be edited [1,2 increased with sequential passage and this may be associated with the gain of virulence observed with sequential passage in the guinea pig model. One interpretation of this data is that the amount of GP1,2 was limiting in early passages. As the proportion of edited mRNA increased so more GP1,2 was available for virus assembly and on virus particles and this contributed to the increase in progeny virus observed in the later passages. However, a more stringent analysis of sequence reads that mapped to the relevant region of the glycoprotein gene showed additional A residues at the following rates: 0 out of 2 mapped reads for passage one, 2 out of 3 (66%) mapped reads at passage two, 17 out of 45 (37.8%) reads at passage three, 45 out of 115 (39.1%) mapped reads for passage four and 37 out of 91 (40.7%) mapped reads for passage 5. Thus, an analysis of those reads mapped at a higher stringency indicated an increased rate of editing both overall and increasing with passage. However, the low number of reads overall means these observations must be treated with caution.Sequence analysis identified editing in the mRNA encoding GPe edited . When wee edited  to alignEBOV sequence at each passage was analysed for coverage and variants using QuasiRecom , allowinSome viral proteins accumulated substitutions whereas other did not. No substitutions were observed in either VP40 or VP30 by passage five. VP40 is a viral matrix protein with multiple roles in the virus life cycle, associating with both cellular and other viral proteins including the ribonucleoprotein complex. It is also involved in virus assembly and release , andSome substitutions were present in early passages but were lost by passage five. For example in VP35 at passages one and two, the predominant amino acid at position 84 was a Gly rather than the Glu found in the input sequence. By passages three, four and five this was again a Glu. In VP40 at passages two and three the predominant amino acid at position 16 was a Pro rather than the Ala found at passages one and two and the input sequence. By passages four and five, this had become an Ala again. Some of these changes have been previously associated with virulence. For example, in VP24, the predominant amino acid at position 163 changes from a Lys to Arg in passage three (and then Lys becomes dominant again in passages four and five), which is a conserved substitution. This substitution was described previously by Kugelman et al., who postulated that this amino acid change in VP24 might modulate interaction with other proteins instead of having an effect on structural stability .A number of amino acid substitutions became established during adaptation and were present in passage five Table . For exa1,2 mRNA. This editing activity of the polymerase complex maybe associated with and mediated by a host cell factor, although both cis-acting factors on the EBOV genome and VP30 have been implicated in this process [As noted by Ebihara et al., additional mutations in the L protein are likely to contribute to virulence by affecting viral RNA synthesis . These m process .In this study, a virus (EBOV) that was not initially virulent in guinea pigs was serially passaged and became more pathogenic in its new host. Several alterations in the amino acid coding sequence were associated with this increase in virulence. A reverse genetics approach would precisely characterise these changes and their linkage with pathogenicity and virulence. Interestingly, this approach was used in a mouse model study of EBOV to investigate the molecular determinants of virulence . There aEbolaviruses are zoonotic, and when certain species such as EBOV infect humans, they can have high case fatality rates. The implication of this is that transmission of the virus is contained when humans live in isolated villages. However, in the advent of large-scale outbreaks in higher population densities there is a greater chance of the virus becoming more adapted for growth and transmission in the human population. Transmission studies with influenza virus using animal models have identified key viral mutations involved in this process and have illustrated how these may be used as predictors during an influenza infection/pandemic –40. Suchad libitum. All procedures were undertaken according to the United Kingdom Animals (Scientific Procedures) Act 1986. A power calculation along with Fisher’s exact test were performed using software G*Power ver.3.0.10 to determine group sizes for the experiments. A minimum group size of six met a power of 0.8 and alpha at 0.05. We also note that from previously published work in this area, that all animals become infected with EBOV at later passages. There were 10 guinea pigs depending on the group for each passage of the virus and a control group. From a practical standpoint of working at CL4 this number also represented the maximum number of animals that could be processed at the time. Of these animals, four were killed at day 7 post infection for preparation of virus and six to eight were carried on and used to measure clinical parameters. The study was performed under a UK Home Office Project License conforming to the Animal Procedures Act. Ethical review was performed by the Public Health England Animal Welfare and Ethical Review Board.Female Dunkin-Hartley guinea pigs were used for animal infection studies, with weights of 250 g to 350 g . Before procedures involving the manipulation of animals, guinea pigs were anesthetised with 1.5% to 2% isofluorane in an induction change until full sedation was achieved. Animals infected with EBOV were housed within an isolator under climate-control conditions in an animal containment level 4 (CL4) room. Food and sterile water were available 2 tissue culture flasks and left to absorb for 1 h. A volume of 5 mL media was then added and cells were incubated at 37°C for 7 to 8 days. Cytopathic effects were determined by microscopy, and the results from each dilution were used to calculated 50% tissue culture infective dose (TCID50) using the Reed-Muench method [The EBOV Zaire ME718 strain was used in this work. This was originally isolated during an outbreak in October 1976  in Yambuh method .4 TCID50. Surplus inoculation was made to confirm concentration via back titration in cell culture. Guinea pigs were sedated, and subcutaneously inoculated with the virus suspension in the lower right quadrant of the back, then returned