The Swedish control program for salmonella includes restrictions and on-farm control measures when salmonella is detected in a herd. Required control measures are subsidised by the government. This provides an opportunity to study costs for on-farm salmonella control. The aim of this study was to describe the costs for on-farm salmonella control in Swedish cattle herds and to investigate the effects of herd factors on these costs in dairy herds.During the 15 years studied there had been a total of 124 restriction periods in 118 cattle herds; 89 dairy herds, 28 specialised fattening herds and three suckler herds. The average costs per herd for on-farm salmonella control was 4.60 million SEK with a median of 1.06 million SEK corresponding to approximately 490 000 and 110 000 EUR. The range was 0.01 to 41 million SEK corresponding to 1080 EUR to 4.44 million EUR per farm. The costs cover measures required in herd-specific control plans, generally measures improving herd hygiene. A mixed linear model was used to investigate associations between herd factors and costs for on-farm salmonella control in dairy herds. Herd size and length of the restriction period were both significantly associated with costs for on-farm control of salmonella with larger herds and longer periods of restrictions leading to higher costs. Serotype detected and administrative changes in the Swedish Board of Agriculture aiming at reducing costs were not associated with costs for on-farm salmonella control.On-farm control of salmonella in Swedish cattle herds incurred high costs but the costs also varied largely between herds. Larger herds and longer restriction periods increased the costs for on-farm control of salmonella in Swedish dairy herds. This causes concern for future costs for the Swedish salmonella control program as herd sizes are increasing. Salmonella has long been recognised as an important food-borne zoonotic pathogen of economic significance. During the last decade increasing efforts have been made to control salmonella along the food chain within the European Union . In SwedWhen salmonella is detected in a Swedish herd, the holding is put under restrictions and a control plan is put in place. The government partly subsidises the farmers for measures that are required during the restriction period. The costs for on-farm control of salmonella in cattle herds is the second most costly part of the Swedish salmonella control program despite only a small number of positive herds detected each year. Only the feed control incurs higher costs . Cattle Economic losses caused by salmonella infections in cattle herds have been well investigated by others . HoweverThe aim of this study was to describe the costs for on-farm salmonella control in Swedish cattle herds and to investigate the effects of herd factors on these costs.This paper includes a description of all Swedish cattle herds that have been under restrictions due to salmonella during the last 15 years and a regression analysis of the associations between herd factors and costs for on-farm control of salmonella in dairy herds.Within the Swedish program, infected cattle herds are detected by faecal sampling on clinical suspicion, sampling of dead calves at necropsy, sampling of lymph nodes at slaughter and through tracings from different sources such as contaminated feed, infected herds, contaminated meat and infected humans. A culture positive on-farm sample results in the herd being put under restrictions and a herd-specific control plan is drawn , 8. The The farmer can claim financial compensation for costs caused by measures stated in the herd specific control plan. Examples of costs that will be compensated are costs for increased working hours, culled animals in cases of overcrowding, production losses due to decreased number of animals, costs for thorough cleaning of stables and replacement of materials that cannot be properly cleaned as well as costs for hygienisation of manure and compensation for loss of its value as fertilizer. The financial compensations paid by the SBA cover either 50 % or 70 % of the eligible claimed costs . The higIn 2009 changes in the administration in the SBA were initiated. The purpose of these changes were to reduce costs. These changes included a more restrictive approach to financial compensations, less focus on major cleaning and more focus on daily hygiene routines in the herds.All cattle herds put under restrictions due to positive on-farm samples during the last 15 years (1999–2013) were included in the study.Data on herd identities, geographic location (county), type of production , salmonella serotype detected and dates when restrictions were initiated and removed were retrieved from records kept at the National Veterinary Institute (SVA). In most of the herds that were put under restrictions only one serotype was detected, isolated findings of a second serotype was not included. If restrictions had been partially removed, the date when restrictions on all animals had been removed was used.Data on herd size at the time restrictions were initiated, financial compensations paid by the SBA and the SBA level of compensation were compiled from documentation at the SBA, available from 1999 and onwards. Only herds where the deadline for claiming financial compensation had been passed were included.When the number of cows, but not the total number of animals was known, the herd size was estimated by multiplying the number of cows by two according to documented herd structure in Swedish dairy herds .i.e. dividing the actual costs by 0.5 and 0.7 respectively. Thereafter, in order to compensate for inflation, the costs for each herd were recalculated to a value corresponding to year 2013, using the official Swedish consumer price index [In order to get comparable costs between herds that had received 50 % and 70 % compensations, all compensations paid by the SBA were recalculated as if all herds had received 100 % compensation, ce index  at the mIn the regression analysis only dairy herds were included, because conditions in non-dairy herds are different in a way considered likely to affect costs. Only dairy herds that had received financial compensations were included. The number of non-dairy herds that had received financial compensations was too small for a separate analysis. Three dairy herds had repeated periods of restrictions, in these herds one randomly selected restriction period was included in the statistical analyses.A causal diagram Fig.  was drawS. Dublin, S. Typhimurium (including monophasic S. Typhimurium), S. Reading and “other serotypes”. “Other serotypes” included seven serotypes with one or two observations each. A categorical variable was created for herds being put under restrictions before and after changes in administration at the SBA in January 2009 as a way to evaluate if these changes had resulted in lower costs as intended. County and year for initiation of restrictions were included as random effects to adjust for possible differences but not estimate their effects.A mixed linear model was used to evaluate potential associations between herd factors and the costs during the restriction period in dairy herds. The recalculated costs was the outcome variable and the fixed explanatory variables included in the model were herd size, length of the restriction period, serotype detected in the herd and changes in the administration at the SBA. Herd size  and restriction period (number of days) were included as continuous variables. Serotype detected in the herd was included as a categorical variable with four levels, et al. [All continuous variables  were log-transformed in order to achieve normally distributed and homoscedastic residuals and to improve model fit. Confounding was tested according to Dohoo et al. . In brieet al.  by compaPredictions were made to illustrate the effect of herd size and length of restriction period on costs. Predictions were obtained as linear combinations of the parameter estimations from the model evaluated at herd sizes of 50–1150 animals with 100 animal intervals and length of restriction periods at 50–1450 days with 150 day intervals. Predictions were made with serotype set to Dublin, county set to Kalmar (H), year set to 2013 and changes in administration at the SBA set to after changes were performed in 2009.All statistical analyses were performed in R using the R package “lmerTest” for the regression analysis , 15During the 15 years 1999–2013 there has been a total of 124 restriction periods in 118 herds with positive on-farm samples. In Fig. Four herds, three dairy herds and one specialized fattening unit, had repeated periods of restrictions. One of the dairy herds had four separate periods of restrictions, the other three herds had two restriction periods each.Compensations were not paid for twelve of the 92 restriction periods in dairy herds (13 %) and for 21 of the 29 restriction periods in specialized fattening units (72 %). Based on experience the most common explanation for this would be that the specialized fattening units were not eligible for compensations due to purchase of too many animals. For dairy herds a likely reason would be a low grade of infection in the herd and consequently only very limited control measures undertaken.S. Dublin were detected in the county of Kalmar (H) and all herds with S. Reading were detected in the county of Skåne (M) (Table S. Reading were detected only during the years 2007 to 2011 during an outbreak in the county of Skåne (M). Salmonella Dublin, S. Typhimurium and “other serotypes” were detected throughout the 15 years studied.In total 75 dairy herds were included in the statistical analysis. Serotype was clustered on county, more than