Lactobacillus acidophilus DMSZ20079. Twelve different formulations containing alginate (ALG) and alginate-psyllium (ALG-PSL) were prepared using extrusion technique. The prepared beads were characterized in terms of size, morphology and surface properties, encapsulation efficiency, viabilities in acid  and bile  conditions, and release in simulated colon pH conditions. The results showed that spherical beads with narrow size distribution ranging from 1.59 ± 0.04 to 1.67 ± 0.09 mm for ALG and from 1.61 ± 0.06 to 1.80 ± 0.07 mm for ALG-PSL with encapsulation efficiency higher than 98% were achieved. Furthermore, addition of PSL into ALG enhanced the integrity of prepared beads in comparison with ALG formulations. The results indicated that incorporation of PSL into alginate beads improved viability of the bacteria in acidic conditions as well as bile conditions. Also, stimulating effect of PSL on the probiotic bacteria was observed through 20-hour incubation in simulated colonic pH solution. According to our in vitro studies, PSL can be a suitable polymer candidate for partial substitution with ALG for probiotic coating.This paper describes preparation and characterization of beads of alginate and psyllium containing probiotic bacteria of  These btivities –4.6-7 CFU/g or mL of viable probiotic bacteria [Probiotics can be bacteria, moulds, and yeasts. However, most of probiotics are bacteria; among them lactic acid bacteria (LAB), typically associated with the human gastrointestinal tract, are the most widely used probiotic microorganisms. In order to exhibit their potential benefits, probiotics need to pass the harsh conditions of gastric tract and colonize and grow on the epithelium of colon in appropriate population . It is sbacteria . To impr2+ chelating agents, and harsh chemical environments [Alginate, a commonly used material to encapsulate probiotics, is a naturally occurring biocompatible and biodegradable linear anionic polysaccharide. Preparation of alginate bead, with well retained bacteria in their matrix, can be easily achieved by simple techniques like extrusion or emulsion methods. In spite of the wide application of alginate microcapsules in this area, some problems related to protection efficiency of them have been reported including susceptibility to disintegration in the presence of excess monovalent ions, Caronments .Plantago, is gel-forming mucilage composed of a highly branched arabinoxylan. The backbone consists of xylose units, while arabinose and xylose form the side chains [Psyllium, the common name used for several members of the plant genus e chains , 8. Psyle chains . Moreovee chains –13. Prebe chains  as “non-L. acidophilus DMSZ20079. Having in mind the pharmacological benefits of psyllium in digestive system as well as its potential to stimulate probiotic growth in the colon, here we aimed to incorporate psyllium in alginate beads containing probiotic bacteria To this end, different formulations containing ALG and/or ALG-PSL were prepared using extrusion technique and characterized in terms of size, morphology and surface properties, encapsulation efficiency (EE), viabilities in acid  and bile  conditions, and release in simulated colon pH conditions.L. acidophilus DSMZ20079 was obtained from DSMZ (Germany), pepsin, pancreatin, sodium alginate, oxgall from Sigma-Aldrich (Germany), MRS broth and MRS agar, sodium hydrogen phosphate, calcium chloride, sodium hydroxide and hydrochloric acid from Merck (Germany), and psyllium seed husk was supplied from Sidpur Sat Isabgol (India).L. acidophilus was cultured in MRS broth at 37°C for 18 hours. Culture was harvested by centrifugation at 700 RCF at 4°C for 7 min and washed twice with saline and collected by centrifugation as above. The washed bacterial cells were resuspended in 7 mL saline, and the cell count was determined using pour plate technique in MRS agar in triplicate. The cell suspension divided in some equal parts and consequently was used to prepare different formulations.Psyllium husk was extracted by a method described by Guo et al.  with som2). The beads were shaken at 150 rpm for 40 min, isolated by aseptic filtration (Whatman No.1), washed twice with sterile water, and kept in 0.1% w/v peptone solution at 4°C. The prepared formulations are shown in The extrusion technique was used to prepare ALG and ALG-PSL beads . Sodium The particle size of beads was assessed using optical microscopy  by Scion image analyzer software. Data were collected from 60 beads in each sample, and mean particle size was reported.The topographical properties of prepared beads were investigated by scanning electron microscopy (SEM)  at an accelerating voltage of 20 KV. Prior to examination, samples were prepared on aluminum stubs and coated with gold under argon atmosphere by means of a sputter coater.To determine the encapsulation efficiency, firstly prepared beads were mechanically disintegrated in phosphate buffer (pH = 6.8), then the number of entrapped cells after adequate dilution were measured by pour plate method, and counts were expressed as number of colony forming units (CFU), and calculated asN is the number of viable entrapped cells released from the beads and N0 is the number of free cells added to the biopolymer mixture immediately before the production procedure.Low pH conditions were produced using 9 g/L sodium chloride and 3.0 g/L of pepsin and pH adjusted to 1.8 with hydrochloric acid . 100 mg The survival (%) of the bacteria was calculated as follows:Prepared beads after 2-hour acid exposure were washed with distilled water, removed, and incubated in 50 mL of high bile condition, containing 6.8 g of monobasic potassium phosphate, and 10 g/L of pancreatin with pH adjusted to 6.8 ± 0.1 using sodium hydroxide and 0.5% w/v oxgall for 2 hours at 37°C with constant agitation at 50 rpm . SamplesThe release of the prepared beads was examined at simulated colonic pH solution as described by Mandal et al. . The beaP < 0.05 considered as a statistically significant difference.Statistical testing was carried out using SPSS19. All of the experiments were performed in triplicates. Data are presented as mean ± SD. The One-Way ANOVA test was performed to assess the difference between different beads and control groups and 2 as hardening solution (1 to 6% w/v) were examined. According to the results of this step, it was found that uniform and spherical bead preparation by ALG concentrations less than 1% (w/v) was quite difficult because of decreased viscosity and less ion sites for cross-linkage [2 produced the best result and chosen as optimum hardening solution.In the preliminary experiments, different concentrations of ALG (0.75 to 3% w/v) and CaCl-linkage . Also, AIn the second step of preliminary tests, the concentrations of PSL to be incorporated in ALG beads were optimized. Addition of PSL into ALG gel results in an increase in the viscosity and adherence of resultant gel. Indeed, incorporation of PSL in the concentrations more than 0.3, 0.5, and 0.6% w/v to ALG in the concentrations of 2, 1.5, and 1% w/v, respectively, yields too adherent mixtures to easily fabricate the beads. Consequently, the compositions in P < 0.05) that can be attributed to the viscosity of the resultant gel. According to the studies in this regard, an increase in the viscosity of the starter gel leads to the preparation of bigger beads by the extrusion method [P > 0.05) were observed between beads contained or not L. acidophilus loads. Scanning electron microscopy images of our formulations also show that the resultant beads are in spherical shape Figures  with groL. acidophilus before bead preparation was 9.81 ± 0.02 log CFU/mL. High bacterial cell entrapping in the range of 9.6 ± 0.06 to 9.78 ± 0.06 (log CFU/g beads) was achieved in resultant beads (P > 0.05) regarding the EE were observed between all formulations. The initial cell count of nt beads . The resThe protective effects of different coats of ALG and ALG-PSL after 2-hour exposure to acid conditions (pH = 1.8) are compared to untreated cells, and results are expressed as log CFU/g in L. acidophilus was 9.81 ± 0.08 log CFU/g which declined to 5.06 ± 0.06 log CFU/g after acid exposure for 2 hours . On the other hand, in our prepared beads with the initial cell numbers ranged between 9.6 ± 0.06 to 9.8 ± 0.03 log CFU/g, after 2 h acid exposure, the counts were 7.03 ± 0.1 to 8.43 ± 0.04 log CFU/g indicating more than 70% survival in all formulations. Overall, it is clear that survived bacteria after acid exposure, in all prepared beads were significantly (P < 0.05) higher than those of untreated cells. In fact, around 5 log reduction in bacterial count in the case of untreated L. acidophilus decreased to 1–3 log reduction among our obtained beads after 2 h acid exposure, and it can be concluded that coating of the bacteria as ALG or ALG-PSL beads can improve the viability of L. acidophilus in that conditions. There are numerous studies in this regard to protect probiotics by encapsulation in alginates beads using different techniques [As it can be seen from bar graphs in chniques . Howeverchniques , 22, 23.chniques . L. acidophilus in those beads in acid conditions and this effect is more obvious in higher concentrations of PSL. For instance, incorporation of 0.1 and 0.6% w/v PSL into 2% w/v (F2) and 1% w/v (F12) ALG solutions increased the survival around 1% and 12%, respectively. The increase in viability of the bacteria by addition of PSL is in line with our expectations, and it can be attributed to the total concentration of polymers blend used, as the survival of L. acidophilus in the beads with the same total amount of polymers showed no significant differences (P > 0.05) (F1 and F8 or F5 and F11). Moreover, the rising trend in the viability values by increase in the proportion of PSL can be attributed to the alginate concentration. As in the lower concentrations of alginate (1% w/v), the PSL effect is more dominant probably due to the fact that ALG concentration is insufficient for protection and addition of PSL increases the total polymer concentration and brings it to the appropriate point to remarkably increase the protection of the cells against acid conditions. Polymer blending is a simple yet attractive method to provide combined properties of polymers to a system and overcome their limitations [On the other hand, incorporation of PSL into alginate beads resulted in a rise in the viability of itations . This kiitations . Moreoveitations  also shoL. acidophilus (passed through acidic conditions) in prepared beads and untreated cells is demonstrated in The effect of 2 h exposure to 0.5% w/v oxgall on the survival of L. acidophilus encapsulated in either ALG or ALG-PSL beads showed better survivability (less than 2.5 log reduction) after 2 h bile exposure compared to those of untreated (P < 0.05) that is dropped by around 4.5 log CFU/mL after 2 h bile exposure. According to the previous studies, survivability of encapsulated probiotics against harsh environmental conditions especially bile tolerance is highly dependent on the strain type. Our finding also concurs with the studies of Sohail et al., who reported that encapsulation of L. acidophilus in extruded macrobeads was effective in maintaining cell viability [It is clear that P < 0.05) and the positive effect of PSL on the survivability of the bacteria is more evident in the lower concentrations of ALG. As a same manner with acid exposure results, we can discuss here that the rise in the total amount of polymer can be probably the reason of this phenomena. For instance, incorporation of 0.6% w/v PSL into 1% w/v ALG solution in F12 results in the fabrication of beads that were significantly more protective for the bacteria against bile condition compared to beads prepared just with 1% w/v ALG solution (F9). However, between the prepared beads, addition of less than 0.4% w/v PSL into ALG did not show significant changes in the viability of the bacteria in the presence of 0.5% w/v oxgall . It can be concluded that the ALG concentrations had no considerable effect on the release of L. acidophilus from the prepared beads. This finding also is in good agreement with Mandal et al. [L. casei in simulated colonic pH solution rose to its highest point in 60 min and after that remained constant. Moreover, according to Picot and Lacroix, a progressive release of coated cells in whey protein in simulated intestinal conditions occurred [Among beads prepared