to their cages and monitored for adverse effects caused by the injection of the anaesthetic until the animals fully recovered. Negative control groups were injected with the same volume of PBS.EBOV stock was diluted in sterile PBS to prepare the relevant dose of virus in a 0.2 mL volume. For passaging experiments (required for virus adaptation), the dose delivered was 10Animals were monitored at least twice daily, and observations  recorded for the duration of the study. A set of humane clinical end points were defined  which indicated that the animal would be euthanised to prevent any unnecessary suffering. Weights of the animals were taken daily, and temperatures recorded using a pre-inserted temperature chip.in vitro CL4 laboratory and thawed at room temperature. Spleens were homogenised by vigorously passing through a 500 mm cell strainer . The resultant suspension was clarified by centrifugation at 400 g for 10 min to remove cell debris. The supernatant was collected, aliquoted and stored at -80°C. One vial was used to assess virus concentration by TCID50 assay.Animals were humanely euthanised by intraperitoneal injection of 200 mg/mL pentobarbital sodium. Necropsies were performed within a flexible-film isolator in the animal CL4 facility. Spleens were removed from four out of the 10 guinea pigs at each passage and stored at -80°C. For processing, samples were transported to the Spleen homogenate from each passage was added to AVL buffer  and removed from the CL4 laboratory for processing. RNA was isolated using a viral RNA extraction kit following the manufacturer’s instructions . Confirmation of EBOV-specific RNA extraction was achieved by use of a block-based PCR assay. The RNA was then pooled from individual guinea pigs for each passage for sequencing analysis.Extracted RNA was DNase treated with Turbo DNase (Ambion) using a rigorous protocol. RNA–Seq libraries were prepared from the resultant RNA using the Epicentre ScriptSeq v2 RNA-Seq Library Preparation Kit. RNA (50 ng) was used as input and following 12 cycles of amplification, libraries were purified using AMPure XP beads. Each library was quantified using Qubit and the size distribution assessed using the Agilent 2100 Bioanalyser. These final libraries were pooled in equimolar amounts using the Qubit and Bioanalyzer data. The quantity and quality of the pool was assessed by Bioanalyzer and subsequently by qPCR using the Illumina Library Quantification Kit from Kapa on a Roche Light Cycler LC480II according to manufacturer’s instructions. The pool of libraries was sequenced on one flowcell of the MiSeq at 2 × 150 based paired-end sequencing with v2 chemistry. Due to the high-expected levels of polyA RNA introduced during the RNA extraction 15% of phiX library was loaded onto the flowcell for sequencing to produce a more balance base composition. The Fastq files were QC filtered as follows. Initial processing and quality assessment of the sequence data were performed using an in-house pipeline (developed by Richard Gregory). Briefly, base calling and de-multiplexing of indexed reads was performed by CASAVA version 1.8.2 (Illumina). The raw fastq files were trimmed to remove Illumina adapter sequences using Cutadapt version 1.2.1 . The opt


Subtype C avian metapneumovirus (aMPV-C), is an important pathogen that can cause egg-drop and acute respiratory diseases in poultry. To date, aMPV-C infection has not been documented in Muscovy ducks in China. Here, we isolated and characterized an aMPV-C, designated S-01, which has caused severe respiratory disease and noticeable egg drop in Muscovy duck flocks in south China since 2010. Electron microscopy showed that the isolate was an enveloped virus exhibiting multiple morphologies with a diameter of 20–500 nm. The S-01 strain was able to produce a typical cytopathic effect (CPE) on Vero cells and cause death in 10- to 11-day-old Muscovy duck embryos. In vivo infection of layer Muscovy ducks with the isolate resulted in typical clinical signs and pathological lesions similar to those seen in the original infected cases. We report the first complete genomic sequence of aMPV-C from Muscovy ducks. A phylogenetic analysis strongly suggested that the S-01 virus belongs to the aMPV-C family, sharing 92.3%-94.3% of nucleotide identity with that of aMPV-C, and was most closely related to the aMPV-C strains isolated from Muscovy ducks in France. The deduced eight main proteins  of the novel isolate shared higher identity with hMPV than with other aMPV . S-01 could bind a monoclonal antibody against the F protein of hMPV. Together, our results indicate that subtype-C aMPV has been circulating in Muscovy duck flocks in South China, and it is urgent for companies to develop new vaccines to control the spread of the virus in China. Flavivirus named BYD virus, which was recently isolated from Peking ducks in China, was reported to cause a reduction in egg production in ducks . Bas in 1980 ,11]. aM. aMPneum in 1980 . To date in 1980 . Turkeys in 1980 . Mallard in 1980 ,14]–[16–[16Pneum in 1980 .aMPV-C infection in chickens was first reported in China in 2013 . InfectiThe animal slaughter experiments were conducted in accordance with the guidelines of the Guangdong Province on the Review of Welfare and Ethics of Laboratory Animals approved by the Guangdong Province Administration Office of Laboratory Animals (GPAOLA). All the animal procedures were conducted under the protocol (SCAU-AEC-2010-0416) approved by the Institutional Animal Care and Use Committee (IACUC) of South China Agricultural University.g for 1 min at 4 °C. The supernatant immediately underwent RNA extraction and virus isolation. The ovary, uterus, larynx, trachea and nasal turbinates were cut into pieces and suspended with a mixture of Phosphate Buffered Saline (PBS) containing penicillin-streptomycin. The suspension was centrifuged at 12 000 × g for 1 min at 4 °C, and the supernatant was used immediately for RNA extraction and virus isolation.Clinical samples were collected from infected ducks  from 2010 to 2012. A total of 60 duck flocks (50 layer and 10 breeder flocks) with clinical signs were examined. Nasopharyngeal swabs, cloacal swabs, ovary, uterus, larynx, trachea and nasal turbinate specimens were collected. The nasopharynx and cloacae of eighteen birds were swabbed per flock. The nasopharyngeal swabs or cloacae swabs were pooled and suspended in minimum essential medium with penicillin-streptomycin, followed