half the herds with M) Table . Herds wResults from the model are shown in Table et al. reported economic losses caused by S. Dublin infection during a 10-year period in a dairy herd with 200 cows adding up to 27 600 EUR in a herd with good hygiene and 415 800 EUR in a herd with poor hygiene [S. Dublin would have costs for controlling salmonella ranging from 170 000 to 510 000 EUR with the lower costs for a shorter restriction period of 200 days and the higher costs for a longer restriction period of 650 days, a timespan that covers the length of the restriction period for most herds being put under restrictions. These predicted costs are largely within the range of previously reported production losses.The results from this study provide opportunities for a rough comparison of costs for on-farm control of salmonella versus production losses reported by others. Nielsen Herd size was the variable with the largest effect on costs Table . Increaset al. showed that herd size, abundance of birds and rodents and several farm sites were associated with a longer restriction period [A longer restriction period was associated with higher costs, which was quite as expected. The effect was significant also after correcting for herd size. The length of the restriction period probably reflects several other important factors. Boqvist n period . Other in period . The drin period , 21.S. Dublin compared to all other serotypes the model was also run with serotypes divided in only two categories. Serotype still had no significant effect on costs. One explanation for the lack of significant differences could be a small number of herds infected with some of the serotypes (Table S. Dublin may be expected to persist longer in a cattle herd than other serotypes [S. Dublin infections either. An explanation for this might be a lower diagnostic sensitivity for S. Dublin versus other serotypes [S. Dublin. Serological testing in herds where restrictions have been lifted suggests that this may be the case.Serotype had no significant effect on costs. In order to evaluate if the costs were different for herds with es Table  which reerotypes  and thererotypes –25 resulDifferences in herd size in herds infected with different serotypes are not considered to contribute to the lack of significant differences in costs between serotypes, as herd size was corrected for in the analysis.As serotype was clustered on county and county was included as a random factor there was concern that the effect of serotype might be underestimated. However, the proportion of variance explained by county was reduced from 30 % to 8 % when the fixed part of the model was added. This indicates that a large part of the variation on county level is explained by the fixed variables. The effect of serotype is therefore, not considered likely to be underestimated.e.g.in the administration at the SBA was not evaluated.The change of administrative routines at the SBA in 2009 showed no significant effect on costs in this study. It shows that despite intense efforts by the SBA it was difficult to achieve changes in the established system that would reduce the costs for on-farm control of salmonella. In this study, only subsidises paid to the farmers were evaluated, other possible benefits S. Dublin, may not be free from salmonella when restrictions were lifted. We therefore use the term on-farm control of salmonella, even if the long term aim is eradication from the herd.Removal of restrictions from a herd is based on negative culture results. Due to poor diagnostic sensitivity of culture on individual animals  culture The costs shown in this study might be higher than necessary for on-farm control of salmonella. When designing a herd-specific control plan it is difficult for the appointed veterinarian to decide which measures are necessary and which are not, and this may lead to application of the precautionary principle. Consequently excessive measures might have been included in the herd-specific control plans. Moreover, during the 1990s companies handling cleaning and disinfection of stables entered the market and with time most farmers engaged these companies to perform the cleaning and disinfection required in the herd-specific control plan. These companies often performed meticulous cleaning procedures excessive to what is necessary for on-farm control of salmonella in cattle herds. This could also have caused higher costs than necessary.Data was only available on a few factors when in reality there is a large number of factors that will affect the costs for control. Examples of such factors are within-herd prevalence at detection, herd hygiene and type of stalling. However, many of these factors are likely to be reflected in the length of the restriction period which was included in the regression analysis.Mandatory on-farm control of salmonella in Swedish cattle herds incurs high costs but also varies largely between herds. Larger herds and longer restriction periods increased the costs for on-farm control of salmonella in Swedish dairy herds. This causes concern for increasing costs for the Swedish salmonella control program in the future as herd sizes are increasing.


This study was undertaken with the aim to compare and establish the genetic relatedness between classical swine fever virus (CSFV) genogroup 2.2 isolate and pestivirus reference strains.The available complete genome sequences of CSFV/IND/UK/LAL-290 strain and other pestivirus reference strains were retrieved from GenBank. The complete genome sequence, complete open reading frame, 5’ and 3’ non-coding region (NCR) sequences were analyzed and compared with reference pestiviruses strains. Clustal W model in MegAlign program of Lasergene 6.0 software was used for analysis of genetic heterogeneity. Phylogenetic analysis was carried out using MEGA 6.06 software package.The complete genome sequence alignment of CSFV/IND/UK/LAL-290 isolate and reference pestivirus strains showed 58.9-72% identities at the nucleotide level and 50.3-76.9% at amino acid level. Sequence homology of 5’ and 3’ NCRs was found to be 64.1-82.3% and 22.9-71.4%, respectively. In phylogenetic analysis, overall tree topology was found similar irrespective of sequences used in this study; however, whole genome phylogeny of pestivirus formed two main clusters, which further distinguished into the monophyletic clade of each pestivirus species. CSFV/IND/UK/LAL-290 isolate placed with the CSFV Eystrup strain in the same clade with close proximity to border disease virus and Aydin strains.CSFV/IND/UK/LAL-290 exhibited the analogous genomic organization to those of all reference pestivirus strains. Based on sequence identity and phylogenetic analysis, the isolate showed close homology to Aydin/04-TR virus and distantly related to Bungowannah virus. Flaviviridae comprised of bovine viral diarrhea virus-1 (BVDV-1), BVDV-2, classical swine fever virus (CSFV), and border disease virus (BDV) [viz. pestivirus of giraffe [The genus pestivirus of the family  giraffe ,4, prong giraffe , Bungowa giraffe  and Tuni giraffe . Further giraffe .Pestiviruses have single-stranded, positive-sense RNA genome of approximately 12.3-12.7 kilobases (kb) that contains only one open reading frame (ORF). The ORF translated into a polyprotein about 3,900 amino acids (aa) flanked by highly conserved non-coding regions (NCRs) at 5’nd 3’ends. The ORF encodes a polyprotein that is co- and post-translationally processed into twelve structural and non-structural proteins. These proteins arranged from 5’ to 3’ ends are: N-terminal protein (Npro), capsid protein (C), envelope glycoprotein , protein 7 (p7), and the non-structural proteins (NS) NS2, NS3, NS4A, NS4B, NS5A, and NS5B [Molecular epidemiology based on complete nucleotide sequence data has broadened the scope for genetic heterogeneity and phylogenetic grouping of isolates existing in field condition. The genetic relatedness of pestivirus isolates, including their relationship to the type viruses of the species, is one of the important parameters for classification of pestiviruses . During Ethical approval was not required for this type of study as live animal is not used anywhere in this study.http://www.ncbi.nlm.nih.gov/genbank/) on 2 April 2015  and ovine (BDV and Aydin strain) origin . This coet al. [et al. [Based on sequence analysis using different region and strategies, different workers have proposed their classification of pestiviruses. Analysis of the genetic and antigenic similarities, Becher et al.  have suget al. . Finding [et al. ; howeverIn the present study, it was observed that CSFV/IND/UK/LAL-290 showed a comparable genomic organization with that of other pestiviruses. The isolate was closely related to Aydin/04-TR virus and distantly with Bungowannah virus. Phylogenetic analysis revealed that BVDV-1, BVDV-2, BVDV-3 sharing a common ancestor, while Bungowannah and pronghorn antelope formed a single separate clade in all the trees. In contrary to the previous findings, pestivirus of giraffe was clustered with CSFV and BDV. Various researchers have classified the pestiviruses in independent manner and availability of the virus sequences. Proposal of nine species in novel pestiviruses also held true in the present study; though, grouping of viruses was different. This study provides a guideline for the classification of newly detected pestiviruses and has a potential application in genetic analysis of other related viruses in future.RK, KKR, and TC conceptualized the work. RK, DM, DC, AS and AK carried out the work and bioinformatics part. KKR, ABD, and RK drafted the manuscript. KKR, DM, ABP, and TC reviewed and edited the manuscript. All authors read the manuscript and approved the final manuscript.


