Comparison of the ability of bovine antisera to neutralize a panel of serotype O FMDV identified three novel putative sites at VP2-74, VP2-191 and VP3-85, where amino acid substitutions correlated with changes in sero-reactivity. The impact of these positions was tested using site-directed mutagenesis to effect substitutions at critical amino acid residues within an infectious copy of FMDV O1 Kaufbeuren (O1K). Recovered viruses containing additional mutations at VP2-74 and VP2-191 exhibited greater resistance to neutralization with both O1K guinea pig and O BFS bovine antisera than a virus that was engineered to include only mutations at the five known antigenic sites. The changes at VP2-74 and VP3-85 are adjacent to critical amino acids that define antigenic sites 2 and 4, respectively. However VP2-191 (17 Å away from VP2-72), located at the threefold axis and more distant from previously identified antigenic sites, exhibited the most profound effect. These findings extend our knowledge of the surface features of the FMDV capsid known to elicit neutralizing antibodies, and will improve our strategies for vaccine strain selection and rational vaccine design.Five neutralizing antigenic sites have been described for serotype O foot-and-mouth disease viruses (FMDV) based on monoclonal antibody (mAb) escape mutant studies. However, a mutant virus selected to escape neutralization of mAb binding at all five sites was previously shown to confer complete cross-protection with the parental virus in guinea pig challenge studies, suggesting that amino acid residues outside the mAb binding sites contribute to antibody-mediated  Picornaviridae. It exists as seven immunologically distinct serotypes . The disease is widespread across the world, especially in Africa and Asia, and is able to spread rapidly (Foot-and-mouth disease (FMD) remains one of the most economically important diseases of cloven-hoofed animals worldwide. The causative agent, FMD virus (FMDV), is an aphthovirus that belongs to the family The virus consists of a non-enveloped capsid of icosahedral symmetry containing a single-stranded positive-sense RNA genome. A single ORF encodes all the capsid proteins (VP1–4), together with a total of nine additional mature, non-structural proteins, including two proteases (L and 3C) and an RNA-dependent RNA polymerase (3D). Only VP1, VP2 and VP3 are exposed on the outer surface of the virus particle and, thus, these three proteins determine both the specificity of virus-neutralizing antibodies and also the ability of the virus to interact with its receptors on host cells. The three-dimensional structures of several serotypes of FMDV have been determined  was reported to resist neutralization by bovine polyclonal sera raised against the parent virus . The deletion of the irus 5M,  by cloniirus 5M, . The pT7tructure . A singltructure . The capFollowing electroporation, live infectious viruses were successfully recovered from all the cDNA clones except the two (pT7S3/O1K-wt26n and pT7S3/O1K-wt26a) containing 26 amino acid substitutions in the capsid. FMDV-specific cytopathic effects (CPE) were observed 24–36 h post-electroporation. Extensive CPE were observed at both the first and second passages. At least two independent clones of each virus were rescued. However, only one clone in each case was used for further characterization. In order to prove that the expected viruses had been rescued, reverse transcriptase (RT)-PCR was carried out on the RNA extracted from infected BHK-21 cells using primer pair O1K-AflIIF and O1K-SpeIR that produced a 2500 bp-long fragment  of expected size (data not shown). No PCR products were generated in parallel reactions in which the enzyme reverse transcriptase was omitted, indicating that the amplified products were not generated from the transfected plasmid DNA. The PCR products were sequenced on both the strands and they contained no additional mutations. The genetic conformations of these viruses were stable on passage in cell culture and no reversion was noticed after sequential passages of the recombinant viruses for at least four passages in BHK-21 cells.in vitro . BHK-21 cells infected with the parent or recombinant viruses were stained following infection, and photographed. Both the parent and the recombinant viruses exhibited variable size plaques with no clear differences between them (data not shown).Standard multi-step growth curves were carried out to compare the growth of the recombinant viruses with the parent viruses. All the viruses grew at a similar rate and to a similar titre, indicating the mutations in the antigenic sites had no adverse effects on the replication efficiency of these viruses An indirect ELISA was carried out to study the binding of the antigenic site-specific mAbs with the parent rO1K-wt and the mutant viruses. As expected, all the mAbs used to generate the mar-mutant virus  recognized the epitopes on the parent virus whereas none of the mAbs bound to the mutant viruses , confirmThe 5M virus did not react with any of the O Lausanne murine neutralizing antigenic site-specific mAbs that were used to generate the mar-mutant viruses. Further, we studied the reactivity of this virus with other available well-characterized neutralizing murine and bovine mAbs (in ELISA) to determine whether there were neutralizing epitopes accessible for binding of mAbs recognizing residues in the same region. Unfortunately, there were no additional mAbs available against antigenic site 1. Some of the mAbs recognizing antigenic site 2 exhibited substantial residual binding with the 5M virus , indicat50), for which a two-dimensional micro-neutralization test was carried out using five different doses of the virus encompassing 100 TCID50 for this purpose. The resultant VN titres at each virus dose were used to calculate the serum titre at 100 TCID50 by regression analysis, and the results using pooled guinea pig antisera are shown in The main aim of this study was to quantify the reduction in neutralization following mutations in the antigenic sites of FMDV. Therefore, it was crucial to determine the VN titre of the sera against all the mutant viruses at a fixed virus dose  and at VP2-74 and VP2-191 (5M2/5) had no additional effect on the efficiency of neutralization of the resultant mutant viruses .As bovine sera against the parental rO1K-wt virus were not available, sera against a very closely related vaccine strain, O BFS, were used to study the impact of the mutations on neutralization by antibodies from a FMD target species. O BFS is 99.2 % identical to O1K-wt at the capsid amino acid level and has the identical amino acid residues as O1K-wt at the critical positions described for the five neutralizing antigenic sites. Analysis of the VN data revealed similar findings as with guinea pig sera, with a 48 % drop in neutralization for the 5M virus, a further 10–20 % diminution with additional single mutations, and no additional impact resulting from double mutations .The rO1K-VP2-191 virus was generated to determine the involvement of VP2-191 with neutralizing antigenic site 2. This virus exhibited a 30 % reduction in VN titre when compared with the parent rO1K-wt virus using guinea pig polyclonal sera . It alsoThe reactivity profile of the mutant viruses with two bovine mAbs, C96 and C2, was studied and the results are presented in r1-values are presented in To determine the threshold of reduction in VN titre of the guinea pig sera, VN tests were also carried out using diverse field isolates from serotype O belonging to topotypes other than Europe–South America represented by O1K  and othein vitro neutralization by guinea pig antiserum raised against the parent virus (and vice versa); nevertheless, guinea pigs were protected by antibodies in two-way cross-challenge studies using the parent and mutant viruses as vaccine antigens or challenge viruses. Adoptive transfer of antiserum was used in these studies to confirm the role of antibody in the demonstrated protection. This indicated the existence of other, as-yet-unidentified, protective epitopes. In fact, analysis of their figures reveals that there was a considerable amount of residual neutralization/antibody binding detected by VN titre and ELISA, respectively, when the guinea pig antisera raised against either wt or mutant antigens were tested in vitro. Therefore, a recombinant copy of the quintuple mutant studied by The mechanism of protection afforded by viral vaccines is not very well understood, although the critical role of neutralizing antibodies in protection induced by FMD vaccination is well established. In the case of FMDV (O1K), a quintuple mutant (G67+OC3) was generated by successive single-step selection with mAbs involving neutralizing antigenic sites 1–5  either individually or in combinations to make a total of ten single or double mutants.To completely block binding of antibodies, it was decided to mutate a total of 26 capsid amino acids  associated with the known antigenic sites. The capsid coding regions of the pT7S3-Some of the additional mutations added to the 5M construct increased resistance to neutralization by guinea pig or bovine antisera, but complete resistance to neutralization was not observed. The mutation introduced at VP3-85 had little effect on the efficiency of neutralization. However, VP2-74 and VP2-191 mutations increased resistance to neutralization by 15–22 % compared with the 5M virus. This change is similar to the findings of VP2-191 is located at the threefold axis of the capsid, close to VP2-188 (only 9.6 Å apart). VP2-188 has an intermediate position between VP2-191 and antigenic site 2 (VP2-70-79), VP2-72 being 5.6 Å away from VP2-188 versus 17 Å from VP2-191. VP2-188 has been reported to be a part of antigenic site 2, based upon studies using bovine mAbs  there was an 88 % reduction in neutralizing antibody titre, whereas the titre reduction with O/ITL/1/93 (ME-SA topotype) was less than half (~40 %) although both of them have ~10 % difference in capsid amino acids when compared with the parent rO1K-wt virus. Similarly, both the Cathay topotype viruses and two viruses of different serotypes (A22/IRQ and Asia 1 Shamir) were not neutralized at all when tested against guinea pig antisera, even though the viruses from different serotypes have 20 % more amino acid changes in the capsid region than the Cathay viruses. This implies that the position of the capsid amino acid substitution is more critical than the total number of changes, which is in line with other studies  virus were used in this study. This virus was originally isolated in southern Germany in 1966. The FMDV O1K-wt is a plaque-purified virus that has been passaged three times in IB-RS2 (pig kidney) cells and is the parent virus that was used to derive the four- and five-site mar-mutant viruses described by g for 10 min. All the viruses were stored at −70 °C until use. IB-RS2 and BHK-21 cells were used for virus titration and electroporation, respectively, to recover virus from cDNA clones.The other serotype O, A and Asia 1 isolates used in this study were obtained from the World Reference Laboratory for FMD (WRLFMD) at Pirbright. Either IB-RS2 or BHK-21 cells were used to grow wt or recombinant viruses. When the CPE was almost complete , virus stocks were prepared by a single freeze–thaw cycle followed by removal of cell debris by centrifugation at 1280 Sucrose gradient-purified FMDV antigens were prepared for use in ELISA and also for use as vaccine antigen for generation of antisera. The parent and mutant viruses generated in this study were grown on BHK-21 cells. When CPE was complete, the culture supernatant was harvested, clarified by centrifugation and inactivated with 5 mM binary ethylenimine at 25 °C for 24 h , 1990. TRabbit and guinea pig anti-FMDV polyclonal sera used as trapping or detecting antibodies in ELISA were prepared against purified serotype O FMDV, O1K. These antisera are routinely used in the WRLFMD for the detection of FMDV in an indirect sandwich ELISA, and were raised in rabbits and guinea pigs by immunizing the animals with inactivated, purified 146S FMD virus particles in Freund’s complete and/or incomplete adjuvant as previously described , which is the parent to all other mutant viruses generated in this study. The vaccine was prepared as a water-in-oil-in-water emulsion with Montanide ISA 50 (Sepic) as recommended by the manufacturer (1 : 1 ratio). The guinea pigs were housed in the high-containment isolation facility of the Pirbright Institute and were observed for 7 days before the beginning of the experiment, to