by centrifugation at 12 000 × Statistical analysis was performed on daily egg production rate represented by 6 different flocks of diseased Muscovy ducks from three different farms. Daily egg production rate of two normal flocks from another farm was analyzed as the control. The birds in these four farms were 38 (Farm 1-F1), 35 (Farm 1-F2), 43 (Farm 2-F1), 45 (Farm 2-F2), 45 (Farm 3-F1), 36 (Farm 3-F2), 35 (Farm 4-F1) and 43 (Farm 4-F2) weeks old, respectively.Total RNA was extracted directly from the treated samples using the RNeasy kit  according to the manufacturer’s instructions. The TaKaRa PrimeScript one-step RT-PCR Kit Ver.2  was used for RT-PCR. All PCR reactions were carried out using the PCR machine . aMPV-C was detected by RT-PCR as described by Ali and Reynolds ; BYD vir2O, 1 μL of each primer in a total reaction volume of 50 μL containing 10 μL of RNA. The reaction mixtures were subjected to amplification with the following temperature conditions: 50 °C for 30 min for reverse transcription (RT), 95 °C for 5 min for initial denaturation, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 2 min. The final extension was carried out at 72 °C for 10 min. We designed 9 pairs of overlapping primers . Multiple-sequence alignments were generated with ClustalX 2.0, and nucleotide sequence homologies were further obtained through the Clustal W method of DNAStar . All seq50 assay was conducted to determine the titer of the virus as described by Brian et al. [The aMPV-positive samples were inoculated in 11-day-old duck embryos via the yolk sac. The yolk fluid was collected 6 days post-inoculation. The yolk fluid collected from the inoculated embryos was further examined for the presence of aMPV by RT-PCR as described by Ali and Reynolds . The embn et al. .g for 1 h at 4 °C. The virion particles in the pellet were visualized by electron microscopy (EM) as described by Kwon et al. [Virus-infected cells were harvested and concentrated via ultracentrifugation at 40 000 × n et al. .5 cells/well) were infected with the isolated virus (105 TCID50) for 48 h. The cells were then fixed with 100% cold methanol for 10 min at 4 °C. The fixed cells were then incubated with a monoclonal antibody  against the F protein of hMPV as described by Luo et al. [Vero cells in 24-well plates  at 37 °C. At the designated time points, the cell culture supernatants were harvested and titrated by a standard TCIDOD) values at 405 nm, sample to positive (S/P) ratios were calculated, and the average S/P ratio was used to evaluate the level of antibodies in each group. Serum samples with S/P ratios greater than 0.2 (titers greater than 396) were considered to possibly contain anti-aMPV antibodies, and a ratio of 0.2 or lower  were considered negative, based on the manufacturer’s recommendations.Sera were collected from convalescent laying Muscovy ducks and control birds. Sera were tested for the presence of aMPV antibodies using the Avian Pneumovirus Antibody Test Kit  according to the manufacturer’s instructions. In order to make the kit suitable for detection of duck immunoglobulins, we replaced the Anti-Chicken HPRO (which was provided with the kit) with Anti-Duck HPRO conjugate for the ELISA test. Based on the optical density , −Newcastle disease virus, −goose parvovirus, −avian pneumovirus, and -BYD virus were used to interact with the S-01 virus at 4 °C for 3 h. The virus-antibody mixtures were transferred into Vero cells in 24-well plates and incubated at 37 °C. When CPE was evident, the cells infected or mock-infected with S-01 or S-01/ anti-serum were detected by IFA, as described by Luo et al. .ad libitum. After acclimatization to the new environment for 7 days to minimize the effects of shipping stress, the ducks were carried out for two series of experiments.To evaluate the pathogenicity of the S-01 virus, thirty-five 20-day-old breeding Muscovy ducks, and 200 300-day-old laying Muscovy ducks were obtained from Rencun and Zhejiang Subsidiaries of Guangdong WENS Foodstuff Group Co. Ltd . All ducks were housed and bred in a negative pressure and pathogen-free Animal Isolation Facility at room temperature (20 ~ 25 °C), relative humidity of 60%, subjected to a 16 h-light per day with average of 20 ~ 35 lux, supplied with water and feed 5 EID50) and cloacal (105 EID50) injections. The ducks in group 4 were mock-infected with 1.5 mL of sterile DMEM containing 4% FBS in the same manner as the control. Ducks in group 5 did not receive any treatment as the negative control. The 35 breeding ducks were divided into four groups. Ducks in groups 1 and 2 were infected respectively with 105 EID50 of F5-embryo-passaged and 105 TCID50 of F5-cell-passaged S-01 virus by intranasal injection. Ducks in group 3 were mock-infected with 1.5 mL of sterile DMEM containing 4% FBS in the same manner as the control. Group 4 had 5 ducks without any treatment as the negative control. The laying ducks were monitored daily for egg production and clinical symptoms, while breeding ducks were monitored only for clinical signs (e.g. respiratory symptoms). On day 7 post-infection, eighteen ducks with typical respiratory symptoms from six inoculated afore-mentioned groups (3 ducks/group) were killed. Meanwhile, six ducks from three control groups (2 ducks/group) were killed. Nasopharyngeal swab & cloacal swab, ovary & uteru, larynx & trachea, nasal turbinates from each duck were collected. Totally 90 tissue samples were collected from 9 experimental groups for pathological examination and virus isolation. Duck embryos and Vero cells were used to recover the virus from tissue samples. The recovered virus was subsequently confirmed by RT-PCR and sequencing.The 200 laying Muscovy ducks were randomly divided into five groups (40 ducks/group). The ducks in groups 1, 2 and 3 were infected with F1-, F5-, F10-embryo-passaged S-01 virus, respectively, and every laying duck was infected with S-01 by intranasal , and the differences between means were compared by the Duncan’s multiple range test (DMRT) using SPSS 17 . The data, including aMPV-C antibody titer and daily egg production between naturally infected and inoculated laying Muscovy ducks, was analyzed by the Student’s From July 2010 to July 2012, four farms consisting of 50 flocks of egg-laying Muscovy ducks and 10 flocks of breeding Muscovy ducks were monitored and investigated. An outbreak in Muscovy ducks, with severe symptoms of coughing and egg-drop, occurred in these