Cryptococcus gattii is a recognized pathogenic fungus along the Pacific coast of the United States from California to Washington. Here we report that C. gattii may also be endemic to the southeastern United States and has probably been present there longer than in the Pacific Northwest. Cryptococcus gattii, recently designated as a separate species from Cryptococcus neoformans, is now recognized as a separate species complex   states of Washington and Oregon, where it is now considered endemic  has been performing passive surveillance for escribed . Five isBecause the population of VGI isolates from the southeastern states (VGI-SE) was only recently identified, it might be a newly emerging population similar to the independent emergences of VGIIa and VGIIc in the PNW . We sequAlthough the overall topology of the tree we generated remained almost identical to that seen by MLST, we were able to separate each of the isolates within the cluster using WGST . A totalC. gattii molecular type VGII exclusively from the PNW. Results showed that the VGII isolates consisted of 3 different highly clonal and recently emerged populations with only 107, 132, and 137 SNPs identified within the VGIIa, VGIIb, and VGIIc populations, respectively  had a primary pulmonary infection (All of the patients for whom disease manifestation was known had meningitis . This fiC. gattii VGI-SE clade in the southeastern United States is an order of magnitude greater than that seen in isolates from the PNW. Gillece et al. hypothesized that the emergence in the PNW began in the mid-1990s in British Columbia, Canada, and spread to the United States in the early 2000s (Our data indicate that the clonal diversity of the C. gattii in the United States was reported from West Virginia in 1924 (C. gattii VGI until decades later (C. gattii isolates from the southeastern United States exist: the first, from 1968, describes clinical and environmental isolates from Savannah, Georgia; the other, from 1982, describes isolates from Alabama, Tennessee, and Louisiana from patients with no travel history to a C. gattii–endemic region (C. gattii, C. neoformans serotypes B and C, their results clearly indicate that C. gattii has existed in the southeastern United States for >40 years, as opposed to the recent emergence in the PNW during the past 2 decades. A more recent report describes a C. gattii VGI isolate from an HIV-positive patient in North Carolina (The first identified isolate of C. gattii indicates the time required to generate the level of diversity seen in the VGI isolates. However, our findings lend credence to the hypothesis that C. gattii has circulated in the southeastern United States long enough to be considered endemic.No current molecular clock for


Emerging tospoviruses cause significant yield losses and quality reduction in vegetables, ornamentals, and legumes throughout the world. So far, eight tospoviruses were reported in China. Tomato fruits displaying necrotic and concentric ringspot symptoms were found in Guizhou province of southwest China.Capiscum annuum, Datura stramonium, Nicotiana benthamiana, N. rustica, N. tabacum and Solanum lycopersicum. The 3012 nt full-length sequence of 2009-GZT S RNA shared 68.2% nt identity with that of Calla lily chlorotic spot virus (CCSV), the highest among all compared viruses. This RNA was predicted to encode a non-structural protein (NSs)  and a nucleocapsid protein (N) . The N protein shared 85.8% amino acid identity with that of CCSV. The NSs protein shared 82.7% amino acid identity with that of Tomato zonate spot virus(TZSV).ELISA experiments showed that crude saps of the diseased tomato fruit samples reacted with antiserum against Tomato zonate spot virus (TZSV). Electron microscopy detected presence of quasi-spherical, enveloped particles of 80–100 nm in such saps. The putative virus isolate was designated 2009-GZT. Mechanical back-inoculation showed that 2009-GZT could infect systemically some solanaceous crop and non-crop plants including Tospovirus, which is named Tomato necrotic spot virus (TNSV). This finding suggests that a detailed survey in China is warranted to further understand the occurrence and distribution of tospoviruses.Our results indicate that the isolate 2009-GZT is a new species of  Thrips-transmitted tospoviruses cause significant economic losses in tomato, chilli and many other important crops worldwide . At leasGroundnut yellow spot virus (GYSV) [Impatiens necrotic spot virus (INSV) [Tomato spotted wilt virus (TSWV) [Watermelon silver mottle virus (WSMoV) [Tospovirus species, provisionally named Tomato necrotic spot virus (TNSV), as the causal agent of this tomato disease.Among the more than 20 Tospoviruses reported so far -10, 8 wes (GYSV) , Hippeass (GYSV) , Impaties (INSV) , Tomato s (INSV) , Tomato s (TSWV)  and Wate (WSMoV) . In 2009Nicotiana benthamiana and N. rustica and in local lesion host Chenopodium quinoa by mechanical inoculation as described previously [A virus (isolate 2009-GZT) was isolated from naturally infected tomato fruits exhibiting necrotic and concentric ring spots in Wengan County, Guizhou province, China in 2009. The virus was maintained in systemic hosts eviously .TZSV-specific antiserum (produced in our laboratory) and ELISA kits for detection of GRSV/TCSV, INSV, IYSV, and TSWV  were used in ELISA to test saps collected from symptomatic tomato fruits and plants inoculated with isolate 2009-GZT.Crude plant saps, extracted from natural symptomatic tomato fruits and from plant leaves mechanically inoculated with isolate 2009-GZT, were stained with 2% Ammonium Molybdate for electron microscopy as previously described .Watermelon silver mottle virus (WSMoV) were used to obtain the 3ʹ-end sequence of about 700 nt of 2009-GZT. The cDNA was synthesized using AMV reverse transcriptase . The primer pairs J13 and UHP were used to amplify the NSs and N genes following the protocols described previously [Total RNA was extracted from symptomatic tomato fruits using TRIzol Reagent  according to the manufacturer’s instructions. The universal primer pairs (TospS-3 W:GC(a/t)GTTCCAGGGTT(a/g)CT(t/c/g)TC, and Tosp-3: AGAGCAATCGAGGCGCTAATAA) designed based on the 3ʹ-end sequences of S RNAs of the members of eviously ,19. The Groundnut bud necrosis virus, GBNV), AF080526 , L12048 , AF013994 , KC290943 , NC_003624 , AF001387 , EU275149 , AB038343 , KF383956, EF445397 , HQ728387 , NC_002051, AY686718 , NC_010489 , EU249351 , NC_003843  for S RNAs; GQ478668 , AF067068 , S54325 , FJ946835 , and AF067069  for N genes.The complete sequence of 2009-GZT S RNA was assembled and analyzed with the aid of DNAMAN version 5.0 . Phylogenetic trees were constructed using the neighbor-joining method with 1000 bootstrap replications in MEGA 6.0 [C. quinoa and in systemically infected N. rustica after mechanical inoculation. Further infection studies were used to determine the experimental host range of 2009-GZT. Systemic infection was observed in C. annuum, D. stramonium, N. benthamiana, N. debneyi, N. rustica, N. tabacum and S. lycopersicum, with the infected plants showing leaf chlorosis, necrosis, ringspot and/or fruit deformation. Local lesion infection was found in C. amaranticolor, C. quinoa and N. glutinosa. The virus did not infect Cucumis sativus . It had NSs and N open reading frames (ORFs) in an ambisense orientation, separated by a 670 nt intergenic region (IGR) with an AU content of 72.9%. The 1380 nt NSs ORF, from nt 67 to 1446 of the viral RNA (vRNA) strand, potentially encoded a protein of 459 amino acids. The 828 nt N ORF, from nt 2112 to 2945 of the viral complementary RNA strand, potentially encoded a protein of 276 amino acids. The predicted molecular masses of NSs and N proteins were 51.6 kDa and 30.2 kDa, respectively. The 5′- and 3′-untranslated regions were 66 and 68 nt long, respectively.The 2009-GZT S RNA sequence shared the highest (68.2%) and lowest (28.9%) nucleotide identities with those of CCSV and GYSV, respectively. The N gene and protein shared higher nucleotide (60.2-76.3%) and amino acid (69.9-85.8%), respectively, sequence identities with those of the WSMoV serogroup  than with those of the IYSV serogroup  (53.5-55.7% nucleotide and 46.1-48.9% amino acid identities). It shared relatively low nucleotide (36.1-45.3%) and amino acid (16.4-36.1%) identities with those of GYSV, GCFSV, INSV, and those of the TSWV serogroup members (Table The NSs gene shared relatively higher identities (58–76.3% nucleotide and 61.6-80.8% amino acid) with those of the WSMoV serogroup than those of the other tospoviruses Table . The S RTospovirus with a provisional name Tomato necrotic spot virus (TNSV). During the field survey, Thrips tabaci and T. palmi were found in tomato fields and the surrounding weeds. These two thrips species may represent potential vectors of TNSV.Although 2009-GZT was originally isolated from tomato, it could infect other solanaceous species. The viral symptoms in those host plants were similar to those described for other tospoviruses such as TSWV, TZSV and HCRV. The virion morphology, ELISA results, and S RNA sequence characteristics indicate that isolate 2009-GZT is a tospovirus. The S RNA sequence divergence between 2009-GZT and the other tospoviruses is at the inter-species level  [Infection by tospoviruses has emerged as an important constraint to the productivity and quality of crops in many regions of the world ,21,22. S tabaci)  and larg