ensure they were in good health. At the beginning of the experiment all the guinea pigs were sero-negative for FMDV. A group of nine guinea pigs were injected subcutaneously with 1 ml of an emulsion containing 5 µg of the FMDV antigen. All the animals received a booster dose at 28 days post-vaccination. The experiment was terminated 2 weeks later and all the blood collected for separation of serum. The serum was stored at −20 °C until use. A pool of sera from nine animals was used in serological tests. The O BFS bovine antisera (21 days post-vaccination) used in the micro-neutralization assay were derived from cattle potency tests carried out at Pirbright. A pool of sera from five animals was used for this purpose.The mAbs used in this study had been produced against the O Lausanne or O Manisa vaccine strains . The O LTotal RNA from cell culture-grown viruses was extracted using RNeasy Mini kits (Qiagen) as recommended by the manufacturer. Reverse transcription and PCR to amplify the capsid-coding region were carried out essentially as described previously  and a SpeI site introduced at the beginning of the 5′ end of the 2B-gene (nt position 6798–6804) were used to facilitate substitution of the capsid-coding genes from other viruses . The pT7S3 cDNA clone originally had a SpeI site at nt position 2756–2762 (at the beginning of the vector harbouring the full-length of FMDV); this SpeI site was first deleted by inverse PCRs (IPCR) using overlapping primer sets (Table S1) to make the introduced SpeI site at the beginning of the 2B-gene unique. In order to introduce the SpeI site, the 5 kb XbaI digestion product of pT7S3 plasmid (flanking the 5′UTR to the coding region for 3A) was cloned into pGEM9zf (Promega) to make pGEM9zf/FMDV, which was used as a template for introduction of the SpeI restriction site by IPCR using overlapping primer sets (Table S1). Final IPCR products were digested with DpnI enzyme and cloned to produce the pGEM9zf/FMDV1 plasmid. The 5 kb XbaI digestion product of plasmid pGEM9zf/FMDV1 was used to replace the corresponding fragment of pT7S3 to generate pT7S3-SpeI. Infectious virus was recovered from this cDNA clone and was sequenced to confirm the presence of the unique SpeI site in the 2B region. This pT7S3-SpeI plasmid was subsequently used in all other experiments.An infectious cDNA (pT7S3) containing the B64 strain of O1K that had been previously described . O/ITL/1/93 was used as a control to study the threshold of neutralization of virus from a different FMDV O topotype. For this, total RNA was extracted from cell culture-grown viruses of each donor strain, and PCR products of the capsid-coding genes were generated from cDNA templates using appropriate primer sets (Table S1). The PCR products containing the capsid-coding sequences for O1K-wt, O/ITL/1/93 and G67 were cloned into the pT7Blue (Novagen) to produce pT7Blue/O1K-wt, pT7Blue/O/ITL/1/93 and pT7Blue/G67, respectively, using standard cloning procedures. The AflII and SpeI-digested products of pT7Blue/O1K-wt, pT7Blue/O/ITL/1/93 and pT7Blue/G67 plasmids were inserted into similarly digested pT7S3-SpeI to generate pT7S3/O1K-wt, pT7S3/O/ITL/1/93 and pT7S3/G67 plasmids, respectively. In an attempt to block the antibody binding on the surface of FMDV capsid completely, 26 residue positions  to make the five-site mutant pT7S3/5M (Table S1). Further mutations in pT7S3/5M either individually or in combination were introduced to make recombinants containing single or double amino acid substitutions in the capsid  was generated in this study by substituting the critical amino acid residues representing the five neutralizing antigenic sites. G67 had six substitutions  at neutre capsid . Three rHpaI enzyme and full-length RNA transcripts were synthesized by in vitro transcription with T7 RNA polymerase  as per the manufacturer’s instructions. The RNAs were electroporated into BHK-21 cells using Genepulse (Bio-Rad), essentially as described previously . The antigenic relationship of viruses based on their neutralization by antibodies is given by the ratio r1, the neutralizing antibody titre against the heterologous virus/neutralizing antibody titre against the homologous virus. The significance of differences in the values of r1 obtained by the polyclonal antiserum was evaluated according to the criteria of Tests for FMD virus-specific neutralizing antibodies were carried out in microtitre plates by two-dimensional VN testing using the guinea pig or bovine antisera following established methodology  was used for the data analysis.


The viral encephalitides of Eastern, Western, and Venezuelan equine encephalitis viruses have been of public health concern for years. Over the last decade, several outbreaks caused by emerging zoonotic viral encephalitides, such as West Nile virus in North America and Nipah virus in Malaysia and Singapore in 1999, resulted in serious illnesses and deaths in persons, domesticated food animals, and wildlife. The Institute of Medicine has cited a number of factors that have led to these and other emerging disease outbreaks: 1) a growing human population that is moving into habitats of wildlife and domesticated livestock and poultry; 2) global climate changes that have caused changes in arthropod vector and rodent reservoir populations; 3) rapid travel and movement of people and animals worldwide; and 4) changing human behaviors (This book is a special issue of the Archives of Virology, and its 244 pages comprise 21 presentations that were made at a symposium on "Emergence and Control of Zoonotic Viral Encephalitides." The symposium was held April 6–8, 2003, in Les Pensieres, Veyrier du Lac, France. The first presentation gives an overview of the emergence of zoonotic viruses maintained by wildlife reservoir hosts and describes a conceptual model of processes that would account for the transmission of viruses among species. The second presentation describes the role of disease surveillance in polio eradication and the identification of emerging viral encephalitides. The third presentation gives an overview of the mechanisms of genetic changes and neurovirulence of encephalitogenic arboviruses.The following 13 presentations include overviews of molecular determinants of virulence of West Nile virus in North America, genetic determinants of Venezuelan equine encephalitis virus, evolution and dispersal of encephalitic flaviviruses, and West Nile and other zoonotic viruses in Russia. Presentations that follow address lyssaviruses and henipaviruses transmitted by frugivorous bats, host-management strategies of novel viral encephalitides associated with bats, regulation of transcription and the nature of the cell receptor with regard to henipaviruses, and entry machinery of flaviviruses. Also included are presentations on persistent infection and suppression of host response by alphaviruses, subversive neuroinvasive strategy of rabies virus, neurovirulence and host factors in flavivirus encephalitis, regulation of apoptosis by viruses infecting insects, and Semliki Forest virus infection of laboratory mice as models to study the pathogenesis of viral encephalitis.The book finishes with presentations on a novel principle of attenuation for developing new generation live flavivirus vaccines, on tick-borne encephalitis, and on a recombinant vaccine developed from a canarypox virus carrying the prM/E genes of West Nile virus that will protect horses against a West Nile virus–mosquito challenge, and on diagnosis of zoonotic viral encephalitis.The book will be worthwhile to virologists and other infectious disease researchers and practitioners interested in the biology, virulence, and genetic evolution of viral encephalitides, and the factors involved in their emergence.


Virus shedding from IHNV infected Atlantic salmon was detected before the onset of visible signs of disease with peak shed rates averaging 3.2×107 pfu fish−1 hour−1 one to two days prior to mortality. Once shed into the marine environment, the abundance of free IHNV is modulated by sunlight (UV A and B) and the growth of natural biota present in the seawater. Virus decayed very slowly in sterilized seawater while rates as high as k =  4.37 d−1 were observed in natural seawater. Decay rates were further accelerated when exposed to sunlight with virus infectivity reduced by six orders of magnitude within 3 hours of full sunlight exposure. Coupling the IHNV transmission parameter estimates determined here with physical water circulation models, will increase the understanding of IHNV dispersal and provide accurate geospatial predictions of risk for IHNV transmission from marine salmon sites.Understanding how pathogenic organisms spread in the environment is crucial for the management of disease, yet knowledge of propagule dispersal and transmission in aquatic environments is limited. We conducted empirical studies using the aquatic virus, infectious hematopoietic necrosis virus (IHNV), to quantify infectious dose, shedding capacity, and virus destruction rates in order to better understand the transmission of IHN virus among Atlantic salmon marine net-pen aquaculture. Transmission of virus and subsequent mortality in Atlantic salmon post-smolts was initiated with as low as 10 plaque forming units (pfu) ml Infectious hematopoietic necrosis virus (IHNV) is an aquatic rhabdovirus that causes devastating epizootics in salmon and trout species in the Western N. America, Europe, and Asia. With the appropriate virus strain, host condition, and environmental parameters, IHNV can infect and cause an acute systemic disease in all five species of Pacific salmon, Atlantic salmon, and rainbow trout In British Columbia, IHNV was first identified from sockeye salmon in 1959 at Cultus Lake Important questions concerning outbreaks in farmed Atlantic salmon are: what is the risk of virus dispersion from an IHNV infected site and what factors influence the spread of virus? Studies investigating spatial and temporal patterns of the IHNV outbreaks suggested that farming practices, such as boat movements and the use of shared personnel and contractors contributed to farm to farm spread of disease both within and between areas Laboratory studies have clearly indicated that Atlantic salmon are susceptible to IHNV through waterborne exposure of virus Salmo salar) from Discovery Islands, British Columbia in 1993 (Epithelioma papulosum cyprini (EPC) cells −1).The virus isolate, BC93-057, was used for all stability and challenge trials described herein and originates from pen-reared Atlantic salmon  was 31±1 ppt. An aliquot of the natural seawater was autoclaved at 121°C for 30 minutes at 15 psi (BetaStar) to generate sterile seawater.Viral decay in the absence of sunlight. Natural seawater samples (30 ml) contained in sterile, capped (denoted as closed) or uncapped (denoted as open) 50 ml tubes were seeded with 3×106 pfu of IHNV (isolate 93-057) following a 30 minute acclimation to either 8, 10, or 12°C. Fetal bovine serum (0.08%) was present in the seeded sample due to carry over from cell culture media present in the virus stock. Consequently, the stability estimates obtained herein may be conservative due to the enhanced stability afforded by low levels of serum. Temperatures were chosen to encompass the annual temperature range recorded from an Atlantic salmon farm site in Okisollo Channel of the Discovery Islands, British Columbia, Canada (0). Thereafter sampling was performed at various intervals (between 8 and 16 hours) for up to 6 days. To stabilize samples, an antibiotic/antimycotic mixture  was added at 1∶100 to all individual sub-samples and stored at 4°C until analysis by plaque assay which occurred within 4 hours of collection., Canada . The ‘opViral decay in the presence of sunlight. Aliquots (30 ml) of natural seawater and sterile seawater seeded with 3×106 pfu of IHNV (isolate 93-057) where individually contained in sealed transparent plastic bags. To expose samples to sunlight, the sealed bags were either placed on the water surface of a 2300L outdoor tank or affixed to a rack submersed at 0.5 m depth. As a negative control samples were covered with black bags to eliminate sunlight exposure. In total, 4 treatment groups were tested and are hereafter designated as ‘dark surface; DS’, ‘dark depth; DDp’, ‘light surface; LS’ or ‘light depth; LDp’. Separate bags for each treatment timepoint were placed in the tank such that at designated timepoints individual bags were removed. Prior to sample collection, all samples were shielded from the sun by covering them with a large black bag. To initiate sunlight exposure (T0), the plastic bag was removed exposing all ‘light’ samples. For the LS group, samples were removed every 15 minutes while for the LDp group, samples were removed every half hour. The samples for DDp and DS groups were removed every hour. Upon removal of the sample from the tank, each was placed on ice and in the dark and immediately taken to the laboratory for analysis by plaque assay. During each experiment, water flow was turned off in consideration of biosecurity and surface temperature ranged from 12 to 14°C, increasing 2°C over the duration of the experiment. The experiment was carried out in an unshaded area on the grounds of the Pacific Biological Station  on July 27 2010 from 1400 to 1700hrs local time.Sunlight measurements. A major agent for the decay of infectivity of viruses in the environment is solar radiation Data analysis. In the viral decay studies without sunlight, the time rate of virus inactivation was modelled as a simple temporal exponential decay (Eq. 1), where 0V is the initial concentration of virus and V(t) the concentration at time t and k is the decay rate constant.