with ALG , in 3 h, the counts hit their maximum level and beyond that no significant changes were observed in cell numbers , lowest amount of growth was observed (under 2 log). The stimulation effect of PSL on tuations –13. For tuations . Howeverlleagues , and theL. acidophilus improved its delivery to the active site. In summary, whatever the prebiotic activity officially accepted or not for PSL, it is important to note that based on our data, it is assumable that PSL can potentially act as a prebiotic and preparation of PSL-ALG beads containing L. acidophilus in the size range of 1.61 ± 0.06 to 1.80 ± 0.07 mm with EE higher than 98% were prepared using extrusion method. Inclusion of PSL into ALG beads maintained the survival rate of L. acidophilus in acid and bile conditions as well as considerable stimulation effect on the bacteria in simulated colon pH solution. Psyllium as a pharmacologically active ingredient for gastrointestinal disease and a potential prebiotic can be a suitable candidate to partially replace with alginate for encapsulation of probiotic bacteria. ALG-PSL beads encapsulating probiotic


This novel virus is related to arenaviruses that cause hemorrhagic fever. Peromyscus mexicanus) captured near the site of a 1967 epidemic of hemorrhagic fever in southern Mexico. Analyses of nucleotide and amino acid sequence data indicated that the deer mice were infected with a novel Tacaribe serocomplex virus (proposed name Ocozocoautla de Espinosa virus), which is phylogenetically closely related to Tacaribe serocomplex viruses that cause hemorrhagic fever in humans in South America.Arenavirus RNA was isolated from Mexican deer mice ( Arenaviridae, genus Arenavirus) comprise Bear Canyon virus, Tamiami virus, and Whitewater Arroyo virus in the United States; Tacaribe virus (TCRV) on Trinidad; Chaparé virus (CHPV) and Machupo virus (MACV) in Bolivia; Guanarito virus (GTOV) in Venezuela; Junín virus (JUNV) in Argentina; Sabiá virus (SABV) in Brazil; and 9 other species  in western Venezuela is the principal host of GTOV  in central Argentina is the principal host of JUNV  cause hemorrhagic fever in humans  and 0 of 29 other cricetid rodents captured in the municipality of Ocozocoautla de Espinosa, State of Chiapas, Mexico (P. mexicanus deer mice in western Chiapas.A recently published study reported antibody against a Tacaribe serocomplex virus in 3 (25.0%) of 12 Mexican deer mice (Baiomys musculus), and 11 Jaliscan cotton rats (Sigmodon mascotensis) were tested for arenavirus by cultivation in monolayers of Vero E6 cells  and 38 antibody-negative rodents were captured on July 16, 2000, at a locality  in the municipality of Ocozocoautla de Espinosa  and 19C-cons and either AVNP42 (5′-GCCGCGGACTGGGAGGGCA-3′) or AVNP122 (5′-GCCGCGGACTGGGGAGGCACTG-3′). The second-round (heminested) PCR used MasterTaq Kit and either AVNP42 and oligont 1010C  protein gene RNA. First-strand cDNA was synthesized by using SuperScript III Reverse Transcriptase  and oligont 19C-cons (p) distances.Analyses of GPC sequences, N protein sequences, and nucleotide sequences included AV B1030026, 8 other viruses from North America, and 15 viruses from South America . Sequenc>0.95 were considered supported by the data  was 96.4% identical to the nucleotide sequence of the homologous region of the N protein gene of AV B1030026.Bayesian analyses of complete GPC gene sequences , panel ANonidentities between amino acid sequences of the GPC and N protein of AV B1030026 and amino acid sequences of homologous sequences of the 8 other viruses from North America ranged from 47.8% to 52.1% and from 44.3% to 45.4%, respectively. Similarly, nonidentities between the amino acid sequences of the GPC and N protein of AV B1030026 and homologous sequences of the 8 other members of group B ranged from 24.7% to 46.8% and from 16.1% to 32.3%, respectively . NonidenArenaviridae, genus Arenavirus  in the family The hallmark of the arenaviruses is their ability to establish chronic infections in their respective principal hosts. The failure to isolate arenavirus from Mexican deer mice in this study could be caused by small sample size or poor specimen