Rabies is a fatal encephalitis caused by viruses belonging to the genus Lyssavirus of the family Rhabdoviridae. It is a viral disease primarily affecting mammals, though all warm blooded animals are susceptible. Experimental rabies virus infection in birds has been reported, but naturally occurring infection of birds has been documented very rarely.Gallus domesticus), which had been bitten by a stray dog one month back, was brought to the rabies diagnostic laboratory. A necropsy was performed and the brain tissue obtained was subjected to laboratory tests for rabies. The brain tissue was positive for rabies viral antigens by fluorescent antibody test (FAT) confirming a diagnosis of rabies. Phylogenetic analysis based on nucleoprotein gene sequencing revealed that the rabies virus strain from the domestic fowl belonged to a distinct and relatively rare Indian subcontinent lineage.The carcass of a domestic fowl (Gallus domesticus, being reported for the first time in India, was identified from an area which has a significant stray dog population and is highly endemic for canine rabies. It indicates that spill over of infection even to an unusual host is possible in highly endemic areas. Lack of any clinical signs, and fewer opportunities for diagnostic laboratory testing of suspected rabies in birds, may be the reason for disease in these species being undiagnosed and probably under-reported. Butchering and handling of rabies virus- infected poultry may pose a potential exposure risk.This case of naturally acquired rabies infection in a bird species,  Rabies is a fatal viral disease affecting humans and other animals. Though all warm blooded animals are susceptible to this disease, rabies is commonly observed in mammals. Birds can be experimentally infected with this virus; however, naturally occurring rabies infection in birds has been reported very rarely. We report an unusual case of natural rabies infection in a domestic fowl from India. The bird was bitten by a stray dog and succumbed after a month. The brain tissue from the carcass was tested at a laboratory and was found to be positive for rabies virus antigens. This report indicates that rabies is a disease that can affect birds. Most often birds succumb due to shock or complication of animal bite injury and may not survive until the development of clinical signs of rabies infection. Moreover, fewer opportunities for diagnostic laboratory testing of suspected rabies in a bird may be a reason for the disease in these species being underestimated. Butchering and handling of virus-infected poultry may pose a potential biohazard. Lyssavirus of the family Rhabdoviridae. It is a viral disease primarily affecting mammals, though all warm blooded animals are susceptible. In India and other Asian countries more than 90% of human infections occur due to exposure to rabid dogs, while cats, monkeys and other wild animals are reported to transmit the infection in the rest of the cases . Perusal of available literature revealed that natural infection of birds with rabies virus has been documented uncommonly. The occurrence of rabies in a chicken under natural conditions is considered extremely rare . The present case of clinical rabies in a domestic fowl was identified from an area which has a significant stray dog population and is highly endemic for canine rabies.Rabies is a fatal encephalitis caused by the viruses belonging to the genus This study included samples received for diagnostic confirmation from a domestic fowl that died naturally. No tissue or clinical sample was obtained from the dead fowl specifically for the purpose of this study. No human subject or human clinical samples were included in this study. Hence, ethical clearance from the institutional review boards  was not required.Gallus domesticus) was brought to the rabies diagnostic laboratory at the Chief Disease Investigation Office, Department of Animal Husbandry, Kerala, India for diagnosis of rabies. The bird had been bitten by a stray dog one month back in its breast muscle. The wound was treated locally. After one month, the bird appeared droopy and off-feed for a day and succumbed. As rabies was frequently reported in the locality among dogs and other domestic animals, the owner brought the carcass for ruling out rabies. The stray dog that bit the fowl could not be traced and laboratory confirmation of its rabid status could not be done.The carcass of a domestic fowl  and custom sequenced from Amnion Biosciences Pvt. Ltd, Bangalore, India using gene specific primers. The sequence was deposited in the GenBank database under accession number KP316199. Partial nucleoprotein gene sequences of rabies virus isolates from GenBank representing various geographical regions in India and two other countries from the Indian subcontinent i.e Sri Lanka and Nepal were used to investigate the phylogenetic relationship with the present rabies virus strain. The sequences were aligned using ClustalW, and a maximum likelihood phylogeny tree was constructed using MEGA5 software  with bootstrap replication values of 1000.Touch impressions of the brain were stained by Seller’s stain and examined for the presence of Negri bodies. Impressions, fixed in high grade chilled acetone at -20°C, were subjected to the fluorescent antibody test (FAT) for detection of rabies virus nucleoprotein antigens No significant lesions could be observed in any of the visceral organs on post-mortem examination. The dog bite wound on the breast muscle was fully healed. The brain impressions tested positive for rabies virus antigens by FAT, however Negri bodies could not be demonstrated. The brain tissue sample was positive for rabies viral RNA by TaqMan real time PCR.. The lower cluster comprises of most of the isolates from the northern and southern part of India belonging to the Arctic/Arctic-like lineage, along with the Arctic Fox isolate from Canada (included for comparison). The upper cluster includes the present strain (NNV-RAB-FOWL) and other rabies virus strains from Southern India, Sri Lanka and Nepal which belong to the distinct Indian subcontinent lineage.The phylogenetic tree comprising of various nucleoprotein gene sequences of rabies virus isolates from India can be divided into 2 divergent clusters , 8; however they remain uncorroborated by lack of additional reports with robust laboratory evidence of naturally acquired rabies in birds. Gough and Jorgenson (1976) examined 343 serum samples of birds for antibodies against rabies virus and found that 23 of them had low passive haemagglutination titers . However, in another serological survey in captured birds, no significant titers were detected . A variety of birds have been infected experimentally with development of clinical signs, often without development of neurological features, or with recovery from clinical signs ,11.Rabies has been conventionally considered as a disease of mammals and clinical cases of naturally acquired rabies has been reported in birds infrequently. A few anecdotal reports were published in the late 1950’s, indicating the rare occurrence of rabies in birds  and the presence of an easily accessible diagnostic facility may be the reason the disease could be identified in poultry. Most often birds succumb due to shock or complication of animal bite injury and may not survive until the development of clinical rabies infection.The present report indicates that rabies is a disease that can affect birds. Lack of obvious clinical signs and fewer opportunities for diagnostic laboratory testing of suspected rabies in a bird, may be the reason for the disease in these species being undiagnosed and probably underestimated. The locality of the bird in the present case is highly endemic for rabies –15. Other studies confirmed the circulation of this distinct variant found only in Sri Lanka and mostly the southern part of India . However, recently isolates from Nepal have also been reported to be phylogenetically related to these distinct variants and this lineage has been designated as the Indian subcontinent rabies virus clade . This clade with phylogenetically related strains from South India (including the present strain), Sri Lanka and Nepal is also evident in  Sequencing and phylogenetic analysis of additional isolates representative of various geographical areas in India and other countries in the Indian sub-continent may aid in further elucidation of the epidemiological significance of these variants.Phylogenetic analysis based on partial nucleoprotein gene sequencing revealed 98% homology of the present rabies virus strain (NNV-RAB-FOWL) to a canine rabies virus isolate (AF374721) from Chennai, in Tamil Nadu state in south India. Interestingly, it clustered with another Indian isolate from Goa, a southern state and also with distinct isolates from Sri Lanka, rather than with other Indian isolates in the Arctic/Arctic-like virus cluster, the extensive circulation of which has been reported from India. Earlier reports had speculated the presence of this distinct Sri Lankan variant in India and suggested that movement of humans and their animals between Sri Lanka and India, particularly within the southeastern coastal area of the mainland, may have resulted in the movement of this variant between these geographically separate regions Gallus domesticus is reported in India for the first time. It indicates that spill over of infection even to unnatural hosts is possible in highly endemic areas. The risk of exposure through consumption of infected meat, though unlikely, and butchering/handling of rabies virus-infected poultry can potentially pose a risk of transmission of rabies to humans, although never reported to date.In conclusion, a case of naturally acquired rabies infection in a bird species,


Our study indicates that transcriptome data are useful for identifying a complete viral genome.The hop latent virus is a single-stranded RNA virus that mainly infects hop plants. Here, we report the complete genome sequence of a hop latent virus, which was  Carlavirus in the family Betaflexiviridae are single-stranded positive-sense RNA viruses. Of them, the hop latent virus (HpLV) is one of three carlaviruses that also includes hop mosaic virus and American hop latent virus (–Humulus lupulus L.) cultivars, which are important bitter flavoring agents used in the production of beer  to remove redundant transcripts (To determine the causal HpLV agents of hops, we searched for publicly available hop transcriptome data . Hop sam version .5 to rem version .5 to remKP861891.The genome sequence of hop latent virus isolate Taurus has been submitted to GenBank under the accession no.