−1) versus time. The slope of the line is the decay rate constant k (per unit time). The correlation coefficient (R2) was calculated to provide information on how well the exponential model fitted the data and 95% confidence intervals were calculated for decay rate constant. Regression analyses excluded data points below 1000 PFU ml−1Viral decay rate constants were calculated by fitting a least-squares method linear regression to a plot of the natural logarithm of virus concentration D(t (J m−2) is calculated as the time integral of UV (A&B) radiation, UV(t)I (W m−2) (Eq. 2).The degree of inactivation of virus exposed to UV light is dependant on the exposure dosage of UV radiation received by the viral particles 2-t1),D(t where UVk is the decay rate constant (J−1 m2).The first order the simple exponential decay rate model (Eq.3) quantifies the survival of viral particles exposed to UV radiation dosage Ethics statement. All fish studies were carried out in accordance with the recommendations in the Canadian Council on Animal Care (CCAC) Guide to the Care and Use of Experimental Animals. The protocols were approved by the Pacific Region Animal Care Committee . All fish handling was performed under Aquacalm™ (Syndel Laboratories Ltd) or tricaine methanesulfonate (MS222) anesthesia.Fish. Atlantic salmon  pre-smolts (Mowi strain) reared at a freshwater hatchery on Vancouver Island with no previous occurrence of IHNV were transported to the Pacific Biological Station and held on UV treated 10°C seawater (32 ppt). Fish were fed daily at 1.5% body weight a commercial diet (Ewos). All challenge studies were conducted in 10°C seawater (32 ppt) with Atlantic salmon varying in smolt age and size as specified in Minimum infectious dose. To determine the lowest concentration of IHNV necessary to cause mortality in Atlantic salmon smolts, a waterborne immersion challenge was conducted at various doses ranging from 101 – 104 PFU/ml. Treatment groups of 40 fish each (average weight 122 g) where either immersed for one hour in aerated 100 L static seawater bath containing virus or cell culture media (negative control group). Each of the three highest virus doses  were conducted in triplicate tanks while the lowest dose (101 PFU/ml) was conducted in duplicate tanks. Immediately after addition of virus, a 1 ml water sample was taken to quantify virus dose in each tank. After the one hour immersion challenge, saltwater flow was resumed at a rate of 8L/min with each treatment group containing a fish density of 4.88 kg/m3. Fish were monitored daily for mortality and signs of disease for a period of 51 days post challenge where upon all remaining fish were euthanized with an overdose of MS222 (250 mg L−1). Virus titers were determined by plaque assay in a minimum of 50% of dead fish from each tank.Viral shedding rate: Experiment #1-Population level IHNV immersion exposure3 PFU/ml and fish were maintained with aeration for 1 hour after which saltwater flow was resumed and the tank was filled to capacity. As a negative (mock) challenge control, fish at an equivalent stocking density were immersion challenge with HBSS.Atlantic salmon (n = 90) were acclimatized for 10 days prior to experimental infection in oval tanks containing 2000 L of 10°C sea water at a flow rate of 28 L/min. After the acclimation period, the water flow was stopped and the volume was reduced to 1000L. Virus was added to a final concentration of 4.6×10To determine the shedding profile, two samples of seawater were collected daily from the experimental tank. For water collection, the flow was stopped and fish were maintained with aeration. At 0 and 1 hour, 50 L of seawater was removed from the tank and placed in sterile containers at 4°C. Following the 1 h sample, the water flow was resumed. Water samples were processed immediately or within 48 hours of collection. Samples that were not processed immediately were stabilized with Antibiotic-Antimycotic  and stored at 4°C until assayed. Each sample was filtered according to Grant et al (2010) using the FlexStand Cartridge Holder system (Amersham Biosciences) fitted with a hollow-fiber column with a surface area of 1.15 m2 . Retentate samples were assayed immediately for viable virus using plaque assay as described above.−1 hour−1) were calculated using the following formula:The viral shedding rates (pfu fish1 waterborne virus titer – T0 waterborne virus titer)/fish countShedding rate  =   and end (T1) of a one hour cessation in water flow. Water samples were collected up to and including the day of fish mortality or time of euthanasia which encompassed a maximum of 30 days post injection. Water samples were immediately stabilized by the addition of Antibiotic-Antimycotic  and quantified within 48 hours of sampling. Virus concentration in the stabilized water samples was determined using plaque assay on PEG pretreated EPC cells as described above. The shedding rate was calculated using the equation as written above with fish count equating to one for each treatment.To obtain a more precise quantification of viral shedding per individual fish, 10 Atlantic salmon were placed in static water (250 L) water containing 4.6×10Viral shedding rate: Experiment #3 –Individual Fish virus injection exposure−1) and then exposed to IHNV via intraperitoneal (i.p.) injection of 5×104 pfu per fish delivered in a single 100 ul dose. As a negative control, one fish was i.p. injected with virus-free medium. All six fish were housed individually in 4 L tanks containing UV treated 10°C saltwater at a flow rate of 1 L min−1. Seawater samples were collected and processed for shed virus titer as described in experiment #2.Atlantic salmon were first anesthesized with MS222  is shown in 2, and 95% confidence intervals for both open and closed samples of natural seawater at three different temperatures. An exponential decay rate provides a good description of the inactivation of the virus in the open containers as is evident by the high R2 value (>0.9) for the least square fits. For the samples in closed containers, R2 values of the fit are lower and more variable (0.6 to 0.9).−1) and not significant at the 95% level. For those samples incubated in closed containers, the temperature effect on virus inactivation appears to be more pronounced (1.35 to 2.99 d−1) than for the samples in open container but the temperature variation in the decay rates for closed samples is not significant given the wide confidence intervals.Virus decay appears to be more rapid for the samples incubated in open containers as compared to the samples incubated in containers that were capped to minimize air exchange . Decay rThe decay rate of IHNV was very sensitive to UV radiation. Virus infectivity was reduced by six orders of magnitude for raw seawater samples held at the surface over the 3 hour exposure to direct sunlight while over the same time period infectious virus concentrations remained stable in samples covered with black bags to eliminate sunlight (Dark control) . A slighTo quantify the effect of UV A&B radiation on virus survival it was first necessary to use the observed UV measurements to compute the UV dosage received by samples held at different depths (surface and 0.5 m depth). For the samples submersed at 0.5 m depth the observations from the Solar Light UVA+B underwater sensor placed next to the samples were integrated to compute UV dosage (Eq. 3). For the samples of virus held at the surface, the Davis Instruments UV sensor data were adjusted and then integrated to compute the UV dosage. It was necessary to adjust the Davis Instruments Erythmal UV sensor measurements because the sensor by design attenuates the UVA radiation (320–400 nm) 2 for the raw seatwater samples were 4.85×10−5 ±1.12×10−5 J m−2 and 0.91, and 3.43×10−5 ±0.71×10−5 J m−2 and 0.93 for the sterile seawater samples. The correlation coefficient for both groups was high (>0.9) suggesting a good model fit. Although the UV decay rate constant was higher in the raw seawater samples than in the sterile seawater samples, c.f. 4.85×10−5 vs 3.43×10−5 J m−2 the overlapping confidence intervals indicates that the UV decay rates are not significantly different at the 95% confidence level  was applied, and the UV decay rate was estimated (least squares fit) separately for the raw and sterile seawater sample groups . The comce level .1, 102, 103, and 104 PFU ml−1treatment groups was 18, 240, 2400, and 47,000 PFU ml−1, respectively. Discrepancy between calculated dose and observed dose is due to inherent variability of the plaque assay procedure. Among the treatment groups, no mortality was observed in the mock challenge group while mortality was observed at all IHN virus exposure levels tested with cumulative mortality ranging from 17.5 to 50 %  while in the lowest virus dose (10 PFU/ml), mortality was only observed in one of the duplicate tanks. Differences in mortality rate were evident between the virus exposure levels, such that both an earlier onset and faster rate of mortality coincided with higher viral dose exposures (4 virus exposure level while longest mean day to death (36 days) was observed at the 101 virus dose . It is noteworthy that this maximum shedding rate is likely a conservative estimate as rates per fish assume that all Atlantic salmon in the tank are virus infected and shedding equivalent quantities of virus. Consequently, experiment #2 and #3 were performed to better quantify viral shedding per individual fish as well as assess shedding variability between fish.An epizootic event was initiated in a laboratory population of Atlantic salmon after a 1 hour immersion exposure to IHNV at a dose of 4.5×10hallenge . Viral s6 to 4.8×107pfu fish−1 hour−1 (> 1.6×107pfu g−1) while the remaining virus exposed and negative control fish had no detectable virus titers.Four of the ten fish (40%), immersion exposed to IHNV and subsequently held in individual tanks, succumbed to mortality or morbidity during a 30 day holding period while no mortality or signs of disease were observed in the negative control fish exposed to media in lieu of virus. Among the virus challenged fish, shed virus was detected in the water of 80% (8/10) of the tanks while no waterborne virus was detected in the negative control tanks. The highest levels of waterborne IHNV were detected in tanks whereupon fish succumbed to mortality  or were euthanized due to extreme morbidity (#11) with peak shedding rates ranging from 8.8×101 hour−1 . In the 7 to 1.1×108 pfu fish−1 hour−1  mortality or extreme morbidity. Shed virus was detected in each of the five tanks housing the virus exposed Atlantic salmon while no virus was detected in the negative control. In tanks whereupon fish succumbed to mortality  or were euthanized due to extreme morbidity (#5) detectable quantities of virus were present in tank water over six to seven day duration. The largest increase in waterborne virus concentration during a one hour timeperiod (peak virus shedding rate) occurred one (tank 3 and 4) or two (tank 2) days prior to mortality. In tank 5, peak virus shedding was observed on the day of euthanasia. Peak shedding rates for fish #2-5 that exhibited IHN disease signs over the 14 day experiment ranged from 2.3×101 hour−1 . Virus t−1 for one hour to induce clinical disease and mortality in a population. As host susceptibility to IHNV is undoubtedly influenced by multiple factors including virus strain This study provides the first quantitative estimates of biological