flocks in Guangdong, China. The outbreak occurred in a seasonal pattern, mainly in early spring and late autumn. Only Muscovy ducks were infected, indicating that the pathogen was species-specific. We observed upper respiratory symptoms and reduction of egg production by approximately 40-85% in the sick Muscovy ducks   . Interestingly, the aMPV-C M gene was detected by RT-PCR using total RNA extracted from nasopharyngeal swabs, cloacal swabs, ovary, uterus, larynx, trachea and nasal turbinates of the diseased ducks as a template (data not shown). Using ELISA, we detected the presence of an aMPV-specific antibody in the convalescent sera. The ucks n = 5  were 102.83±0.30, 104±0.51, 106.47±0.45, 104.75±0.33 and 104.6±0.42 TCID50/0.1 mL, respectively.The isolate was further confirmed by an immunofluorescence assay. There was no antibody available for the subgroup C aMPV, but our amino acid sequencing revealed that the F protein of S-01 was identical to that of aMPV-C and hMPV by 98.5-99.1% and 81.6-81.8%, respectively. Because Luo et al. used recombinant fusion protein  to confiA sera neutralization test was used to further analyze whether there were other viruses in the isolate. When the Vero cells were inoculated with the viruses that interacted with the antisera to duck reovirus, avian influenza virus (H5N1 and H9N2), Newcastle disease virus, goose parvovirus, and BYD virus, CPE occurred in the cells at day 3 post-inoculation (data not shown). CPE was also observed in the cells inoculated with the S-01 virus. When the Vero cells were infected with the S-01 virus in the presence of the corresponding antibody, the cells grew as well as the control cells (without infection) (data not shown). In conjunction with the sera neutralization test, the virus suspension was tested by RT-PCR and PCR for miscellaneous potential viruses, such as the duck reovirus, avian influenza virus (H5N1 and H9N2), Newcastle disease virus, goose parvovirus, and BYD virus. We found that only aMPV-C-was detectable (data not shown). Taken together, our findings revealed that the novel isolate, which was able to bind to the monoclonal antibody to F protein of the hMPV and produce a typical CPE in Vero cells, was an aMPV-like enveloped virus, and the best viral replication phase was at approximately 72 hpi.To confirm the identity of the etiological agent of the outbreak, it was essential to reproduce the disease by experimental infections of breeding and laying ducks with the S-01-virus strain. Initially, the infected laying ducks only exhibited symptoms of coughing, which disappeared in 6–7 days, and only a slight egg-drop was observed in the first three reproductions. We adjusted cell-passage strain to the duck embryo-passage strain. The laying duck groups that were infected with embryo-passaged strain of S-01 experienced serious upper respiratory symptoms and egg drop Figure C and D. We used a pair of primers specific for the M gene of subgroup C aMPV to detect aMPV-C. The PCR amplification produced a 631-bp fragment, which was consistent with previously published data . Sequenca, AY590688) . The sequence of S-01 shared 92.3-94.3%, 69.6-70.4%, 60.4%, 60.2%, 46.6%, 46.2%, 46.1% and 46.4% nucleotide identities with that of aMPV-C, hMPV, aMPV-A, aMPV-B, RSV, BRSV, MPV and HRSV, respectively  of 1185 nt, which encodes a protein of 394 aa. The length of the deduced aa of the S-01 N gene was the same as that of aMPV-C, whereas the length of the same region in aMPV-A or -B was 391 aa. The P gene contained 885 nt in a single ORF encoding a protein of 294 aa, which was 15 aa longer than that of aMPV-A and 14 aa longer than that of aMPV-B. The M gene contained 765 nt in a single ORF encoding a protein of 254 aa. All the metapneumoviruses had an M gene encoded by a single ORF of 254 aa. The F gene sequence of S-01 had 1614 nt in a single ORF encoding a protein of 537 aa. The S-01 L protein gene contained 6018 nt in a single ORF encoding a protein of 2005 aa, which had the same length as most aMPV-C, but was longer than that of the goose strain, aMPV-A, or aMPV-B . The varying mortality rates may be related to the different main secondary pathogens isolated from the two periods. In addition, the therapeutic effects of vaccines and antibiotics might also play a role in the different mortality rates.The recent aMPV-C epidemic, which has been circulating in southern China for about two years, was more complex and worse than previous outbreaks, such as the outbreak occurring in France . Once thinvasion . PasteurIsolating aMPV from field samples is not easy . Kwon eta and aMPV-Cb, according to the different lengths of the G protein  with North-American isolates, including aMPV-Cb . S-01 resembled French isolates (France/duck/AJ811991&AJ811992) the most and had higher identity with them than with North-American isolates. North-American and French isolates of aMPV-C belonged to significantly different genetic lineages [aMPV-C can be divided into two sublineages, aMPV-Cn Figure . Intereslineages . This maa, aMPV-A, aMPV-B, aMPV-D and hMPV was shorter than that of aMPV-Cb. All of the deletion regions may be related to host specificity, but more experimental evidence is needed to confirm this hypothesis. It was estimated that cross-species transmission of metapneumoviruses from birds to humans occurred approximately 200 years ago [b may be the ancestor of metapneumoviruses.The sequence analysis revealed that the G gene sequence of aMPV-Cears ago . At the ears ago . Our resPrior to our study, there was no published report that successfully replicated the disease with experimental aMPV-C infection in Muscovy ducks. Although Toquin et al. effectively isolated aMPV-C from Muscovy ducks, they did not mention to reproduce a clinical case of aMPV-C infection in Muscovy ducks . We initHoogen et al. published the genomic sequence of hMPV (hMPV-001 strain), allowing for detailed comparison of aMPV and hMPV . CompariDucks seem to be susceptible carriers for many zoonotic viruses, such as avian influenza virus and BYD virus, and they play an important role in the transmission and evolution of these viruses . Ducks wThe authors declare that they have no competing interests.SKS, FC and HYL conceived and designed the study. SKS, SC, WL, JJL, GWL, SYF, CL generated the raw data. SKS, JPL and JPQ analyzed raw data. SKS performed statistical analysis. SKS wrote paper. HYL revised the paper. All authors read and approved the manuscript.