Lactobacillus plantarum I-UL4 is a novel multiple bacteriocin producer that harbours both plw and plnEF structural genes simultaneous which has not been reported elsewhere. Therefore, molecular characterisation of bacteriocin genes that harboured in L. plantarum I-UL4 was conducted in this study.Bacteriocin-producing Lactic acid bacteria (LAB) have vast applications in human and animal health, as well as in food industry. The structural, immunity, regulatory, export and modification genes are required for effective bacteriocin biosynthesis. Variations in gene sequence, composition and organisation will affect the antimicrobial spectrum of bacteriocin greatly. brnQ1, napA1, plnL, plnD, plnEF, plnI, plnG and plnH) of plnEF locus and 2 genes (plw and plwG) of plw locus were amplified successfully from genomic DNA extracted from L. plantarum I-UL4 using specific primers designed from 24 pln genes selected randomly from reported plw, plS, pln423 and plnEF loci. DNA sequence analysis of the flanking region of the amplified genes revealed the presence of two pln loci, UL4-plw and UL4-plnEF loci, which were chromosomally encoded as shown by Southern hybridisation. UL4-plw locus that contained three ORFs were arranged in one operon and possessed remarkable amino acid sequence of LMG2379-plw locus, suggesting it was highly conserved. Interestingly, the UL4-plnEF locus appeared to be a composite pln locus of JDM1-plnEF and J51-plnEF locus in terms of genetic composition and organisation, whereby twenty complete and one partial open reading frames (ORFs) were aligned and organised successfully into five operons. Furthermore, a mutation was detected in plnF structural gene which has contributed to a longer bacteriocin peptide.Under optimised conditions, 8 genes (plnEF and plnW loci are classified as class I bacteriocin and class II bacteriocin molecules respectively. The concurrent presence of two pln loci encoding bacteriocins from two different classes has contributed greatly to the broad inhibitory spectrum of L. plantarum I-UL4. The new genetic composition and organisation of plnEF locus and concurrent presence of plnEF and plnW loci indicated that L. plantarum I-UL4 is a novel multiple bacteriocin producer that possesses vast potentials in various industries.Plantaricin EF and plantaricin W encoded by  Lactococcus, Leuconostoc, Pediococcus, Lactobacillus, and Streptococcus  were different from the reported plnEF loci.The rted plw , plS  respectively.The DNA sequences for both


Invasive pests impose a major burden on the agricultural industries and the natural environment, threatening some native species with extinction. Although chemical agents are often deployed to eliminate pests, unspecific toxicity makes their use undesirable. Biological agents are of interest as an alternative method to cull specific pest species with little residual environmental harm. The deliberate release of microbial pathogens, referred to as biological control (biocontrol), has, to date, largely involved viruses. Here, we summarise the past and proposed applications of the biocontrol of vertebrate pests. Despite understandable fears, vertebrate biocontrol has yet to be associated with adverse effects, and provides large-scale natural experiments by which to understand the intimate interactions between hosts and their pathogens.Aedes aegypti mosquitoes artificially infected with Wolbachia bacteria [Wolbachia can strongly inhibit DENV in laboratory experiments, and can spread among wild mosquitoes, whether it will lead to a reduction in human DENV prevalence and incidence has yet to be determined [Biocontrol, in which a biological agent is used to reduce the population size of a target pest organism, is widely used in agriculture to protect plants from arthropods and fungal infections . Althougbacteria . While Wtermined ,3.Cyprinus carpio), an invasive fish species that comprises up to 90% of fish biomass in the major Murray-Darling river system of southeast Australia and the cause of environmental degradation and loss of native fish biodiversity [The use of pathogens as biocontrol agents to control vertebrate populations has caused more concern, and the fear of inadvertent infection of other species, even humans, persists. In the 1950s, classical swine fever virus was released on small islands in California in an attempt to reduce the wild swine population, but the virus did not establish persistent transmission . Indeed,iversity .Marion Island is a small and remote island in the Southern Indian Ocean with a large seabird population. In 1949, cats were brought to the island to hunt mice in the island’s meteorological station. By 1975, the island had an estimated feral cat population of over 2,000, which posed a major threat to local avian species, with more than 45,000 birds predated each year. Feline parvovirus is a highly contagious (DNA) virus that is transmitted via body fluids or contaminated surfaces and resistant to temperature changes, making it ideal for a sub-Antarctic environment .Oryctolagus cuniculus). Following importation for hunting purposes in the 19th century, rabbits rapidly spread all across much of Australia, forming a “grey blanket” that caused major damage to vegetation [Australia’s unique fauna and flora are highly sensitive to imported species, and the deliberate or accidental introduction of feral animals has posed an enormous problem to local wildlife. Historically, the most important of these has been the European rabbit , but highly virulent in the European rabbit, resulting in myxomatosis and death within a few days [MYXV belongs to the family few days . In 1950few days . Fortuitfew days . After iInterestingly, MYXV was also released in New Zealand, but it failed to establish there, likely due to the lack of appropriate insect vectors . The NewAlthough a native species, the European rabbit is regarded as a pest in some parts of Europe. Hence, two years after the successful use of MYXV in Australia, the virus was privately released as a biocontrol in France. The impact was enormous. The virus spread rapidly through France and England, reducing the size of the rabbit population by more than 90% . ImportaThe epizootics of MYXV and RHDV in Australia constitute a unique natural experiment in how viruses establish themselves in a novel and naïve host population, including the subsequent evolutionary “arms race” between hosts and pathogens and the evolution of virulence . As suchTo be effective as a biocontrol, a virus needs to be species-specific, readily transmissible, and highly virulent. The breadth of host range of some viruses is so broad that they can be regarded as species “generalists,” with parvoviruses serving as an important example . Indeed,Sustained high virulence (with high transmission intensity) is also critical to effective biocontrol. Ideally, the virus should spread quickly and kill rapidly, but retain the ability to readily transmit to new hosts. MYXV and RHDV are both highly virulent in European rabbits, killing within a few days. For reasons that are currently unclear, these viruses have experienced strikingly different trajectories of virulence evolution: the mean virulence of field strains of MYXV declined , while there is no evidence that RHDV has attenuated . UnfortuIf performed safely, biocontrol interventions are a potentially powerful way to control invasive pest species and have provided important insights into the patterns, dynamics and processes of virus—host coevolution. We await the release of koi herpersvirus with interest.


Attention Deficit Hyperactivity Disorder (ADHD) is associated with considerable burden of illness at a patient, family and societal level. Although pharmacological treatment is recommended by authoritative guidelines, evidence on its influence on the broader burden of illness is limited. As treatment induces costs, proper healthcare decision making requires evidence on the associated societal costs or benefits and particularly the difference that response to treatment can make.Data on ADHD related resource use of patients 8–18 years and parents were collected by means of a cross-sectional, online survey amongst members of the Dutch parent association. Children were stratified to responders and non-responders to treatment according to pre-defined expert definitions.Analyses were performed on 618 questionnaires . Children were 11.8 years on average and mainly boys (82 %). Total monthly costs for children were €578 and €839 for responders and non-responders, respectively (p = 0.021), with a breakdown to direct medical costs , direct non-medical costs , and indirect non-medical costs . For parents, total costs were €246 vs. €399 for the responding and non-responding children, respectively (p = 0.006), with a breakdown to direct medical costs  and indirect non-medical costs . Total monthly costs of children and their parents together were €824 and €1228 for responders and non-responders to treatment, respectively (p = 0.002).These results stress the importance of a focus on response to treatment, not only beneficial for patients and their family, but also resulting in considerable societal benefits. Attention Deficit Hyperactivity Disorder (ADHD) is a heterogeneous psychiatric disorder characterized by a chronic pattern of age-inappropriate inattention, hyperactivity and impulsivity with a worldwide-pooled prevalence of 5.3 % Asherson . AdverseADHD is associated with a considerable burden of illness at both the patient, family, and societal level , and conduct disorder (CD)). Parents of children with ADHD were asked via the magazine of the association to join the study online by means of a personal login code. Inclusion criterion: being a parent of a child aged 8–18 years diagnosed with ADHD. Because of this membership, it was assumed valid to rely on the parents’ knowledge about the ADHD diagnosis of their child. Due to anonymity it was not possible to verify whether the diagnosis was indeed valid.The study sample was derived out of the members of the Dutch association The questionnaire was available from 01-09-2010 until 03-10-2010. The parent taking care of the child most of the day was asked to complete the questionnaire. Parents were not reimbursed for their participation in any way. In case the parent had more than one child with ADHD, parents were asked to answer the questions for their youngest child. Since the study did not interfere with clinical practice, according to Dutch regulation no approval of an ethics committee was needed. Also, with the methodology of this study focusing on parents instead of patients, it was not obligatory to use informed consent forms. Nevertheless, the protocol took into account all privacy aspects and all necessary efforts were taken in this study to protect privacy. Participants were informed on the purpose of data collection (scientific publication), respondents were fully free to decide to join and were able to exit the questionnaire at any time. Due to the login code that was distributed by Balans magazine, only members of Balans were able to access the questionnaire. The questionnaire could be completed only once per IP address, but these IP addresses were not saved and/or traceable so participants’ anonymity was guaranteed. Also, adequate security measures were employed in order to prevent unauthorized access, manipulation to or disclosure of the personal data although these were already unidentifiable when stored.