parameters associated with waterborne transmission and dispersion of IHNV within net-pen reared Atlantic salmon. Our results demonstrate that Atlantic salmon smolts are highly susceptible to IHNV, requiring a minimum virus exposure of 10 PFU ml−1 established IHN disease demonstrates that at this exposure level there is a degree of stochasticity in transmission of virus and that this exposure is near or equal to the minimum infectious dose. Nonetheless, the observation of high viral titers in mortalities from the 10 PFU ml−1 exposure group reveals that this low virus dose was sufficient at initiating an epizootic cascade and provides a quantitative estimate of the extreme susceptibility of Atlantic salmon to IHNV. As fish were exposed to higher virus levels, more consistent and rapid infections were established suggesting that virus transmission and infection prevalence was dose-dependent and likely a function of a higher percentage of individuals becoming infected during the period of initial viral exposure. As viral levels exceeded 103 PFU ml−1, dose effects were less evident suggesting a threshold challenge level was reached above which little evidence of faster or more severe mortality events where observed. The high level of susceptibility observed in our studies corroborates previous transmission studies which demonstrated Atlantic salmon to be more susceptible to IHNV than sockeye (Oncorhynchus nerka), and Chinook salmon (Oncorhynchus tshawytscha) The observation that only 50% of the replicate groups of fish treated with 10 PFU ml7 pfu fish−1 hour−1, 6 vs 107 pfu fish−1 hour−1) in comparison to those calculated from individual fish (exp #2 & 3) due to the fact that shedding rate calculations presume a 100% infection prevalence in the population when in essence only a small portion of the host population were shedding virus.Once infected with IHNV, Atlantic salmon shed increasingly higher quantities of virus with the progression of acute IHN disease. Atlantic salmon succumbing to IHN disease, shed peak levels of virus  the capacity for peak virus amplification remains similar. However differences in the kinetics of virus shedding were observed between experiments 2 and 3, both in the onset and duration of virus shedding. Atlantic salmon waterborne exposed to IHNV (exp #2) exhibited a later onset of virus shedding (an average of 3.5 day later) and shed for a longer duration than fish exposed to IHNV via ip injection (exp #3). These observed differences in virus shedding kinetics are likely consequences of the severity of the initial virus exposure route. As the injection challenge proved to be a more severe challenge than an immersion exposure  it is probable that ip injection establishes a more rapid systemic viraemia by circumventing the natural sequential progression of virus to the organs via the circulatory system 11 pfu in one hour during peak virus shedding. Such a scenario illustrates that if an IHNV epizootic is initiated among high densities of highly susceptible animals that massive quantities of shed virus can result. However to appreciate the role of shed virus in the dispersal and subsequent transmission of IHN disease it is crucial to understand the infectious capacity of the virus when shed from its host.Using the laboratory estimates of IHNV shedding capacity of Atlantic salmon as derived herein, it is possible to quantify virus amplification potential from infected sites. For instance if 1% of 500,000 Atlantic salmon contained in an open net-pen site are undergoing acute IHN disease there is a potential for this outbreak to generate upwards of 1.6×10Achromobacter sp and Pseudomonas sp. Virus decay studies presented herein demonstrated that the infectivity of IHNV is markedly reduced over periods of hours when held in natural seawater. The most consistent decay of infectivity is likely biologically mediated as virus decay is more rapid in natural seawater than sterilized seawater . These rThe infectivity of IHN virus was also significantly reduced via sunlight, specifically UV A and B radiation. Previous work has demonstrated the extreme susceptibility of IHNV to the germicidal effects of UV-C As IHN virus is a significant threat to both cultured and wild salmonid fish, either through hindering enhancement efforts of endangered species or causing significant economic losses within aquaculture operations, there is a need to understand the processes controlling the abundance, infectivity, and spread of this virus. When applied collectively, the quantitative estimates determined in our study form the basis of a simple virus transmission model  which il


To the Editor: Recently West Nile virus (WNV) infection has been reported in three alligators (Alligator sp.) from central Florida  with neurologic signs from the District of Columbia and Maryland area (Clemmys caspica) was seropositive (Rana ridibunda) with a Russian strain of WNV resulted in high levels of viremia (Crocodylus niloticus). To assess the potential role of crocodiles in the life cycle of WNV in Israel, serum specimens were collected from 20 healthy crocodiles on a commercial farm in the Negev Desert, in southern Israel . The crocodiles came from two separate breeding farms  in the Syrian-African Rift Valley, which is on the main route of bird migration from Africa to Europe. Five males and 15 females, 1–2.5 years of age, were examined. Blood was withdrawn from the crocodiles’ ventral caudal vein, separated by centrifugation, and kept at –20°C until analyzed. Neutralizing antibody titers were determined against WN-goose-98 (-4.2 50% tissue culture infective dose) were diluted 1:400, and virus neutralization titers were checked 3 days later.We report, for the first time, WNV infection in crocodiles  , according to the manufacturer’s protocol and resuspended in 30 μl of RNase-free water. The primer pair WN240-Kun848  was used to synthesize an 800-bp product in the E gene region . No differences were discernible in either the seroprevalence rate or in the average titers of crocodiles from two different breeding farms. In BHK cells, a similar seroprevalence rate was observed, with titers ranging from 1:40 to 1:1,280 . All serum samples, except one, were <1:10 against ITV virus, which had a titer of 1:640 against WNV and 1:10 against ITV. Viremia was not detected in any of the 20 samples in Vero cell culture or by RT-PCR.These results demonstrate a high rate of infection with WNV in crocodiles in Israel. The crocodiles may have been exposed to the virus during the summer at their present location, since no difference in prevalence was seen between the two groups (which differed only in the farm of origin) and since the younger crocodiles had been hatched in the spring of 2002. Furthermore, a cross-reaction with the other prevalent flavivirus in Israel, ITV, was ruled out. Preliminary results from an equine seroprevalence study (involving 800 horses over a 3-year period) of virus neutralization antibodies to WNV collected during fall 2002, indicate that most horses sampled in Israel’s Arava Valley (a desert in the Syrian-African Rift near the Jordanian and Egyptian borders) and the Gulf of Aqaba/Eilat   were positive . WNV was also isolated from mosquitoes in the same region (WNV has been endemic in Israel since the early 1950s (The role of various reptile species in the epidemiology of other arboviruses such as western equine encephalitis, eastern equine ecephalitis, and Venezuelan equine encephalitis is well documented (


Plant made biologics have elicited much attention over recent years for their potential in assisting those in developing countries who have poor access to modern medicine. Additional applications such as the stockpiling of vaccines against pandemic infectious diseases or potential biological warfare agents are also under investigation. Plant virus expression vectors represent a technology that enables high levels of pharmaceutical proteins to be produced in a very short period of time. Recent advances in research and development have brought about the generation of superior virus expression systems which can be readily delivered to the host plant in a manner that is both efficient and cost effective. This review presents recent innovations in plant virus expression systems and their uses for producing biologics from plants. It takes just a quick glance at the number and variety of novel biologics that are currently emerging from the commercial sector to realize that the use of plants as production platforms for vaccines and other therapeutic proteins has over time moved from mere theory and into actual practice. Plant-derived biologics are safe, efficacious, and easy to mass produce. They can offer new solutions to the challenge of providing inexpensive medicines that lack cold chain requirements or an established medical infrastructure for the world's rural poor. While assisting developing countries has always been a fundamental driving force for the development of plant-made biologics, other uses have also been presented and range from facilitating the stockpiling of vaccines against pandemic infectious diseases to the applications of plant-made proteins in the field of personalized medicine , 2.Originally, plant-made biologics were engineered to be expressed from stably transformed plants. As this research area progressed to the point of determining plant-based pharmaceutical performance in actual clinical trials, it became increasingly apparent that lengthy tissue culture and plant regeneration procedures and concerns regarding public perception over the use of GM technology were all major stumbling blocks with regard to the use of transgenic plants that needed to be addressed. The design and implementation of novel transient expression systems through the use of plant virus expression vectors has been one technology that circumvents many of these hurdles. Not only are plant viruses able to produce extremely high levels of foreign protein at low cost and in a matter of a few days postinoculation, but also their use as a technology is more appealing to the public because it lacks the negative connotations that are often associated with GM plants. Indeed, removal of the genes encoding virus movement and/or coat proteins prevents plant viruses from moving from plant to plant and as a result reduces concerns about transmission and cross-contamination of pharmaceutical proteins to weedy relatives.As research and development of plant virus expression vectors progress, the means of introducing them to the host plant has also advanced. Rather than merely infecting plants with the appropriate virus vector, plant leaves can now be inoculated by agroinfiltration, that is, by incorporating the virus vector into Agrobacterium tumefaciens and infiltrating the leaf using a syringe or a vacuum. Both methods have their own advantages, and both are rapid and scalable and lack the requirement of sophisticated equipment. The universality of these technologies makes them amenable for both commercialization and classroom alike .Over the past decade, plant viruses have been developed both as vectors for biopharmaceutical protein production and also as research tools for plant functional genomics studies, by incorporating virus induced gene silencing (VIGS) as a means to target and downregulate specific host transcripts. The latter technology has great implications for the development of crops with improved characteristics, while the former has the potential to launch a new era for the production of biologics. This review will discuss several of the plant virus expression systems that have been developed to date and will provide selected examples of their application in the field of plant-made biologics. A. tumefaciens using a vacuum into all mature leaves of a tobacco plant, thus infecting the whole plant in its entirety   . The autrequired .CMV has also been utilized as an antigen presentation system to express epitopes of porcine circovirus type 2 (PCV2) capsid protein . ChimeriThe icosahedral, positive sense RNA virus Cowpea mosaic virus (CPMV) has also been designed for the generation of vaccine and other therapeutic proteins in plants. Initially designed as an epitope presentation system, CPMV chimeric particles have been shown to elicit a robust immune response for a variety of diseases . CPMV haMedicago, Inc., has also developed their own CPMV vector-based technology which produces virus-like particles (VLPs) carrying influenza virus antigens . These VNonreplicating CPMV expression systems have also been developed. These involve the positioning