quality. Alternatively, a cricetid rodent other than the Mexican deer mouse is the principal host of Ocozocoautla de Espinosa virus (OCEV).Members of the rodent family Cricetidae, subfamily Sigmodontinae (Baiomys taylori) captured in Texas and northern Mexico (The presence of OCEV in Mexico suggests that the last common ancestor of the 9 viruses in group B  emerged P. mexicanus deer mouse is a relatively common species in forests of southern Mexico (The history of the State of Chiapas includes an outbreak of a highly lethal hemorrhagic fever in 1967 (It is generally accepted that humans usually become infected with arenaviruses by inhalation of virus in aerosolized droplets of secretions or excretions from infected rodents. Another source of infection may be ingestion of infected rodents (Human consumption of wild rodents is common in rural areas in some regions of Mexico. For example, Mexican deer mice and other cricetid rodents are consumed by the Tzeltal Indians in the highlands of Chiapas (


El Moro Canyon virus and Limestone Canyon virus are widely distributed and may cause hantavirus pulmonary syndrome. To increase our knowledge of the geographic distribution of hantaviruses associated with neotomine or sigmodontine rodents in Mexico, we tested 876 cricetid rodents captured in 18 Mexican states (representing at least 44 species in the subfamily Neotominae and 10 species in the subfamily Sigmodontinae) for anti-hantavirus IgG. We found antibodies against hantavirus in 35 (4.0%) rodents. Nucleotide sequence data from 5 antibody-positive rodents indicated that Sin Nombre virus (the major cause of hantavirus pulmonary syndrome [HPS] in the United States) is enzootic in the Mexican states of Nuevo León, San Luis Potosí, Tamaulipas, and Veracruz. However, HPS has not been reported from these states, which suggests that in northeastern Mexico, HPS has been confused with other rapidly progressive, life-threatening respiratory diseases. Analyses of nucleotide sequence data from 19 other antibody-positive rodents indicated that El Moro Canyon virus and Limestone Canyon virus are geographically widely distributed in Mexico. Bunyaviridae, genus Hantavirus) that are principally associated with members of the rodent family Cricetidae, more specifically, members of the subfamily Neotominae or Sigmodontinae  is a potentially fatal zoonosis caused by hantaviruses  in Honduras, and Bayou virus is associated with the marsh rice rat  in the southeastern United States  are the principal hosts of the hantaviruses, for which natural host relationships have been well characterized. The current principal host relationships of some hantaviruses seem to represent a long-term association between viruses in the genus Peromyscus boylii), the deer mouse (P. maniculatus), the western harvest mouse , the hispid cotton rat (Sigmodon hispidus), the fulvous pygmy rice rat (Oligoryzomys fulvescens), and 122 other species in the Neotominae or Sigmodontinae , harvest mice (Reithrodontomys spp.), or cotton rats (Sigmodon spp.)—are widely distributed in northern Mexico, and the hantavirus assemblage of southern Mexico includes CHOV or hantaviruses that are phylogenetically closely related to CHOV.The rodent fauna of Mexico comprises the brush mouse  captured in Morelos (R. sumichrasti) and MTNV RNA in an Orizaba deer mouse (P. beatae) from Guerrero (Oryzomys mexicanus) and Jaliscan cotton rat (S. mascotensis) from Colima (P. levipes) captured in Tamaulipas (P. maniculatus), transvolcanic mice (P. hylocetes), black-eared mice (P. melanotis), and Sumichrast’s harvest mouse captured in the state of Mexico  strain VHV-574 by using an ELISA in which CADV can be highly cross-reactive with SNV, BCCV, and other North American hantaviruses  in conjunction with oligonucleotide 5′-GGTGGTTGTGGTAGTAGTAGACTCC-3′ (C glycoprotein.Samples of lung tissue from the antibody-positive rodents were tested for hantavirus nucleocapsid (N) protein gene RNA. Subsequently, we determined the nucleotide sequences of a 1,078-nt fragment of the glycoprotein precursor (GPC) genes of 11 of the hantaviruses associated with the antibody-positive rodents. We chose these 11 