+; F+): Compliance to prescribed daily dose of medication, no problems in functioning.: Non-compliance to prescribed daily dose of medication, problems in functioning.: Non-compliance to prescribed daily dose of medication, no problems in functioning.: Compliance to prescribed daily dose of medication, problems in functioning. and . Experts considered  to describe children not requiring medication, as no problems arise when medication is not taken according to prescription.  was considered to reflect several situations in which proper compliance is not translated into proper functioning: inappropriate dosing, inappropriate medication, or presence of an undiagnosed but relevant comorbid condition. The design of the study precluded to analyze these situations. The two remaining combinations,  and , were the basis for responder group definition. As it was deemed necessary to provide parents with a hands-on description of these groups, the experts operationalized the combinations to the following descriptions based on treatment compliance and functioning in family, school and the environmental level:‘Your child uses the prescribed daily dose of medication. Using this treatment, your child’s functioning is fine and there are no noteworthy problems at home, at school, with peers or during leisure time’.Responder to treatment: The daily dose of medication used by your child differs from the dose prescribed. Using this treatment, your child’s functioning is quite fine, but he/she is hampered by problems at home, at school, with peers or during leisure time for short periods of time’.Non-responder to treatment: ‘The experts decided to exclude combinations  was used, which is designed for self-report and is focused on psychiatric disorders like ADHD  or non-related to ADHD  to aid them in making a correct distinction. This could also mean costs for parents getting coaching and/or parenting tips; this is only necessary because of their child with ADHD and therefore these costs are considered related to ADHD. Costs for parents referred to their own health consumption only plus costs due to productivity losses.Table st and 2nd line care) was assumed to have similar costs as 1st line mental health care in case of ADHD. Unit costs for a visit to a school doctor and a company doctor were assumed to be similar to a general practitioner visit as costs for a school doctor and a company doctor were not defined in the costing manual. These assumptions were confirmed by a specialist in costing studies (LH). Costs for individual and group trainings were not specified in the costing manual. For individual sessions, costs for ambulatory 2nd line care were assigned based on communication by the Dutch mental health association ‘GGZ Nederland’. For group trainings, the association estimated time used by the expert divided by the number of clients per session. For day-care, a weighted average of day-care treatment (8 h a day) and half-time treatment (4 h a day) was used . For parents, the average number of days of medication use in the entire group of parents per month was multiplied by mean public Dutch costs of medication use in adults (€0.66 per day)  . As healthcare cost data tend to be skewed, tests were repeated using Mann–Whitney tests to check if conclusions remained similar using a test for non-parametric data. Multiple regression analysis was used to check for independent influence of other factors than treatment response on the mean total costs. A p-value of <0.05 was considered significant. It was not possible to examine test-retest reliability in this study, since the questionnaire was administered only once. Analyses were performed in IBM SPSS Statistics version 21.Completeness of the data is reported based on numbers completing the questionnaire. Furthermore, the data was reviewed for fraudulent responses, errors , missing data and possible strange patterns indicating programming errors. Study variables are described using absolute numbers (mean and median), percentages and 95 % confidence intervals. Missing data was replaced by means of multiple imputation as is the recommended method to reflect estimates across the range of circumstances considered . Table Data on resource use for children was missing for 19 respondents and data on resource use and production losses for parents for 27 respondents. This missing data was replaced by means of multiple imputation. Data were imputed based on the most optimal regression model. For children, this model was based on responder group (responder vs. non-responder), gender of the parent, >1 child with ADHD in the family and comorbidity. For parents, data was imputed based on a model with responder group, >1 child with ADHD, comorbidity and marital status.Table p = 0.006). Both total direct medical and indirect non-medical monthly costs were higher for parents of a non-responding child with €81 (p = 0.010) and €65 (p = 0.092) respectively. As for children, doctor’s consultations differed largely between responder groups and this difference was significant . Estimated annual costs were €1560 vs. €2532 for direct medical costs (p = 0.010); indirect non-medical costs were €1392 vs. €2172 (p = 0.092) and total costs were €2952 vs. €4788 (p = 0.006) (responder vs. non-responder).For parents, total monthly costs were significantly different between responder groups, incurring higher costs of €153 for a parent when having a child non-responding to treatment. Estimated annual costs were €2952 for the parents of responding children and €4788 for parents of non-responding children . Estimated annual costs were €9888 for the responder group and €14,736 for the non-responder group . Multiple regression showed that the factors treatment response, gender of the parent, >1 child with ADHD and comorbidity had an independent influence on total costs for children. For total costs of parents, the factors treatment response, >1 child with ADHD, comorbidity and marital status of the parent were independent influencers.The purpose of this study was to estimate ADHD related costs for children and their parents taking a societal perspective associated with treatment response. Non-response to treatment was shown to be associated with significant higher direct and indirect costs both inside and outside healthcare for children as well as their parents. Estimated annual costs differed between response groups, with extra costs of €4848 for non-responders. Costs for special education were very low relative to the other cost items in this study. This could reflect little need for extra attention, but it can also be a reflection of the general national Dutch trend towards less use of special education Dekker . A studyRecently, a systematic review was performed on European studies of ADHD-related costs published between 1990 and 2013  treatment for ADHD. Direct medical costs for children - specifically day-care treatment and trainings - were identified to be the most disparate with higher costs in the non-responder group. Direct non-medical costs were also unequal between groups with the highest difference in coaching at school. For parents of children with ADHD, doctor’s consultations were higher in the responder group. Treatment pathways should incorporate a focus on achieving response to treatment reflected in recovery both at home and at school and proper compliance to prescription. Also family involvement should be a standard part of the pathway.The results of this study contribute to the large evidence gap in societal cost calculation related to ADHD and emphasize the need for a broad perspective when analyzing problems related to ADHD since non-medical costs have a large contribution to total ADHD-related costs. With the trend of cost-effectiveness studies being used increasingly in guideline development and clinical decision-making, there is a clear need for comprehensive cost studies like the study at hand (Marsden & Wonderling


Carya illinoensis) showing leaf mosaic symptom through the use of deep sequencing of small RNAs. The complete genome of this virus was determined to comprise of 9,310 nucleotides (nt), and shared 24.0% to 58.9% nucleotide similarities with that of other Potyviridae viruses. The genome was deduced to encode a single open reading frame (polyprotein) on the plus strand. Phylogenetic analysis based on the whole genome sequence and coat protein amino acid sequence showed that this virus is most closely related to Lettuce mosaic virus. Using electron microscopy, the typical Potyvirus filamentous particles were identified in infected pecan leaves with mosaic symptoms. Our results clearly show that this virus is a new member of the genus Potyvirus in the family Potyviridae. The virus is tentatively named Pecan mosaic-associated virus (PMaV). Additionally, profiling of the PMaV-derived small RNA (PMaV-sRNA) showed that the most abundant PMaV-sRNAs were 21-nt in length. There are several hotspots for small RNA production along the PMaV genome; two 21-nt PMaV-sRNAs starting at 811 nt and 610 nt of the minus-strand genome were highly repeated.A novel potyvirus was discovered in pecan ( RNA silencing is an antiviral immunity and fundamental cellular mechanism that regulates gene expression through the production of small RNAs (sRNAs). Based on the finding that the viral small silencing RNAs (vsRNAs) can be overlapping in sequence and assembling into long contiguous fragments of the invading viral genome234Potyviridae is the largest and most economically important family of plant