of the foreign gene between the 5′ leader sequence and 3′ untranslated region (UTR) of RNA-2 . DeletioPlant DNA viruses, including geminivirus and Cauliflower mosaic virus (CaMV), have also been designed as pharmaceutical protein production platforms. Geminiviruses are small single stranded DNA viruses and are named for their twinned capsid morphology; they infect a broad range of plants and can replicate to extremely high copy numbers. Two geminiviruses have been developed for biopharmaceutical production, Bean yellow dwarf virus (BeYDV), a mastrevirus, and Beet curly top virus, (BCTV), a curtovirus , 28. Botowpea mosaic virus (CPMV) have been designed which can incorporate fluorescent dyes, polyethylene glycol (PEG) polymers, and various targeting moieties on their surfaces for the purpose of creating novel tumour-targeted molecular imaging agents [Besides offering a novel production platform for plant-made biopharmaceuticals, plant viruses have been engineered to provide other medical applications. For example, virus-like particles based on Cg agents , 30. Theg agents .As another example, Cowpea Chlorotic Mottle Virus (CCMV) has been shown to stably assemble in vitro and package the RNA derived from a mammalian virus, Sindbis virus. The hybrid VLPs were able to deliver and release their RNA contents within the cytoplasm of mammalian cells. The CCMV-based VLP was shown to protect against RNA degradation by cellular nucleases. By conjugating subcellular targeting moieties, these hybrid VLPs could be directed toward distinct sites within the cell .Plant viruses have also been engineered to act as adjuvants to elicit an immune response that is more potent and effective. Recently, nanoparticles that are constructed from the coat protein of the rod-shaped Papaya mosaic virus (PapMV) have been shown to be highly immunogenic and are taken up by dendritic cells . These nThe development of plant virus vectors continues to evolve with respect to their ease and breadth of use in the field of plant-made biologics. Improved expression vectors based on deconstructed viruses will most likely remain a trend in the near future. Efforts will be made to address other challenges including enhanced expression levels, the generation of proteins with more complex quaternary structures, and issues concerning host specificity. With the advancement of techniques to facilitate their introduction and transient expression in plants, plant virus expression vectors and components derived from them offer strategic advantages for the rapid and cost-effective production of biologics for the world's poor. The fact that this technology lends itself to other applications as well, including the development of vaccines to combat global pandemics and even cancer targeting therapeutics, offers much needed vigor to address a broadening spectrum of needs for medical researchers for many years to come.


To the Editor: The globalization of food production and trade has increased the potential risk for infectious foodborne diseases. Hepatitis A virus (HAV) and norovirus (NoV) constitute the most important foodborne pathogens of humans in terms of numbers of outbreaks and persons affected in industrialized countries  imported from Peru occurred in 1999 and 2008. In both situations, the Spanish Ministry of Health activated the National System of Epidemiologic Surveillance and the European Community Rapid Alert System for Foodstuffs. The implicated shellfish batches were immobilized or removed, and all the shellfish from Peru were banned from the European Union , oysters , cockles , and razor clams . Digestive tissue was dissected from duplicated samples  and homogenized with 0.1% peptone water (pH 7.4), centrifuged at 1,000 × g for 5 min, and supernatant recovered. RNA was extracted by using both Total Quick RNA extraction Cells and Tissue kit  and Nucleospin RNA Virus Kit .Fifty mollusk samples imported into Spain during September 2006–March 2009 were analyzed for NoV genotype I (GI) and GII, HAV and astrovirus (AsV). Countries of origin were Morocco, Peru, Vietnam, and South Korea . The spe<41 was considered positive. AsV was detected by standard RT-PCR  by using the Platinum Quantitative RT-PCR Thermoscript 1-step system   with 5 µL of template RNA, and primers, probes, and conditions as described (0) (103 PFU) was used as control for extraction. To calculate the real-time RT-PCR efficiencies, external viral RNA  or synthetic DNA  controls for the respective virus were co-amplified with each template viral RNA as described  of 50 samples were contaminated by >1 virus , althougSix samples (30% of positive samples) were positive for >1 virus. Thus, 2 samples from Morocco showed contamination with NoV GI and AsV. From Peru, 1 sample was contaminated with both genogroups of NoV and another with NoV GI, NoV GII, and AsV. Samples from Vietnam (n = 2) were contaminated with HAV–NoV GI and NoV GI–AsV. Co-infection with different viruses or multiple virus strains could lead to more severe symptoms and the occurence of 2 episodes of the same or different diseases and also be a way to facilitate emergence of new recombinant strains (4 to 7.7 × 107 RNA copies/g digestive tissue; for NoV GII, from 1.03 × 105 to 8.9 ×105 RNA copies/g digestive tissue; and for HAV, from 4.7 × 103 to 4.4 × 107 RNA copies/g digestive tissue (Contamination levels for NoV GI ranged from 3.5 × 10e tissue . For HAV,Determining the association of a viral infection with a particular contaminated product is often complicated, and the epidemiologic investigations necessary for finding this association are time consuming and allow the virus to spread before the problem is recognized. Furthermore, there are analytical difficulties in detecting and quantifying virus in shellfish samples and in monitoring them; other problems include ascertaining the representativeness of the sample (The inadequacy of the bacterial indicators makes it necessary to develop new prevention strategies, based on microbial risk assessment, to ensure the sanitary quality of shellfish, both in production areas and in international trade operations. Implementing these methods and providing training to laboratories in developing countries are essential to achieving these objectives.


Anterior gradient homolog 2 (AGR2) is a functional protein with critical roles in a diverse range of biological systems, including vertebrate tissue development, inflammatory tissue injury responses, and cancer progression. Clinical studies have shown that the AGR2 protein is overexpressed in a wide range of human cancers, including carcinomas of the esophagus, pancreas, breast, prostate, and lung, making the protein as a potential cancer biomarker. However, the general biochemical functions of AGR2 in human cells remain undefined, and the signaling mechanisms that drive AGR2 to inhibit p53 are still not clearly illustrated. Therefore, it is of great interest to develop molecular probes specifically recognizing AGR2 for its detection and for the elucidation of AGR2-associated molecular mechanism.dK values in the nanomolar range after 14 rounds of selections. Aptamer C14B was chosen to further study, due to its high binding affinity and specificity. The optimized and shortened C14B1 has special G-rich characteristics, and the G-rich region of this binding motif was further characterized to reveal an intramolecular parallel G-quadruplex by CD spectroscopy and UV spectroscopy. Our experiments confirmed that the stability of the G-quadruplex structure was strongly dependent on the nature of the monovalent ions and the formation of G-quadruplex structure was also important for the binding capacity of C14B1 to the target. Furthermore, we have designed a kind of allosteric molecule beacon (aMB) probe for selective and sensitive detection of AGR2.Through a bead-based and flow cytometry monitored SELEX technology, we have identified a group of DNA aptamers that can specifically bind to AGR2 with In this work, we have developed new aptamer probes for specific recognition of the AGR2. Structural study have identified that the binding motif of aptamer is an intramolecular parallel G-quadruplex structure and its structure and binding affinity are strongly dependent on the nature of the monovalent ion. Furthermore, with our design of AGR2-aMB, AGR2 could be sensitively and selectively detected. This aptamer probe has great potential to serve as a useful tool for early diagnosis and prognosis of cancer and for fundamental research to elucidate the biochemical functions of AGR2. Anterior gradient homolog 2 (AGR2) was identified initially as a secretory factor expressed in the anterior region of the dorsal ectoderm in Xenopuslaevis embryos, where it was postulated to mediate the specification of dorsoanterior ectodermal fate, particularly in the formation of the cement gland in vitro selection process or SELEX  with high binding affinity and specificity towards defined targets in vitroVarious ligands have been developed for specific molecular recognition, such as small molecules, antibodies, and peptides et al has reported a light-switching excimer aptamer probes for sensitive quantitative detection of PDGF in cell media et al have developed a functionalized polypyrrole nanotube with aptamer to build a VEGF biosensor in vivo characterize the complex pathogenic activities that accompany tumor growth for disease early diagnosis and pathogenesis measurement The possibilities provided by aptamers are enormous, and some aptamers have already shown many important applications in bioanalysisand biomedicine + ion intensively. Furthermore, we designed an allosteric molecule beacon AGR2-aMB based on the identified aptamer, which enables simple, sensitive and selective detection AGR2. The aptamer sequences and AGR2-aMB reported in this study are potentially useful tools for early diagnosis and prognosis of cancer and for fundamental research to elucidate the biochemical functions of AGR2.In this study, using the bead-based and flow cytometry monitored SELEX technology, we aimed to obtain specific aptamers to AGR2 and study theirs structure and potential function. Beads-based SELEX allowed the use of simple, yet effective, flow cytometry analysis to monitor the progress of the selection, avoiding the tedious, time consuming and radioactive EMSA process in vitro sepharose-bead-based SELEX is schematically illustrated in To identify aptamers against AGR2, recombinant AGR2 was fused with glutathione-S-transferase (GST) to facilitate the attachment of the protein to solid supports (Sepharose GSH-beads). The resulting AGR2-GST-beads were used as the positive target in SELEX while the GST-beads as negative control to remove non-specific surface binding sequences. The process of d = 64.1±5.4 nM), while there was no observable binding of the library to control beads (An 87-nucleotide (87-nt) single-stranded DNA (ssDNA) library with 45 random bases flanked by two primer sequences (22-nt and 20-nt) was subjected to the SELEX procedure. The library was first allowed to interact with excess negative control beads, and only the DNA sequences that did not bound to the GST-beads were collected. The collected sequences were then incubated with AGR2-GST-beads. After rigorous washing, those sequences that either did not bind, or only weakly bound to the target were discarded. Only the sequences that bound strongly enough were retained on beads, and the bead-ssDNA complexes were collected and amplified by PCR for the next round of selection. After multiple rounds of selection, the subtraction process efficiently reduced the DNA sequences that bound to the GST beads, while those AGR2-specific aptamer candidates were gradually enriched. The progress of the selection process was monitored by flow cytometry. The stronger binding of DNA library to AGR2, the more FAM labeled sequences bound to the beads, thus the higher fluorescence intensity the beads would emit. With the increasing number of selection cycles, steady increases in fluorescence intensity on the target beads were observed . The binol beads . These rdK = 13.1±7.2 nM). Titration of C14B to GST-beads revealed no observable binding, establishing that the binding target of C14B was indeed AGR2. Other three sequences have similar binding constants to AGR2 but a little weaker than C14B, in which the dK of C14A is 20.9±5.2 nM, C14C is 44.6±7.0 nM and C14D is 48.4±15.6 nM. . The majority of eliminated sequences were primer sequences, implying that the binding region of the aptamer was the middle of random sequences and the primer sequences do not or contribute