viruses to represent the geographic distribution and natural host associations of the hantaviruses in Mexico included in this study. Total RNA was isolated from 30 mg to 45 mg of lung tissue by using Tri Reagent . First-strand cDNA was synthesized from small (S) segment and medium (M) segment RNA by using SuperScript II RNase H>0.95 were considered supported by the data  against hantavirus was found in 35 (4.0%) of 876 rodents captured in 18 states in Mexico . The antHantavirus N protein gene RNA was detected in samples of lung from 24 (68.6%) of the 35 antibody-positive rodents . The BayHantavirus GPC gene RNA was detected in each of the 11 rodents assayed for GPC gene RNA . The topC glycoproteins of the 11 hantaviruses from Mexico in this study, CARV, HUIV, and MTNV ranged from 0% to 18.4% (C glycoproteins of these 14 hantaviruses and the sequences of the homologous fragment of the GC glycoproteins of the other hantaviruses found in North America ranged from 1.1% (H0030073 and SNV strain Blue River-Oklahoma) to 18.9% (M0040049 and BCCV strain SPB 9408076).Nonidentities among the amino acid sequences of the 359-aa fragment of the Gto 18.4% . NonidenHantavirus . There is presumptive evidence for RIOSV or hantavirus(es) that are genetically closely related to RIOSV in Sumichrast’s harvest mice, a Mexican harvest mouse (R. mexicanus), and a Chiriqui harvest mouse (R. creper) captured in Panama  captured in northern Arizona (P. hylocetes) and the black-eared mouse (P. melanotis) in the state of México, the nimble-footed mouse (P. levipes) in Nuevo León, the El Carrizo deer mouse (P. ochraventer) in San Luis Potosí, and the gleaning mouse (P. spicilegus) in Jalisco are natural but not necessarily principal hosts of LSCV.Specific knowledge of the natural host relationships of LSCV previously was limited to LSCV RNA in 6 brush mice  in Mexico includes 23 states . Alternatively, SNV in Mexico is substantially less virulent than SNV in the western United States, or human contact with SNV-infected rodents in Mexico is less frequent or less intimate than human contact with SNV-infected rodents in the western United States.Laboratory confirmation of the diagnoses of most HPS cases in the United States before 2010 was based on the results of serologic assays in which ELMCV and LSCV can be highly cross-reactive with SNV (


Objective(s): Incidence of neurocognitive and psychological disorders may be related to serum homocystein (Hcy), cobalamin (vitamin B12) and folate levels in old people. The aim of this study was to assess the relation between Hcy, cobalamin, folate and neurocognitive and/or psychological disorders in the elderly. Materials and Methods: In this cross-sectional study, 280 subjects with ≥ 65 years old ,were evaluated. The subjects were selected from 12 regions of Mashhad, Iran, over March to October 2009. After blood sampling, data were collected by questionnaire, face to face interview and performing neurocognitive and psychological tests. The sera of 250 persons were analyzed for cobalamin and folate by RIA method. Amongst the aforementioned samples, 78 cases with cobalamin <300 pg/ml and folate <6.5 ng/ml were analyzed for Hcy by ELISA method. Results: Amongst the people, 126 (45%) were male and 154 (55%) were female. The prevalence of hyperhomocysteinemia (HHcy) was 59.5% and 37.1% in male and female respectively . Hcy inversely correlated to cobalamin  and to folate . Hcy, cobalamin and folate correlations to neurocognitive and psychological impairments were not statically significant. Conclusion: Hyper Hcy or low cobalamin and folate in the elderly, are prevalent but their relationships with neurocognitive and psychological impairments is controversial. If these relationships had been confirmed, performing a single serum Hcy or cobalamin test would have been enough enough to diagnose and prevent neurocognitive impairments and inversely, neurocognitive-psychological sign and symptoms could have meant probable tissue vitamin deficiencies. However methods of assessing neurocognitive and psychological markers with validity and reliability of clinical and laboratory tests for finding aforementioned relationships should be revised.  