viruses, accounting for ~30% of the currently known plant viruses and causing significant losses in agricultural, pastoral, horticultural and ornamental crops6Potyvirus is a genus in the family Potyviridae. Most potyviruses are transmitted by aphids in a non-persistent manner. The virions of potyviruses are non-enveloped with a flexuous and filamentous nucleocapsid of 680 ~ 900 nm long and 11 ~ 20 nm in diameter. The typical genomic RNA is a linear positive sense single-strand RNA ranging in size from 9,000 ~ 12,000 nucleotides. It contains a single long ORF, which encodes a large polyprotein that is cleaved into 11 mature proteins including P3N-PIPO (P3 N-terminus-pretty interesting potyvirus ORF)11Carya illinoensis) is native to central and southern North America. It is an excellent multipurpose tree, providing a source of nutritious edible nuts, furniture-grade wood, and aesthetic value for the landscape. Pecan nuts are ranked highest in antioxidant capacitance and are enriched with many nutrients, minerals, and vitamins that are essential for optimum health. Pecans have been extensively cultivated in many countries for their commercial value. In China, pecan is increasingly being grown in many regions, including Hebei, Henan, Jiangsu, Fujian and Zhejiang provinces. Diseases affecting pecans are mainly caused by bacteria, fungi, nematodes, and physiological problems. No viruses have been reported to occur in pecans until now. During our survey of plant viruses of woody plants in southeast China, we found several pecan trees which showed typical symptoms of plant virus infection, with systemic mosaic on leaves. Further experiments were performed and finally a novel potyvirus was identified from these pecan trees. The virus is tentatively named Pecan mosaic-associated virus (PMaV). Furthermore, small RNA profiles of this virus suggest that the RNA silencing-mediated antiviral immunity mechanism is also applicable to pecan. To our knowledge, this the first viral disease found in pecan.Pecan . All thirty-six available complete genome sequences of Potyviridae viruses from GenBank were retrieved, and pairwised genome similarity comparison was performed. As a result   tail, and has been submitted to the GenBank database under the accession number me (ORF) . The ORFme (ORF) , that we143GWSG146 which is typical of potyviruses. The helper component proteinase (HC-Pro) comprises 447 residues (50.7 kDa) encoded by nucleotides located at position 673–2,013 nt, and is cleaved at the sequence YKVG/G, in agreement with the classical motif YXVG/G. The 375FRNK378 box, which is involved in RNA silencing suppression and associated with symptom development, is present246RITC249 and 504PTK506, which are associated with aphid transmission2511GA-AAAAA2750 and with a UAG stop codon at the position 2,751 nt. The cylindrical inclusion (CI) protein comprises 645 residues (71.8 kDa) encoded by nucleotides located at position 3,286–5,220 nt, and is cleaved at the site MTFQ/S. CI contains the 1161GAVGSGKST1169 and 1250DECH1253 motifs typical of the helicases of the superfamily 21423GERIQRLGRVGR1434, 1380ATNIIENGVTL1390 and 1277KVSATPP1283) whose functions are not clear. Nuclear inclusion body ‘a’ protein (NIa) includes the VPg (NIa-VPg) and NIa protein (NIa-Pro). Virus genome-linked protein (VPg) is a basic protein with 191 amino acids (22.2 kDa) encoded by nucleotides located at position 5,380–5,952 nt, and is cleaved at the sequence IQFE/A. Nuclear inclusion protein  comprises 243 residues (27.6 kDa), and is cleaved at the sequence MKFQ/G. NIa-Pro has cysteine protease active site which is responsible for cleavage at the P3/6K1, 6K1/CI, CI/6K2, 6K2/VPg, VPg/NIa-Pro, NIa-Pro/NIb, NIb/CP catalytic site with the consensus 2011H-34X-D-67X-G-X-C-G-14X-H2132. Nuclear inclusion body ‘b’ protein (NIb) comprises 533 residues (61.0 kDa) encoded by nucleotides located at position 6,682–8,280, and was cleaved at the site ASAQ/S. It also contains the typical RdRp motif GDD (2515GNNSGQPSTVVDNTLMVILAIVYSLIKLGHPYRTHKDIIKYFVNGDD2561). Finally, the CP  comprises 285 residues (32.2 kDa), and also contains the highly conserved 2735DAG2737 motif involved in aphid transmissionProteinase P1 comprises 205 amino acids (23.5 kDa) encoded by nucleotides located at position 58–672 nt. The mature protein is predicted to be cleaved at the sequence WRGF/S, which is cut by P1 serine proteinase. P1 of this virus has a motif For phylogenetic analysis, multiple-alignments were first performed using the Muscle programRecombination is a dominant feature of potyvirus evolution. To see whether or not the novel PMaV was generated by recombination, recombination analysis was performed using two different programs (TOPALi and RDP). No recombination events were detected, independently of the data set used for the analysis (data not shown).Potyvirus particles were found in infected pecan leaves with mosaic symptoms . This result is in line with the fact that most potyviruses have narrow host ranges.An experimental host range for PMaV was determined by mechanical inoculation of two plant species in Arabidopsis, the 5′-terminal nucleotide partially determines the preference of sRNAs for AGO proteins. Therefore, the distribution of 5′-terminal nucleotides was determined for the sequenced PMaV-sRNAs  were mostly 21 to 24-nt in length; among them the 21-nt class is most prominent, accounting for 42.22% of the total PMaV-sRNAs, followed by the 22-nt sRNAs . In ArabaV-sRNAs . For allPolarity analysis of redundant PMaV-sRNAs showed that they were derived slightly more often from the positive-sense genomic sequence (57%) than the antisense genomic sequence (43%) . FurtherPotyvirus filamentous particles in infected pecan leaves presenting mosaic symptoms. As a whole, these results support our finding that PMaV is a new species in the genus Potyvirus. During our investigation, we observed very severe mosaic symptoms in PMaV infected leaves; however, its pathogenicity in pecan is still being assessed. Also, this virus cannot be mechanically inoculated onto the two common hosts reported for other members of the family Potyviridae, indicating that PMaV may also have narrow host ranges. Other plants nearby the pecan trees are still under testing.Pecan tree are increasingly being grown commercially all over the world because pecan nuts supply many valuable nutrients for a healthy diet. At present, there are no reported viral diseases found in pecans. In our research, we isolated and identified a new virus, tentatively named PMaV, from pecan in Lin’an, Zhejiang province, China using small RNA deep sequencing technology. The complete genomic sequence of PMaV was cloned and sequenced. The full-length nucleotide sequence only shared 58.9% similarity with its closest relative, LMV. Furthermore, using electron microscopy, we identified the typical To date, several potyviruses have been reported as targets of the RNA silencing machinery of various herbaceous host plants. However, there is little information on this mechanism in woody plants. Here, profiling of small RNAs from PMaV-infected pecan leaves was performed. The presence of large quantities of PMaV-sRNAs  indicates that RNA silencing-mediated antiviral immunity is also utilized by pecan. In contrast to other potyvirus-derived small RNAs in herbaceous plants, which display a clear preference for adenine and uracil bases at the 5′-end20Monellia caryella) and the yellow pecan aphid (Monelliopsis pecanis), but we do not know whether these aphids can transmit PMaV. In contrast to herbaceous plants, woody plants are usually bred by grafting; whether or not this virus can be transmitted to pecan tree through grafting is currently under investigation. Our study, identifying a novel potyvirus that infects pecan trees, will assist in the development of molecular diagnosis of this virus in pecan trees all over the world.There are still many questions regarding PMaV epidemiology that need to be answered. First, pecan trees are native to central and southern North America, but we still do not know the geographic origin of the virus. Second, although most potyviruses in nature are transmitted by aphids, we do not yet know the mode of PMaV transmission. There are several species of aphids that inhabit pecan trees, such as the black margined aphid  following the manufacturer’s instructions. The small RNA library was constructed as describedhttp://www.ncbi.nlm.nih.gov/). Then the whole viral genome was obtained through several RT-PCR amplifications covering all the gaps, using a battery of sense and antisense-specific primers designed based on the contig sequences. The 5′- and 3′-terminal sequences were determined using 5′/3′ RACE PCR . The primer sequences were shown in TM  with Phusion® high-fidelity DNA polymerase . The amplified fragment was recovered with the AxyPrepTM DNA Gel Extraction Kit  and cloned using the ZeroBack Fast Ligation Kit . The positive colonies were further confirmed by DNA sequencing.Assembled contigs were scanned against the GenBank database using BLASTN . The putative cleavage sites and conserved domains of the putative functional proteins in the PMaV polyprotein were determined by comparison of its own sequence with other potyvirusesPotyviridae were retrieved from NCBI (http://www.ncbi.nlm.nih.gov/). Phylogenetic trees were constructed by the Neighbor-joining method using MEGA 6.06 softwareP-value cut-off of 0.05.Thirty-six whole genome sequences and twenty CP amino-acid sequences of representative recognized members of each of the eight genera comprising the family 2O in a mortar, dipped the grid in the serosity for about 10 seconds, then dry with filter paper. The grid was covered with a drop of 2% tungsten phosphate staining solution (pH 7.0) for about 20 seconds, and the copper grid was dried before EM observation.Samples from pecan leaves with mosaic symptoms and non-mosaic leaves were prepared for transmission electron microscopy (TEM). The samples were grinded with 0.5 ml ddHAfter removal of the adapter sequences and low quality reads by in-house Perl script, the resulting clean reads were then assembled into larger contigs using Velvet 0.7.31 with the k-mer value of 17Accession codes: Sequencing data were deposited in NCBI GenBank database with the accession number KT633868.How to cite this article: Su, X. et al. Discovery and small RNA profile of Pecan mosaic-associated virus, a novel potyvirus of pecan trees. Sci. Rep.6, 26741; doi: 10.1038/srep26741 (2016).