little to the binding affinity of aptamer. C14B1 has five portions of poly-G and we designed five truncated sequences by removing one of poly-G portion each . It is well-known that guanine-rich sequences can fold into four-stranded secondary structures called quadruplexes. According to G-Quadruplex prediction formula d(G3+N1-7G3+N1-7G3+N1-7G3+) Further examination revealed that C14B1 has multiple stretches of guanines  by affinity chromatography, the GST-AGR2 fused protein was linked to sepharose beads for positive selection. After being washed several times with W1 buffer , the final purified AGR2-GST-beads were stored in sterilized PBS buffer. Flow cytometry analysis with TAMRA-labeled anti-AGR2 monoclonal antibody (Santa Cruz) and SDS-PAGE indicated that the GST-AGR2 fused protein was successfully linked to the sepharose beads.2HPO4 and 2.0 mM KH2PO4, pH 7.4) was denatured by heating at 95°C for 5 min, then quickly cooled on ice for 10 min, and subsequently incubated for another 10 min at room temperature before binding. The ssDNA pool was then incubated with negative GST beads (1.0×105 beads) for counter selection to remove sequences non-specifically binding to GST beads. After filtering with a homemade filter column, the filtrate was incubated with positive AGR2-GST-beads (1.0×105 beads) at 37°C for 45 min. The unbound or nonspecifically bound oligoes were removed by filtration. The sequences bound to the target-coated beads were then amplified by PCR with FAM and biotin-labeled primers . After denaturation in NaOH (0.1 M), the selected sense ssDNA was separated from the biotinylated antisense ssDNA strand on streptavidin-coated sepharose beads  and used for next round selection. For the first-round selection, the amount of initial ssDNA pool was 5 nmol, dissolved in 500 µL binding buffer, and the counter-selection step was eliminated. To acquire aptamers with high affinity and specificity, the selection strength was enhanced gradually by increasing the number of washes (from three to ten times with 200 µL 1×PBS buffer each) and decreasing the amount of the ssDNA library per round (from 200 to 150 pmol).The procedures of selection were as follows. The ssDNA pool (200 pmol) dissolved in binding buffer . The resulting 62 sequences were subjected to multiple sequence alignment analysis by using Clustal W 6.0 software to discover highly conserved motifs in groups of selected DNA sequences. The discovered consensus sequences with high repeats among selected pools were then chemically synthesized for further testing.The resulting pool from the 144 AGR2-GST-beads or GST-beads in binding buffer (200 µL) at 37°C for 45 min. Beads were washed three times with 200 µL binding buffer by means of filtration, and suspended in binding buffer (250 µL). The fluorescence intensity of the resulting beads was monitored with a FACSAria cytometer (Becton Dickinson Immuno cytometry systems) by counting 10000 events. The binding affinities of aptamers were determined by incubating AGR2-GST-beads (5×104) with various concentrations of FAM-labeled aptamers (pre-heat-treated) in binding buffer (200 µL) at 37°C for 45 min in the dark. Beads were then washed three times with the binding buffer, then resuspended in binding buffer (250 µL) and subjected to flow cytometry analysis. The FAM-labeled unselected ssDNA library was used as negative control for the nonspecific binding evaluation. All binding experiments were repeated two to four times. The mean fluorescence intensity of target protein labeled by aptamers was used to evaluate binding affinity by subtracting the mean fluorescence intensity of nonspecific binding produced by unselected ssDNA library. The dissociation constants (dK) of the fluorescent ligands were obtained by fitting the dependence of fluorescence intensity of specific binding on the concentration of the ligands to the equation (1): Y = BmaxX/(dK+X) using SigmaPlot software.To monitor the enrichment of aptamers after selection, the FAM-labeled ssDNA pool was incubated with 5×10CD measurements were carried out on a Jasco J-810 spectropolarimeter equipped with a programmable temperature-control unit (Julabo HP-4). The concentration of DNA samples were 2 mM. Before the CD spectrum measurement, the DNA samples were annealed by heating to 95°C for 5 min, then rapidly cooled on ice for 10 min, and incubated for another 10 min at room temperature. The spectra from 400 to 200 nm were obtained by using 1 nm slit width and 0.1 nm scanning resolution. Each CD spectrum was an average of 8 scans with the buffer background subtracted.m) were taken as the temperature of half-dissociation of the quadruplex and were obtained from the maximum of the first derivative dA/dT plots at 295 nm. The heat-treated DNA solutions at several concentrations were introduced into a quartz cuvette and overlaid with a thin layer of silicone oil to prevent evaporation. The optical path length was 1 cm. Absorbance and temperature were recorded every 2°C.UV absorbance and melting studies were carried out on an Agilent 8453 spectrophotometer equipped with a programmable temperature-control unit (Agilent 89090A). Melting temperatures (T2 at pH 7.4), and then rapidly cooled on ice for 10 min, and subsequently waiting for another 10 min at room temperature before use. To the solution of AGR2-aMB, various concentrations of AGR2 were added and the resulting solutions were allowed to incubate at 37°C for 30 min. To the mixture, 1 µL of streptavidin beads (about 5×104 beads) were added and the mixture was allowed to set for 45 min in dark. After washing twice with Tris-HCl buffer, SA beads were filtered to eliminate unbound AGR2-aMB and resuspended in Tris-HCl buffer solution 250 µL before flow cytometry analysis. In selectivity tests, BSA (Tagene Biotechnology Xiamen), trypsin (Dingguo Beijing), thrombin  and IgG (Dingguo Beijing) were used.AGR2-aMB (40 nM) was annealed by heating to 95°C for 5 minutes in 200 µL Tris-HCl buffer , and the other is thymine-rich sequences (40 clones).(TIF)Click here for additional data file.Figure S2The dissociation constant measurement of C14A, C14B, C14C and C14D against AGR2 GST and GSH.(TIF)Click here for additional data file.Figure S3Flow cytometry assay to monitor the binding of C14B1 and its five truncated sequences with a) AGR2 (target protein) and b) GST (control protein).(TIF)Click here for additional data file.Figure S4UV thermal-denaturation experiment of C14B1. Denaturation profiles obtained at 295 nm for the aptamer at three different concentrations . The Tm (59°C) at 295 nm is independent of oligonucleotide concentration, indicating that the aptamer forms an intramolecular G-quartet.(TIF)Click here for additional data file.Figure S5The secondary structure of AGR-aMB. Stable hairpin structure is formed by intramolecular hybridization between the SA aptamer sequence and the complementary sequence of C14B1.(TIF)Click here for additional data file.Table S1d to AGR2.The sequences of C14A, C14B, C14C and C14D, and their K(DOC)Click here for additional data file.Table S2The truncated sequences by removing one of poly-G portion each from C14B1.(DOCX)Click here for additional data file.


Bean pod mottle virus (BPMV) based virus-induced gene silencing (VIGS) vectors have been developed and used in soybean for the functional analysis of genes involved in disease resistance to foliar pathogens. However, BPMV-VIGS protocols for studying genes involved in disease resistance or symbiotic associations with root microbes have not been developed.Here we describe a BPMV-VIGS protocol suitable for reverse genetic studies in soybean roots. We use this method for analyzing soybean genes involved in resistance to soybean cyst nematode (SCN). A detailed SCN screening pipeline is described.The VIGS method described here provides a new tool to identify genes involved in soybean-nematode interactions. This method could be adapted to study genes associated with any root pathogenic or symbiotic associations. Virus-induced gene silencing (VIGS) is a reverse genetic tool that exploits RNA-mediated antiviral defense strategies used by plants . VIGS isBean pod mottle virus (BPMV)  was used as an endogenous control gene for normalization of samples.Note 1: VIGS vector requests should be made to John Hill (johnhill@iastate.edu) or Steve Whitham (swhitham@iastate.edu), Iowa State University, Ames, IA.Note 2: Soybean cultivars differ in susceptibility to BPMV infection. Soybean cultivar Williams 82 is highly susceptible. The recombinant inbred lines  used in our study were less susceptible to BPMV than Williams 82. Thus, the silencing efficiency attained can vary between cultivars.Note 3: Preparation of gold particles: Weigh 30 mg gold particles (BioRad cat. No. 165-2263), resuspend in 100% ethanol in a 1.5 ml tube, vortex well and let it sit for 10 min at room temperature. Centrifuge the suspension and discard the supernatant. Add 1 ml sterile water, vortex well and centrifuge again to pellet the gold particles. Repeat this washing 2 more times. Add 0.5 ml sterile 50% v/v glycerol to the final pellet of gold particles and vortex well to re-suspend. Finally, while vortexing at low speed, aliquot 50 μl into several 0.5 ml tubes and store at -20°C. Each aliquot is enough to coat DNA that can be used for three separate bombardments.Note 4: Alternatively, seeds can be germinated, and seedlings grown at 26°C, up until the day of virus inoculation. This will reduce the time to the two-leaf stage by 1-2 days.Note 5: The amount of tissue required depends on the virus titer of the infected leaf tissues used for inoculum preparation. In our experience, a ratio of 1:20 w/v of tissue results in reliable virus infection on plants. Fresh tissues can also be used, however, more tissue per ml of buffer (1:10 w/v) is recommended.6). BPMV carrying a portion of a soybean Actin gene (BPMV-Actin) was detected in roots at 12 dpv . We also examined the timing of GFP gene silencing in composite plants [Note 6: We have carried out experiments to determine the time point at which optimum virus levels are achieved in the roots. In a time-course RT-PCR analysis of root tissues harvested at different days post-virus (dpv) inoculation of leaves, we detected BPMV in roots by 6 dpv Figure ; V6. BPMe plants  expressie plants .SHMT gene mapped to the Rhg4 locus, a major quantitative trait locus for SCN resistance [SHMT-VIGS silenced plants showed a statistically significant increase in susceptibility to SCN compared to control plants inoculated with BPMV empty vector in repeated experiments (Figure SHMT gene has been shown to be required for SCN resistance by TILLING, RNAi, and complementation analysis [Here we present the detailed description of a SCN-BPMV-VIGS pipeline to demonstrate the usefulness of VIGS methodology in studying genes involved in soybean-nematode interactions. A 328 bp fragment corresponding to a sistance , was useanalysis .Although VIGS is routinely used for functional studies of genes involved in aboveground interactions, the use of VIGS to study belowground interactions is limited. The development of an efficient VIGS protocol is challenging, in particular when you are dealing with root-biotic interactions. This is due to the fact that inoculation of the virus needs to be carefully timed with inoculation of the infecting organism so that optimal target gene silencing is achieved at the time of infection. In addition, the temperature optimums for the virus and infecting organism must be considered. While our methodology combined established SCN phenotyping and soybean VIGS methods, several specific modifications were made to overcome these challenges. We first analyzed for the presence of BPMV in roots and then monitored silencing of GFP in transgenic hairy roots and the roots of stable transgenics  utilizinVIGS is a valuable tool for functional genomic studies in crop plants like soybean, where whole plant transformation is difficult and time consuming. This has been validated by recent discoveries of genes involved in Asian soybean rust–soybean interactions ,17. Our The authors declare that they have no competing interests.PKK, PSJ, TJB, and MGM conceived and designed the study. PKK and RH developed the SCN-BPMV-VIGS pipeline. GY performed the qRT-PCR analysis to detect BPMV in roots, PKK and NWG performed the composite plant assays to monitor GFP silencing in roots, PKK and RH carried out the SHMT-VIGS analysis. JH and SAW provided materials and intellectual input for VIGS analysis. PKK and MGM wrote the manuscript. All authors critically revised and approved the final manuscript.