The study was conducted at Artificial Breeding Research Centre, NDRI, Karnal, to determine the sexual behavior and its relationship with semen quality parameters in Sahiwal breeding bulls.A total of 63 ejaculates were collected from six adult Sahiwal bulls (age ~47 mo and bwt ~466 kg), to study the relationship of sexual behavior and semen quality. The degree of association between different variables was estimated by Pearson’s correlation coefficient method.The results depicted that, sexual aggressiveness showed significantly high positive correlation with libido score (LS) and sexual behavior score (SBS). Reaction time (RT) and total time taken in mounts (TTTM) had a significant negative correlation with LS and SBS. Penile erection score and penile protrusion score (PPS) both had a significant positive correlation with ejaculatory thrust score, mating ability score, and SBS. Results of correlation among seminal attributes and with sexual behavior depicted that ejaculate volume had positive significant correlation with initial progressive motility (IPM), sperm concentration (SCON), head abnormality, total abnormality, hypo-osmotic swelling test (HOST), acrosomal integrity (AI) whereas, mass activity had positive significant correlation with IPM, SCON, non-eosinophilic spermatozoa count (NESC), HOST, AI, RT and TTTM and IPM had positive significant correlation with SCON, NESC, HOST, AI, and TTTM, whereas and HOST had positive significant correlation with AI. Among seminal attributes, SCON had a positive significant correlation with PPS where as head abnormalities had a positive significant correlation with RT and TTTM.It can be concluded that the relationship of sexual behavior and semen quality parameters are reflecting that the sexual behavior of individual bulls is important to harvest good quality and quantity of semen as desired type of sexual preparation can be provided. Demand of frozen semen of elite Sahiwal bulls is increasing throughout the country due to its milk productivity, adaptability, disease resistance, heat tolerance, and survivability on poor feed and fodder resources. To achieve the optimum target of quality frozen semen production with better fertility breeding soundness evaluation (BSE) of bulls is important, for which sexual behavior and semen quality of breeding bulls need to be studied along with their relationship.Studies of semen characteristics and correlation among various semen characteristics  of bullsSelection of bulls on the basis of sexual behavior and semen quality are more important and economical . In termThe work was carried out under the institute project (no. B-33) entitled “Management practices to improve semen productivity by mitigating low libido problem in Sahiwal bulls. We have carried out experiment in accordance with the Guidelines laid down by the Institutional ethics committee of ICAR-NDRI with prior approval.Total of 63 ejaculates were collected from six Sahiwal bulls  maintained under identical feeding and management condition during the experiment (19-03-2014 to 05-05-2014) at Artificial Breeding Research Centre, NDRI, Karnal. Healthy, sexually mature and clinically normal bulls showing good libido were selected randomly from the herd.Two successive ejaculates were collected from the bulls using sterilized bovine artificial vagina (IMV model-005417) maintained between 42-45°C, over a male dummy bull. Soon after collection, each ejaculate was placed in a water bath at 30°C and various standard laboratory tests for semen volume and mass activity (MA), individual progressive motility, sperm concentration (SCON) (million/ml) )×100MAS: MAS was scored on a 10 point scale distributed among different behavioral events displayed by bulls during semen collection i.e. MO, PES, PPS, ETS, grasping of teaser at pelvic level (Gr), and penile movement to locate artificial vagina (Pm). After MO  if a bull did not ejaculate, then 1 point was deducted from the total score for each futile attempt. The MAS was determined in successful attempts only as follows:[{(MO+PES+PPS+ETS+Gr+Pm)-Futile attempts}/10)] ×100.SBS: The SBS was calculated from the LS and MAS as follows:(LS + MAS)÷2t-test as suggested [The degree of association among and between sexual behavior and seminal attributes were estimated by Pearson’s correlation coefficient method and significance was tested by Student’s uggested .The overall least squares means of ejaculate volume, MA, IPM, SCON pH, NESC, total morphological abnormality , HOST, and AI were 3.40±0.20 ml, 2.90±0.11, 73.20±2.1%, 1130.10±38.45 million/ml, 6.80±0.04, 84.30±1.63%, 6.59±0.21% , 63.15±1.72%, 89.00±1.44%, respectively. The overall least squares means of SA, RT, DT, TTTM, PES, PPS, ETS, LS, MAS, SBS, were 3.75±0.06, 239.41±28.40 s, 4.41±0.21 s, 243.81±28.41 s, 2.97±0.02, 1.90±0.04, 2.62±0.07, 93.84±1.13%, 46.09±1.80%, 70.37±1.10%, respectively in Sahiwal breeding bulls.The relationships of sexual behavior and semen quality have great significance to predict the quality and fertility of bulls. The results of correlation coefficients among different sexual behavior parameters, semen quality parameters and between them are presented in Tables-The SA showed highly significant (p<0.01) positive correlation with LS, SBS and non-significant negative correlation with RT and TTTM. The result indicates that the bulls showing higher SA normally better in libido. The results are in similar line as reported by Kumar  in Sahiwet al. [The results of relationship of different seminal attributes of Sahiwal bulls depict that ejaculate volume had positive significant (p<0.05) correlation with IPM, SCON, head abnormality, total abnormality, HOST, AI as well as non-significant positive correlation with MA, NESC, and pH. MA had positive significant (p<0.01) correlation with IPM, SCON, NESC, HOST, AI, and CYBR-14. The results are in consonance with the findings reported in crossbred bulls  and in Set al.  in crosset al. ,9 and inet al.  and in Ret al.  in crosset al.  HOST hadThe correlation coefficients between seminal attributes and sexual behavior parameter depicted that MA and IPM both had negative significant (p<0.05) correlation with RT and TTTM which indicates if more RT then there are poor MA and IPM of semen. Similar reports regarding MA and RT was reported by Elrabie . SCON haSahiwal bulls are sexually sluggish and total time required to make breeding bulls donate quality ejaculates is more in indigenous bulls. It is, therefore, necessary to study the sexual behavior of individual bulls critically to take corrective measures for successful collection of semen. The individual preference of the bulls also needs to be studied for maximizing harvest of quality ejaculates as some bulls prefer false mount and some other bulls do not require false mount if they are restrained properly in the semen collection yard. Sometimes in the presence of dominant and adult bulls, the young bulls shows submissive behavior i.e., a form of animal behavior in which one individual attempts through appeasement displays to avoid injury by a dominant member of its own species and may results in reluctance of MO to the dummy. This behavior is more prominent in indigenous dairy bulls. The temperature of artificial vagina, timing of presentation of AV to the penis is very crucial in case of indigenous bulls as a fraction of a second little delay in grabbing the penis for diverting to AV; the bull will dismount early and become reluctant to mount again with a long refractory period. Semen quality and quantity is related with the sexual behavior of bull and specifically MA and volume parameters are more closely related with sexual behavior and ejaculatory thrust. As we are well aware that RT and ejaculatory thrust is important as it is highly correlated with quality as well as the quantity of semen. The standards of SBS and semen quality in comparison to crossbred bulls are lower in indigenous bulls, which reflects that, there is a need of involvement of expert technical persons for semen collection based on the individual preference of the indigenous bulls. Sufficient time should be given for sexual preparation of bulls starting from training of the bulls for semen collection, as it is a learning process. Overview of the results revealed the importance of sexual behavior and its relationship with semen quality to design appropriate management strategies to maximize quality semen production from the individual bulls.The correlation of various sexual behavior and semen quality parameters reflected the importance of sexual behavior in predicting the quality of the semen and their future utility. The overview of the results depicted the need of greater emphasis on management interventions to improve sexual behavior particularly SA and RT, which will be helpful in increasing ejaculate volume and total output of spermatozoa, which will lead to increase in production of frozen semen doses required for breed improvement program. The collection of semen should be based on the individual animal’s sexual behavior and preferences to harvest maximum sperm per ejaculate. The relationship of sexual behavior with semen quality will be helpful in analyzing the behavior of individual bull to provide appropriate management interventions to maximize the semen production of bulls by incorporating those in routine management practices in semen banks. Besides that sexual behavior and semen quality are the important criteria for declaring a bull satisfactory breeding bull during BSE of a male at sexual maturity. It will be helpful in reduction of rearing cost by rejecting the bulls which are not satisfactory in BSE.MB and TKM have designed the study. SS carried out the laboratory work and analysis of data. MB, TKM, AKG and AKC supervised the research and helped in drafting of the manuscript. AK and PS helped with the laboratory work. All authors read and approved the final manuscript.