We analyzed the complete genome sequence containing the 3′ and 5′ noncoding regions (NCRs) of H3N2 canine influenza virus (CIV) with the matrix gene from the pandemic A/H1N1 virus, which will provide a better understanding of the pathogenesis, transmission, and evolution of variant CIV. Orthomixoviridae, family Orthomixovirideae, contains single-stranded, negative-sense RNA of the following 8 gene segments: polymerase basic 2 (PB2), polymerase basic 1 (PB1), polymerase acidic (PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix (M), and nonstructural (NS). To date, two subtypes of CIV, H3N8, and H3N2 have been isolated from dogs (3Canine influenza virus (CIV), which is a member of the genus om dogs 36. Recentom dogs 3. TherefoViral RNA was extracted from the allantoic fluid of virus-inoculated embryonated chicken eggs with the RNeasy MiniKit , and the viral RNAs are circularized with T4 RNA ligase as described previously , 9. One-The complete genome of A/canine/Korea/MV1/2012 (H3N2) is 13,628 nucleotides long. The size of segments 1 (seg-1) to 8 (seg-8) are 2,341, 2,341, 2,233, 1,765, 1,565, 1,467, 1,027, and 889 nt, respectively. They encode 12 viral proteins with the following amino acid lengths: PB2, 759; PB1, 757; PB1-F2, 90; PA, 716; HA, 566; NP, 498; NA, 469; M1, 252; M2, 97; NS1, 230; and NS2 (nuclear export protein [NEP]), 121. The lengths of NCRs of the viral RNA of A/canine/Korea/MV1/2012 (H3N2) were variable  in the different genome segments, but the virus has conserved 12 nucleotides (AGCA/GAAAGCAGG) at 3′ NCR of 8 segments, and 13 nucleotide (CCTTGTTTCTACT) at 5′ NCR, which is consistent with previous studies , 12. HerKF155142 to KF155149.The complete genome sequence of the A/canine/Korea/MV1/2012(H3N2) has been deposited and updated in GenBank under accession numbers


Based upon the principle of incident dark field (IDF) illumination, this hand held computer-controlled device was designed to address the technical limitations of its predecessors, orthogonal polarization spectroscopy and sidestream dark field (SDF) imaging. In this manuscript, we aimed to compare the quality of sublingual microcirculatory image acquisition between the IDF and SDF devices.The ‘Cytocam’ is a third generation video-microscope, which enables real time visualisation of the Using the microcirculatory image quality scoring (MIQS) system, , two independent raters compared 30 films acquired using the Cytocam IDF video-microscope, to an equal number obtained with an SDF device. Blinded to the origin of the films, the raters were therefore able to score between 0 and 60 for each film analysed. The scores’ distributions between the two techniques were compared.p < 0.0001). Of the six categories assessed by the MIQS, nearly one fifth of the SDF videos were scored as unacceptable for pressure (20 %), content (20 %), and stability (17 %), with focus scoring deficiently 13 % of the time. High agreement between the two raters scoring values was evident, with an intra-class correlation coefficient (ICC) of 0.96 .The median MIQS (95 % CI) given to the SDF camera was 7 (1.5–12), as compared to 1 (0.5–1.0) for the IDF device  contains supplementary material, which is available to authorized users. Incident dark field (IDF) imaging is an important technique that allows real time visualisation of the microcirculation . Based u2), which may be magnified by a factor of 211 times on the display monitor [The Cytocam is a lightweight 120 g), fully digitalised pen-like device  that applies the principle of incident dark field microscopy introduced by Sherman and Cook in 1971 . Blood v20 g, fulAlthough the IDF device should have significant superiority, in terms of image quality, over previous technologies this requires confirmation. We therefore set out to directly compare IDF and SDF images in a formalised manner.Five separate image sites per organAvoidance of pressure artefactsElimination of secretionsAdequate focus and contrast adjustmentHigh quality recordingThirty films of human sublingual microcirculation obtained using an SDF video-microscope , were compared to thirty comparable films obtained using the Cytocam IDF video-microscope. The films were picked at random from a database of over 800 SDF and IDF films, all of which were obtained from healthy adult volunteers who had given informed consent. Ethical approval for the study had been obtained from University College London Research and Ethics Committee. Two raters , blinded to the device on which the video was generated, independently graded the films using the Microcirculation image quality score (MISQ) system . These wIn 2013, a more formal approach to grading the quality of image acquisition prior to analysis was described, thereby giving a semi-objective measure of its suitability to be entered for computer analysis and quantification . Six keyEach of the six categories is graded as 0 , 1 (acceptable) or 10 (unacceptable). If the total of the six categories is >10, then the video is unsuitable for Table U-test was used to compare the score’s distribution between the two techniques. The two-tailed significance level was set at 0.05, and R (version 3.1.0) was used for the analyses.The agreement between the two raters was assessed using the intra-class correlation coefficient and Bland and Altman limits of agreement. Over the range of scores given, Spearman’s correlation coefficient was used to assess the degree of over- or under- estimation of the score by either rater. The Mann–Whitney All 60 videos were analysed by both raters and no problems were encountered. The distribution of the individual total scores by rater is shown in Fig. p = 0.21). For each device, the breakdown percentages of films scored as optimal, acceptable or unacceptable is presented in Table p < 0.0001). The distribution of these values may be seen in Fig. Very good agreement between raters’ total scores are evident with an intra-class correlation coefficient of 0.96 . In addition, good agreement is evident in Fig. These results demonstrate for the first time, that the Cytocam IDF video-microscope is superior to the SDF video-microscope in terms of the quality of sublingual microcirculatory image acquisition.High agreement between the two raters scoring values was demonstrated, and whilst it is evident from the Bland Altman plot that some individual variation existed between raters, neither individual demonstrated a trend in over- or under-estimating the score as the total values increased. Using the total score value to determine if an image was deemed suitable for analysis, , there was 100 % exact agreement  between the two raters.As to whether the IDF video-microscope was superior to the SDF video-microscope in terms of providing acceptable images for analysis, the median score of 7 given to the SDF images, as opposed to 1 for the IDF videos, indicates that the SDF camera is more prone to produce unacceptable results. In this instance, 100 % of the images obtained using the IDF video-microscope were judged to be acceptable for data analysis, as opposed to only 50 % of these data collected using the SDF device. Table Although 60 films chosen at random from a large database of images were analysed (30 for each device), a weakness in this manuscript was that no power calculation was performed prior to commencing. This said, the strong statistical significance supports the belief that it was adequately powered. Additionally, as the MIQS still relies on observer input to grade images, it is thus subjective in its film assessment. Nevertheless, it is the most formal approach to image grading we have to date, and the high ICC supports its use.In conclusion, these data demonstrate that the IDF video-microscope provides improved image acquisition of human sublingual microcirculation when compared to the SDF video-microscope. Superior in five out of the six categories comprising the MIQS, the use of IDF offers an advanced insight into the clinical evaluation of the microvasculature.


To the Editor: Zika virus (ZIKV), a member of the family Flaviviridae, is a mosquito-borne virus that is endemic to Africa and Southeast Asia. ZIKV causes illness that is similar to dengue fever, characterized by joint pain, myalgia, headache, and rash (9 cells/L (reference range 3.5–10 × 109/L), with mild lymphopenia of 1.0 × 109 cells/L (reference range 1.5.–4.0 × 109/L) and neutropenia of 1.4 ×109 cells/L (reference range 1.5–7.3 × 109/L). No thrombocytopenia or elevated liver enzyme levels were detected. C-reactive protein levels (1.4 mg/L) were within reference range.The previously healthy 31-year-old woman from Norway was admitted to the Oslo University Hospital, Norway, on December 13, 2013. Six days earlier, she had returned from a 14-day vacation to Tahiti, where she mainly stayed in the capital, Pape'ete, and took a short trip to the island of Mo'orea. One day after her return to Norway, she experienced fever, intense joint pain, and myalgia. Subsequently, a maculopapular rash developed. At the time of admission, her temperature was 37.7°C, and she had enlarged nuchal lymph nodes; injected conjunctivae; and a maculopapular rash on her trunk, extremities, and face . Clinica5 copies/mL; in vitro–transcribed RNA from a reference plasmid was used as a quantification standard.Because of the patient’s clinical picture and travel history, an acute ZIKV infection was suspected and several diagnostic tests were ordered. In a serum sample taken 5 days after symptom onset, no IgM or IgG against ZIKV, dengue virus (DENV), Japanese encephalitis virus, yellow fever virus, or chikungunya virus was detected by in-house indirect immunofluorescence . The partial ZIKV genome  was successfully amplified from the serum sample, and phylogenetic analysis of an ≈200-bp long genomic fragment of the nonstructural protein 3 gene demonstrated that strain Tahiti clusters within the Asian ZIKV lineages and is closely related to a strain from Malaysia . In a follow-up serum sample collected 36 days after symptom onset, IgG and IgM seroconversion against ZIKV was demonstrated; IgM titer was 1:1,280 and IgG titer was 1:2,560 (cutoff <1:20). In the same sample, low IgG titers against tick-borne encephalitis virus and DENV  were noted (cutoffs <1:20). Real-time RT-PCR for ZIKV in this serum sample was negative.Travel-related imported ZIKV infections have been reported after travel from Thailand to Germany . This finding is consistent with previously reported findings of ZIKV RNA loads of 930–728,800 copies/mL  would not be high enough for efficient transmission of ZIKV to susceptible vectors such as Photograph of patient with Zika virus infection.