To the Editor: Cryptococcus gattii was considered to be geographically restricted to countries with tropical and subtropical climates until 1999, when an outbreak of cryptococcosis in humans and animals occurred in the temperate climate of Vancouver Island, British Columbia, Canada  of 255 samples of plant detritus of Eucalyptus camaldulensis trees collected from the residential locality of an autochthonous case of cryptococcal meningitis caused by C. gattii in Apulia (C. gattii from plant debris of trees belonging to Ceratonia siliqua (carob), Pinus halepensis (stone pine), and E. camaldulensis in Spain , Douglas fir , oak , walnut , and mulberry . The main criterion in selecting a tree for sampling was advanced age and presence of large trunk hollows variably sheltered from sunlight. The sampled sites had no bird nests and were apparently free from avian excreta. The decayed wood samples were collected with an in-house swabbing technique by using simplified Staib niger seed agar as described  fingerprinting and multilocus sequence typing (MLST). The MLST loci herlands  .C. neoformans species complex were isolated from the 112 decayed wood samples examined from 52 trees. One strain that originated from an oak tree (Q. macranthera), was identified as C. neoformans var. grubii. The remaining 3 strains, all originating from different hollows in a Douglas fir tree, were identified as C. gattii genotype AFLP4/VGI and mating type α. The strains were deposited at the CBS-KNAW  Fungal Biodiversity Centre , and the sequences were deposited in GenBank (accession nos. JN982044–JN982073).Four strains of C. gattii isolates in our study are more closely related to the clinical isolate from the Netherlands (C. gattii isolates (AFLP4/VGI) reported from the Netherlands and other countries in Europe than to isolates from outside Europe  isolate from the Netherlands was recovered postmortem in 1957 from the lungs of a pregnant woman with cryptococcosis (MLST analysis showed that the C. gattii prevalent in Europe (,,C. gattii has been reported from Douglas fir trees in Vancouver Island, British Columbia, Canada; however, those isolates represented another molecular type, i.e., AFLP6/VGII (C. gattii isolates have been implicated in human infections in that region, but to our knowledge, no environmental isolates have been found until now.Furthermore, genotype AFLP4/VGI appears to be the genotype of C. gattii in the environment and its previous isolation from a clinical case in the Netherlands suggests that this pathogen is endemic to the temperate climate of northern Europe. This suggestion agrees with the concept emerging from a decade of investigations in Canada and the Pacific Northwest that the geographic distribution of C. gattii extends to the temperate region, albeit with another AFLP genotype (,,C. gattii in this continent than what appears in the literature.Our detection of


The recently discovered human parvovirus 4 (PARV4) has been associated with seropositivity for human immunodeficiency virus, hepatitis B virus and hepatitis C virus. High prevalence is seen especially in intravenous drug users. The virus has been detected in blood products and persons who have been repeatedly transfused have shown to be a risk-group. Furthermore, reports from different parts of the world suggesting a prevalence ranging from zero to one third of the healthy population and the virus is thought to cause a latent or persistent infection. We investigated the presence of PARV4 DNA and parvovirus B19 (B19) DNA in serum from 231 severely immunocompromised cancer patients that have been exposed for blood products. Compared to B19, which was found in 3.9% of the patients, we found no evidence of PARV4. Our results may indicate a very low prevalence of the virus in Sweden, and it would be useful to measure the real PARV4 exposure of the healthy population as well as individuals with known risk factors by serology. Parvovirus 4 (PARV4) genotype 1 was discovered seven years ago In a cross-sectional study, we investigated the presence of PARV DNA in serum of severely immunocompromised children and adults with various cancer diagnoses. Because of frequent transfusions of blood products, we assumed these patients to be at high risk of acquiring PARV4 infection. Moreover, due to the immunosuppression they would be less able to control an acute, persistent, or latent infection as seen with parvovirus B19 (B19) www.epn.se). In response to a decline in hemoglobin concentration and/or platelet count, red cell and platelet concentrates, respectively, were administered. In a few cases of sepsis, fresh frozen plasma was used. Coagulation factors were not administered. All patients were sampled in a severely immunocompromised state indicated by an absolute neutrophil count ≤500/mm3 which predominantly was accompanied by severe or moderate lymphopenia. The seroprevalence of HIV, HBV, and HCV was extracted from the patients' medical records. Collected samples were stored at −80°C. Total viral nucleic acids were extracted with QIAamp MinElute Virus Spin Kit  according to the manufacturer's protocol with 200 µL of specimens eluted into 50 µL of extract. A real-time PCR assay designed to amplify an 103 bp product of the region from the gene for VP1 on the three known PARV4 genotypes Adult patients were, after giving their informed written consent, eligible for enrollment. Pediatric patients were enrolled after informed oral consents from the children or their guardians which was documented by the informing physicians. The study and both consent procedures were approved by The Regional Ethical Review Board in Stockholm  and in 2 children 5.0%; . All werTo our knowledge there are no reports on the prevalence of PARV4 in Sweden, and our findings may suggest a low prevalence. Except from being recipients of different blood products, our study cohorts included low-risk individuals regarding reported risk factors such as HIV, HBV, and HCV seropositivity and usage of intravenous drugs. However, there are reports on PARV4 DNA in the blood of the healthy population in our part of the world We used serum samples for detection of PARV4 DNA by PCR. Although plasma has been used in most studies, PARV DNA has been detected in human serum before In conclusion, we failed to observe a high prevalence of PARV4 DNA in these cohorts of highly transfused and severely immunocompromised patients. It would be useful to measure the real PARV4 exposure of the Swedish healthy population as well as individuals with known risk factors by serology.


To the Editor: Hantaviruses (family Bunyaviridae) are transmitted from rodent reservoirs to humans. These viruses cause life-threatening human diseases: hantavirus cardiopulmonary syndrome in the Americas and hemorrhagic fever with renal syndrome in Asia and Europe (Hylomyscus simus) in Guinea . Histologic examination of organs of the animal showed no obvious pathologic findings.One sample yielded a product of the expected size and was subjected to cloning and sequencing. The positive sample (MGB/1209) was obtained from 1 of 18 investigated slit-faced bats (family Nycteridae). The animal was trapped at the Magboi River within Gola National Park, Sierra Leone , and the identification as The obtained 414-nt sequence covers a genomic region, which was found to correspond to nt position 2,918–3,332 in the large segment open reading frame of prototypic Hantaan virus. Bioinformatic analysis on the amino acid level showed highest degrees of identity to shrew- and mole-associated hantaviruses . On the basis of tree topology of a maximum-likelihood phylogenetic tree, the sequence does not cluster with rodent-associated hantaviruses but groups with those found in shrews and moles .Considering that bats are more closely related to shrews and moles than to rodents  for the new virus because it was detected in an animal captured at the Magboi River in Sierra Leone. The MGBV nucleotide sequence is novel and has not been known or handled before in our laboratory. Before this study, hantavirus nucleic acid was found in lung and kidney tissues of bats from the genera N.hispida as a natural reservoir host of the virus. Although the presented bat-associated sequence is obviously distinct from other hantaviruses, which suggests association with a novel natural host, a spillover infection from another, yet unrecognized host cannot be ruled out. However, detection of the virus exclusively in 1 organ  suggests a persistent infection that is typically observed in natural hosts of hantaviruses (Further screening is necessary to confirm ,To date, only a few reports exist on cases of hemorrhagic fever with renal syndrome in Africa (


O’nyong-nyong means “severe joint pain” in the language of the Acholi people of East Africa. O’nyong-nyong virus was first isolated in Uganda in 1959 at the beginning of an outbreak that spread to Kenya, Tanzania, Zaire, Malawi, and Mozambique. One of the largest arbovirus epidemics ever recorded, the outbreak lasted until 1962 and affected >2 million persons. A species of the genus Alphavirus and closely related to chikungunya virus, O’nyong-nyong virus is transmitted by the bite of anopheline mosquitoes and causes an acute, self-limited, febrile illness characterized by lymphadenitis and joint pain.Sources: Dorland’s illustrated medical dictionary. 30th ed. Philadelphia: Saunders; 2003 and wikipedia.org


To the Editor: Bats are hosts for various emerging viruses, including the zoonotic paramyxoviruses Hendra virus and Nipah virus, which occur in Australia and Southeast Asia, respectively, and cause severe disease outbreaks among humans and livestock . Four samples could not be quantified, probably because copy numbers were too low. Virus load in urine was 1.8 × 106 per mL. For 4 of the 14 positive samples, we gathered additional sequence information by using pan-Paramyxovirinae primers targeting the most conserved genomic region  sequences are shown to form at least 3 distinct groups in the Paramyxoviridae family  through bushmeat hunting (using nets or shotguns), handling, and consumption; and 2) through environmental contamination with bat excretions and saliva. Despite the substantial exposure suggested by this study, to our knowledge, no human infection associated with bat paramyxoviruses has been reported in Africa, and elevated numbers of deaths have not been observed in bat hunters. Nevertheless, the existence of isolated cases cannot be excluded because underreporting is widespread, and many cases are undiagnosed. Additional studies on virus-host ecology, along with clinical surveys of exposed persons , are required to assess the zoonotic risk of these viruses and, ultimately, diminish the threat of a novel paramyxovirus entering and spreading in human populations.Details regarding positive samples and dataset composition.