We report on a case of human infection with influenza A(H7N9) virus in Jilin Province in northeastern China. This case was associated with a poultry farm rather than a live bird market, which may point to a new focus for public health surveillance and interventions in this evolving outbreak. Since it was first reported in spring 2013 . FurtherOn February 15, 2014, a 50-year-old man, owner of a small farm in Changchun, Jilin Province, experienced an isolated fever (axillary temperature 38.3°C). He had no history of underlying medical conditions. The man sought medical care that day at Jilin University Third Hospital and returned to the hospital on February 16, 17, and 19 with ongoing fever. Radiographic imaging showed evidence of pneumonia; he received intravenous administration of azithromycin and xiyanping, a traditional Chinese medicine. On February 19, the man sought care at Jilin University First Hospital, where throat swab samples were taken. The same day, testing of the samples at the local and provincial Centers for Disease Control and Prevention yielded positive results for influenza A(H7N9) virus by real-time reverse transcription PCR (RT-PCR); China CDC confirmed results the next day. Treatment with oseltamivir (150 mg 2×/d) and methylprednisolone (80 mg 2×/d) was initiated, and oxygen was administered by nasal cannula. The man recovered and was discharged from the hospital on March 7.The man denied exposure to poultry other than on his farm, including to poultry on other farms or in LPMs. He reported no contact with persons who had similar symptoms before onset of his illness. As part of the case investigation, 68 close contacts of the case-patient were monitored for 7 days; 1 contact had influenza-like illness. Throat swab specimens were collected from this person on days 2 and 3 after symptom onset and tested for influenza A(H7N9) by using real-time RT-PCR; results were negative.The virus from the case-patient’s specimens was isolated in egg culture and designated A/Jilin/10117/2014 (H7N9) . The isolate’s 8 genes were similar to those of the virus A/Anhui/ 02/2013 (H7N9) (GISAID accession no. EPI_ISL_141190); nucleotide/amino acid homology was 99.5%/99.6% for hemagglutinin, 99.2%/98.9% for neuraminidase, 99.3%/99.7% for polybasic 1, 96.4%/98.9% for polybasic 2, 97.9%/99.6% for polymerase acidic, 99.7%/99.6% for nucleoprotein, 97.7%/100.0% for matrix protein, and 99.5%/99.1% for nonstructural protein.Numida meleagris), black-bone silkie chickens , local chickens, and a goose. None of the birds received avian influenza vaccines on the case-patient’s farm; previous vaccination histories were unknown. Birds commingled in an egg production warehouse  virus infection epidemiologically linked to a poultry farm from which samples tested positive for H7N9 and H9N2 viruses. Previous reports of human subtype H7N9 virus infections in mainland China identified a poultry-related exposure for 84% of case-patients, 77% of which were linked to LPMs, but none were linked to farms . According to China’s Ministry of Agriculture, almost all positive samples have been from LPMs; H7N9 virus was initially isolated from farms in Guangdong Province in southern China in mid-March 2014 (Unlike avian influenza A(H5N1) virus, which frequently causes severe disease and death in poultry, influenza A(H7N9) virus is a low pathogenicity virus and has not been observed to cause substantial deaths in avian species. Under experimental conditions, birds exhibited no clinical disease after H7N9 virus challenge, although chickens and quail shed high levels of virus ; in addition, we found that 95% (19/20) of specimens positive for H7N9 virus were also positive for H9N2 virus. The H7N9 virus possesses internal gene cassettes from poultry H9N2 virus, another low pathogenicity virus that circulates widely in poultry in Asia (Public health measures taken to contain the outbreak of H7N9 virus infection have thus far focused on LPMs (


Mycobacterium europaeum, a slow-growing nontuberculous mycobacteria belonging to the Mycobacterium simiae complex, was described after the seminal characterization of five isolates collected from three sputum specimens and a jaw gland biopsy in Italy, Greece and Sweden. Five respiratory tract isolates were further reported in Iran. Here, we report the first isolation of M. europaeum in France, in the respiratory tract of a patient co-infected with human immunodeficiency virus and hepatitis C virus.3 T4 lymphocytes, a human immunodeficiency virus viral load of 1.6 log and a hepatitis C virus viral load of 6 log. Reverse-transcriptase polymerase chain reaction of her nasopharyngeal aspiration confirmed influenza A H1N1. Three sputum specimens lacked acid-fast bacilli but one grew mycobacteria identified by using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry as M. europaeum with a 1.56 log score. A 1,482-bp 16S ribosomal ribonucleic acid gene sequence yielded 99% similarity with both Mycobacterium parascrofulaceum ATCC BAA-614 and M. europaeum DSM 45397T and partial rpoB polymerase chain reaction-sequencing yielded a 725-bp sequence exhibiting 100% similarity with M. europaeum strain DSM 45397T.A 49-year-old Caucasian woman with a 26-year history of human immunodeficiency virus-hepatitis C virus co-infection was admitted for significant influenza-like syndrome in a context of repetitive exacerbations of chronic obstructive pulmonary disease. Significant biological parameters included lymphocytes of 1.6G/L including 237/mmM. europaeum in France, in the respiratory tract of a patient co-infected with human immunodeficiency virus and hepatitis C virus. M. europaeum warrants further attention in immunosuppressed patients with influenza, using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry and rpoB partial sequencing as tools for its accurate identification.We report the first isolation of  Mycobacterium europaeum, a slow-growing nontuberculous mycobacteria (NTM) belonging to the Mycobacterium simiae complex, was described after the seminal characterization of five isolates collected from three sputum specimens and a jaw gland biopsy in Italy, Greece and Sweden [M. europaeum in France, in the respiratory tract of a patient co-infected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV).d Sweden . Five red Sweden  from twod Sweden . In factd Sweden . Here, w3 T4 lymphocytes, a HIV viral load of 1.6 log and a HCV viral load of 6 log. Her chest radiography was normal. Reverse-transcriptase polymerase chain reaction (PCR) of her nasopharyngeal aspiration confirmed influenza A H1N1. At a 6-month follow-up, her clinical outcome was favorable with initial supportive treatment only. Three sputum specimens lacked acid-fast bacilli (AFB) after Ziehl–Neelsen staining and microscopic observation but one grew AFB after 21-day incubation in MGIT  at 37°C in a 5% carbon dioxide atmosphere. After subculture on Coletsos , the isolate was deposited in our collection (CSUR P1344) and tentatively identified by using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS), the bioMérieux extraction protocol, a MicroFlex™ mass spectrometer  and the Bruker MALDI Biotyper procedures as previously described [M. europaeum DSM 45397T listed in the Bruker Mycobacterium Library 1.0 and 2.0 databases. A 1,482-bp 16S ribosomal ribonucleic acid (rRNA) gene sequence (GenBank LN680852) yielded 99% similarity with both Mycobacterium parascrofulaceum ATCC BAA-614 (GenBank GQ153273) and M. europaeum DSM 45397T (GenBank HM022196), two species known to share almost identical 16S rRNA gene sequence [rpoB PCR-sequencing [M. europaeum strain DSM 45397T  commercially available.Here, one d medium , 2, illuDatabase  in orderM. europaeum has not been previously reported in France. Three of the five seminal strains have been isolated from sputum. Of interest, two Iranian patients [M. europaeum lung infection is insufficient, there is not enough known to be sure that colonization is not in fact a slowly progressive infection. In addition, like other species of the M. simiae complex [M. europaeum was here isolated in a patient with laboratory-confirmed influenza, a condition previously associated with Mycobacterium tuberculosis infection [patients  with a hpatients . Here, cpatients . As our  complex , 8, this complex . Of intenfection  through nfection . InteracWritten informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


The first two authors, Katharine Eakin and Renana Baratz-Goldstein, should be noted as contributing equally to this work.


Agaricus subrufescens has a worldwide distribution with a high level of polymorphism in the ITS region. A previous analysis suggested notable ITS sequence heterogeneity within the wild French isolate CA487. The objective of this study was to investigate the pattern and potential mechanism of ITS sequence heterogeneity within this strain. Using PCR, cloning, and sequencing, we identified three types of ITS sequences, A, B, and C with a balanced distribution, which differed from each other at 13 polymorphic positions. The phylogenetic comparisons with samples from different continents revealed that the type C sequence was similar to those found in Oceanian and Asian specimens of A. subrufescens while types A and B sequences were close to those found in the Americas or in Europe. We further investigated the inheritance of these three ITS sequence types by analyzing their distribution among single-spore isolates from CA487. In this analysis, three co-dominant markers were used firstly to distinguish the homokaryotic offspring from the heterokaryotic offspring. The homokaryotic offspring were then analyzed for their ITS types. Our genetic analyses revealed that types A and B were two alleles segregating at one locus ITSI, while type C was not allelic with types A and B but was located at another unlinked locus ITSII. Furthermore, type C was present in only one of the two constitutive haploid nuclei (n) of the heterokaryotic (n+n) parent CA487. These data suggest that there was a relatively recent introduction of the type C sequence and a duplication of the ITS locus in this strain. Whether other genes were also transferred and duplicated and their impacts on genome structure and stability remain to be investigated.The internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster is widely used in fungal taxonomy and phylogeographic studies. The medicinal and edible mushroom  Fusarium  and thus cannot be distinguished from each other. At this locus, the observed homozygosity at ITSII for the null allele was 24% (6/25), which is also in agreement with a rate around 50% of homozygosity at this locus for the two alleles.Among the genotypes of the 25 heterokaryons at the g et al. . At the ITSI locus. ITS of type C is a non-allelic homolog  located at the second locus ITSII that is not linked to ITSI.In conclusion, allelic segregation among both homokaryotic and heterokaryotic offspring agree with the presence of two unlinked ITS loci. ITS of types A and B are two alleles segregating at the Agaricus that we have obtained since over a decade ago, this was the first time we observed such stable and reproducible chromatograms with triple peaks. Then the presence of three distinct ITS sequence types was confirmed by the more reliable method of PCR cloning and sequencing. The observed number of clones bearing three types of ITS  was not significantly different from the 1:1:1 ratio in our PCR clone library. The three types of ITS were also found in the offspring but none of 94 single spore isolates possessed only the type C.The presence of three distinct types of ITS within one strain was supported by data from different methods. First they were inferred by interpreting the shifted sequences in the chromatograms of the isolate CA487 due to length differences among the three types. Similar chromatograms were obtained with CA516, a collection from the same site, indicating that a mycelium possessing these three ITS sequences was persisting at this location. Among the hundreds of ITS sequences of specimens in the genus A. subrufescens and A. bisporus [Among the 94 single spore isolates, 25 (27%) showed evidence of heterozygosity and were considered heterokaryotic. This is lower than the 44% estimated previously among a different set of offspring of the same strain by Thongklang et al. . Howeverbisporus .ITSI locus. ITS of type C segregated with a null allele at the ITSII locus unlinked to ITSI. At ITSI, the rate of homozygosity was close to 50%, similar to the other loci. ITSI and ITSII are not linked to any other marker used in this study.The presence of the different types of ITS and their segregation were separately analyzed