This paper has been retracted as it is found to contain a substantial amount of material from a number of previously published papers. The three most plagiarized papers are 1) K. I. Hidari and T. Suzuki, “Dengue virus receptor,” Tropical Medicine and Health, vol. 39, no. 4, supplement, pp. 37–43, 2011; (2) A. Cabrera-Hernandez and D. R. Smith, “Mammalian dengue virus receptors,” Dengue Bulletin, vol. 29, no. 662, pp. 119–135, 2005; (3) A. Cabrera-Hernandez, C. Thepparit, L. Suksanpaisan, and D. R. Smith, “Dengue virus entry into liver (HepG2) cells is independent of hsp90 and hsp70,” Journal of Medical Virology, vol. 79, no. 4, pp. 386–392, 2007 (see [ K. I. Hi


The aim was to estimate the direct costs caused by ADEs, including costs for dispensed drugs, primary care, other outpatient care, and inpatient care, and to relate the direct costs caused by ADEs to the societal COI (direct and indirect costs), for patients with ADEs and for the entire study population.We conducted a population-based observational retrospective cohort study of ADEs identified from medical records. From a random sample of 5025 adults in a Swedish county council, 4970 were included in the analyses. During a three-month study period in 2008, direct and indirect costs were estimated from resource use identified in the medical records and from register data on costs for resource use.Among 596 patients with ADEs, the average direct costs per patient caused by ADEs were USD 444.9 [95% CI: 264.4 to 625.3], corresponding to USD 21 million per 100 000 adult inhabitants per year. Inpatient care accounted for 53.9% of all direct costs caused by ADEs. For patients with ADEs, the average societal cost of illness was USD 6235.0 [5442.8 to 7027.2], of which direct costs were USD 2830.1 [2260.7 to 3399.4] (45%), and indirect costs USD 3404.9 [2899.3 to 3910.4] (55%). The societal cost of illness was higher for patients with ADEs compared to other patients. ADEs caused 9.5% of all direct healthcare costs in the study population.Healthcare costs for patients with ADEs are substantial across different settings; in primary care, other outpatient care and inpatient care. Hence the economic impact of ADEs will be underestimated in studies focusing on inpatient ADEs alone. Moreover, the high proportion of indirect costs in the societal COI for patients with ADEs suggests that the observed costs caused by ADEs would be even higher if including indirect costs. Additional studies are needed to identify interventions to prevent and manage ADEs. Adverse drug events (ADEs), ”injuries resulting from medical intervention related to a drug”,In a Swedish survey study of the adult general public, respondents reporting ADEs during one month had a 150% higher overall cost of illness (COI) compared to respondents without ADEs, and considerable direct costs caused by ADEs.Thus we conducted a study to examine ADEs identified from medical records in a random sample of the adult general population. The aim of this study was to estimate the direct costs caused by ADEs, including costs for dispensed drugs, primary care, other outpatient care, and inpatient care, and to relate the direct costs caused by ADEs to the societal COI (direct and indirect costs), for patients with ADEs and for the entire study population.The study received ethical approval from the Regional Ethical Review Board in Gothenburg . Informed consent was not obtained since the retrospective study did not affect the healthcare of the included individuals. Although personal identity numbers were used to link data from medical records and registers, data were analysed anonymously. Statistics Sweden replaced the personal identity numbers by a random serial number after the final data linkage.We conducted a population-based observational retrospective cohort study to examine ADEs identified from medical records from primary care, other outpatient care and inpatient care. Identified ADEs included: adverse drug reactions, drug abuse, drug dependence, drug intoxications from overdose, sub-therapeutic effect of drug therapy, and morbidity due to drug-related untreated indication.In depth description of the sample size calculation, identification, exclusion and evaluation process is reported elsewhere.The healthcare encounters of individuals still living in the county during the allotted study period (n  =  5000) were identified from the Care Data Warehouse, the regional patient register in Östergötland County that includes all public and most private healthcare encounters. Data on prescribed and dispensed drugs were obtained from medical records and the Swedish Prescribed Drug Register of the National Board of Health and Welfare. A pharmacist manually reviewed the medical records of individuals with hospitalisations or outpatient physician or nurse encounters during the study period (n  =  2464), including medical records before and after the study period. From the medical records, using triggers and a list of dispensed drugs from four months before to one month after the study period, the pharmacist collected data on suspected ADEs in a standardised data collection sheet. The suspected ADEs were evaluated independently by two experienced professionals, one physician and one pharmacist, according to published criteria for causality,Using personal identity numbers, data from the medical records, the Care Data Warehouse, and the Swedish Prescribed Drug Register were linked to additional register data: demographic and socioeconomic variables  from Statistics Sweden, sick-leave and disability pension from the Social Insurance Agency, and administrative data from the Cost Per Patient Register from Östergötland County Council. Main occupation was derived from register data, assuming that the sources and distribution of income reflected the individuals' occupations (e.g. if the main income was from student grants the occupation was interpreted as student).For patients with ADEs, each healthcare encounter during the study period was evaluated for costs caused by prevalent ADEs. The association between the ADEs and the healthcare encounter was evaluated as dominant (i.e. the main reason for the encounter), partly contributing , less important (i.e. played a minor or uncertain role for the encounter), or not contributing (i.e. other symptoms/circumstances were the main reason for the encounter). The categories were based on Hallas' criteria for drug-related hospitalisations.COI studies measure the economic impact of disease and can be used to inform decision makers of the financial relevance in relation to other health issues.For the societal COI, direct and indirect costs were estimated for all study individuals. The direct costs included costs of healthcare services from the Cost Per Patient Register and dispensed drugs  from the Swedish Prescribed Drug Register. Data for indirect costs were extracted from the Swedish Social Insurance Agency and included individuals' lost productivity due to sick-leave or disability pension, estimated by the human capital approach,Differences in the socio-demographic characteristics of patients with ADEs compared to patients without ADEs, and all patients compared to individuals without any healthcare encounters, were tested for statistical significance (at p<0.05) using Fisher's exact test, or chi square test when exceeding the memory limit of Fisher's exact test. All cost analyses were prevalence-based, i.e. included costs during the study period for each patient, and were translated to United States dollar (USD) using the 2008 exchange rate  and inpatient care. The overall direct costs for ADEs per 100 000 adult inhabitants per year were calculated from the direct costs resulting from all ADEs during the three months, as following: [/all study individuals] * 4 * 100 000. The multiplication by four was for extrapolating three-month costs to annual costs.For patients with ADEs, direct and indirect costs were described and tested for statistical significance (p<0.05) compared to patients without ADEs, using two-tailed t-tests with unequal variances. Since the cost data were skewed, Wilcoxon rank-sum test was also used, but with minimal changes to the statistical significances. For patients with ADEs, the direct costs caused by ADEs and the societal COI, were presented by patient and ADE characteristics. Differences by age categories were tested for statistical significance (p<0.05) using one-way Anova, and by sex using two-tailed t-tests with unequal variances. The average direct healthcare costs per person were also illustrated by care level  for the study population, all patients with healthcare encounters, total costs for patients with ADEs, and for costs caused by ADEs among patients with ADEs.A sensitivity analysis of the direct costs was conducted based on Diagnosis Related GroupsStatistical analyses were made using the STATA/IC statistical software, version 11.2.ADEs were identified among 596 (12.0%) of the 4970 individuals in the study population. The characteristics of the study population are presented in ADEs were assessed as a dominant cause of 991 healthcare encounters , causingAmong the study population, 2560 patients accounted for 13 914 healthcare encounters during the study period. Of all encounters, 1845 (13.3%) were not assigned a cost. The average societal COI for patients with ADEs were USD 6235.0 [5442.8 to 7027.2] , of whicAmong the 596 patients with ADEs, the average direct costs caused by ADEs and the direct and indirect components of the societal COI did not differ by patient characteristics , apart fFor the 2224 healthcare encounters assigned a DRG in the register, our comparison showed that the average DRG weight was higher for encounters among those with ADEs compared to patients without ADEs  during the study period (p = 0.002). Among patients with ADE, the corresponding difference was not statistically significant between encounters dominantly caused by ADEs compared to other encounters .Our population-based study demonstrates that ADEs cause significant costs in healthcare, as the direct costs caused by ADEs during a three-month period were USD 445 per patient with ADEs. We found that 44% of the direct costs caused by ADEs occur outside inpatient care settings. The total societal COI per patient with ADEs, USD 6235 during the study period, was high compared to the COI of patients without ADEs. Indirect costs constituted half of the societal COI for patients with ADEs.The main strengths of this study were the population-based random sample of adults in Östergötland, which is reasonably representative of all adults in Sweden.Our estimated cost per patient caused by ADEs was low compared to the costs identified in previous studies of ADEs in single-episode hospitalisations.There are, to our knowledge, no prevalence- or incidence-based COI studies on ADEs, other than a survey study on the societal COI for survey respondents with self-reported ADEs.In summary, our estimated direct healthcare costs caused by ADEs represented approximately 10% of the total direct healthcare costs in a random sample of adults in a Swedish county. Additional studies are therefore needed to identify interventions to prevent and manage ADEs. This includes research to further analyse the processes involved in interventions for diagnosing, treating and monitoring ADEs during healthcare encounters. Moreover, nearly half of the direct costs of ADEs in the society occurred in outpatient settings. The economic impact of ADEs will hence be underestimated in studies focusing on inpatient ADEs alone. In addition, there is a need for studying a broader variety of costs and outcomes resulting from ADEs, such as productivity loss, to ensure all relevant consequences are acknowledged. The high proportion of indirect costs in the societal COI for patients with ADEs suggests that the observed costs caused by ADEs would be even higher if indirect costs were included in this study.