in the homokaryotic and the heterokaryotic offspring. Based on the fact that the three types of ITS were equally prevalent in the parent strain, one simple hypothesis was that CA487 was a trikaryon, i.e. a heterokaryon composed of three types of genetically different nuclei  with eacITSI may contain hundreds of copies of the ITS and here we assume the simplest explanation that all the copies of the allele ITSI-a were of type A and all the copies of the allele ITS-b were of type B. The hypothesis that a proportion of the copies of the allele ITS-a could be of type B sequence is possible but unlikely for two reasons. Firstly, the chromatograms of type A and type B sequences in types A and B homokaryons were very clean with no obvious minor peaks resulting from the differences between the two sequences. Secondly, wild strains with only type A or type B are found in Europe, North and South Americas and the Caribbean. Therefore, alleles A and B seemed to have diverged a long time ago. If types A and B sequences could be mixed in the same cluster within the same homokaryon, we should expect to find all the intermediate proportions between 100% A 0% B and 0% A 100% B. However, in the different offspring we have studied (present and previous studies), we have never found any homokaryon with both the type A and type B sequences.The locus ITSI-a and ITSI-b, respectively, but it remains unknown in which of the two nuclei the ITSII locus can be found. Unfortunately, attempts to de-heterokaryotize CA487 to obtain the two constitutive haploid nuclei have failed. The methods we used were the same protocols that have allowed us to successfully obtain homokaryons from heterokaryons in A. bisporus through protoplasting.The heterokaryon CA487 has two constituent nuclei bearing alleles ITSI is not linked to the centromere. Specifically, the heterokaryons that are homoallelic at the centromere-linked locus PRS088 are putative sister progeny heterokaryons and thus should be highly homozygous at all centromere-linked loci [ITSI locus since four of these ten heterokaryons are heterozygous ITSI-a/b. As for the second ITS locus, it is possible that the null allele represents undetected ITS that could not be amplified with the current universal primers. However, we believe this is highly unlikely as we have not failed amplification of any samples from the genus Agaricus using the universal primers. The simplest hypothesis is that ITSII is a duplicate locus of the ITS introgressed from a homokaryon closely related to those from an Asian strain and that it is present in only one of the two constitutive haploid nuclei of the parent CA487. ITSII is not linked to ITSI and this genomic composition is persisting in the natural environment.Our data indicated that ked loci . This isA. subrufescens is the earliest species arising in the A. sect. Arvenses approximately 12 Ma ago. The geographical distribution of the different types of sequences suggests that geographical isolation might have contributed to their divergence.ITS sequences similar to or identical with the types A, B, and C sequences of isolate CA487 have been found in field specimens from different continents. The extensive differences among the three sequences suggest that these sequences have likely diverged from each other for a long time. However, the exact reason for their divergence is not known. These divergences may have occurred several millions years ago. Indeed, an approximate date of 2–3 Ma is indicated in the multigene Maximum Clade Credibility tree of Zhao et al.  for the A. subrufescens and noted extensive heterozygosity in American samples. He suggested that hybridization might have occurred between populations. He also noted that the Hawaiian samples were highly diverged from samples in the Americas. Since 2005, many specimens have been collected in Europe and in Asia. Some European samples possessed similar heteromorphisms as the American samples while ITS sequences of the Chinese and Thai samples were similar or even identical to those of the Hawaiian samples [Kerrigan  analyzed samples , 21, 22.A. subrufescens from DR Congo in Africa [A. subrufescens remains sporadic in Europe and to our knowledge all or almost all the cultivated strains originated from America and most of them bear both types of ITS A and B sequences. Alternatively, human assisted migration of the three types of ITS sequences into the same ecological niche could have brought the different types together and resulted in their hybridization and subsequent generation of a strain such as CA487. We would like to note that CA487 was isolated in 2006, before A. subrufescens were formally described based on field specimens in Asia. In addition, to our knowledge, A. subrufescens was not commercially cultivated in Europe, and there was only few imports from Brazil [Our results suggest two possibilities. In the first, the type C sequence might have a distribution much broader than is currently known to cover Eurasia and even Africa. Incomplete sequences of old herbarium samples of n Africa  showed am Brazil .Interestingly, since 2012, the Thai isolate CA918 possessing an ITS of type C has been cultivated and crossed with the Brazilian strain WC837 and the French strain CA487. These crosses generated strains with different combinations of ITS types such as AC or BC and these strains have been cultivated at INRA . These oA. subrufescens, Rocha de Brito et al. [A. bisporus. This result indicated that the position of PR160 and/or of the ITS locus likely differed between these two species, despite their high level of macro-synteny [ITSI is most likely functional because offspring having only the ITSI-a or the ITSI-b alleles are viable. In contrast, the ITSII-c allele was never found alone in the single spore isolates, thus whether it is functional or not remains to be determined. Based on the chromatograms and the relative proportions of clones with the types A, B, and C sequences from strain CA487, the copy number of the type C ITS sequence within strain CA487 is likely very similar to those of types A and B. The long-term effects of such introgression and ITS duplication on the genome structure and function of A. subrufescens remain to be examined.In strain WC837 of o et al.  found th-synteny . In straS1 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.