Annually many old people refer to physicians or clinics with mild and vague symptoms such as anemia and neurocognitive and psychological impairments that may be due to cobalamin and folate deficiency followed by gastric atrophy and malnutrition.Two enzymatic reactions dependent on cobalamin (Cbl), has been identified of which one converts methylmalonyl–coenzyme A to succinyl-CoA using adenosyl-cobalamin as a cofactor. So Cbl deficiency will increase methylmalonyl-CoA as methylmalonic acid (MMA). The other enzyme reaction has role in the synthesis of methionine from homocysteine (Hcy) using methyl-cobalamin as a cofactor whicht is important for definition the neurocognitive and/or psychological aspects of cobalamin and folate deficiency. Both Deficiencies involve in production of tetra Hydrofolaet and cause megaloblastic changes in red blood cells .The common symptoms of cobalamin and folate deficiency are anemia, macrocytosis and neurologic symptoms such as parenthesis, ataxia, dementia, depression and psychosis . FrequenHyper homocysteinemia is caused by various conditions including cobalamin and folate deficiency, renal failure, genetic defects, decreased blood volume and hyperthyroidism. These are independent risk factors for nervous degeneration and cardiovascular and cerebrovascular disorders in old people and thus require special medical management  -6 . Pat12 and B6 whicht are required in methylation of homogystein to methionin) correlate with decline cognitive performance , deep tendon reflex (DTR), Romberg test, geriatric depression scale (GDS), insomnia, motivation, easily crying, pain perception and also persons documents for dementia were recorded in a special form.12, B6 and folic acid) in recent 3 months, smoking, medication with metformin ,trimetoprim and omeprazole. Elderly with more than 65 years and completed data in questionnaires and forms were included in our study. After blood sampling, sera were collected and kept in -20 ˚C until measuring. Serum cobalamin and folate analyzed by RIA method  Serum methylmalonic acid (MMA) often increases with cobalamin deficiency while homocysteine increases with both folate and cobalamin deficiency, conclusively assaying of serum homocysteine was preferred (16-17). 78 cases, who had cobalamin <300 pg/ml and folate< 6.5ng/ml and also no confounders were analyzed for homocystein by Elisa method .Exclusion criteria in participant were creatinine>1.5 mg/dl, B vitamin Supplementation . Pearson coefficient of correlation was used to assess relation between serum cobalamin, folate and homocystein. P-value of less than 0.05 was considered as significant.12), folate and homocystein. Previous studies had shown high prevalence of hyperhomocysteinemia (HHcy), cobalamin and/or folate deficienciy and cognitive impairments in the elderly  was 22.7% and with borderline levels (122-330 pg/ml) was 51.8% and the prevalence of folate deficiency with low levels (<3 ng/ml) was 16.7% and with borderline levels was (<6.5 ng/ml) 64.2 % .et al, 640 patients were studied. 50% of old people present in the population had low or borderline levels of serum cobalamin (120-150 pmol/L) which correlates to both cerebrovascular diseases and cognitive disorders (P=0.046)  . Incidenis group .P = 0.001 for both).Normal serum homocystein has been defined as<14 µmol/l (26), or <15 µmol/l . Serum mCut off points for cobalamin and folate deficiencyMany researchers has determined cut off points for cobalamin and folate deficiency. Figlin et al defined cobalamin deficiency as <220 pg/ml with MMA >0.24 µ mol/l, and folic acid deficiency as <4.9 ng/ml with homocysteine >15 µ mol/l (30) and Rajan et al defined cobalamin deficiency equal to 299 pg/ml . WaltersCorrelation between cobalamin and folate deficiency and HHcyP≤0.001) and a negative association between HHcy and cobalamin  and folate .Our study showed significant association between serum cobalamin and folate . Prevalence of HHcy with this Regimen is reported. As cognCorrelation of HHcy or cobalamin deficiency with neurocognitive dysfunctions is controversial. It is unknown whether HHcy or vitamin deficiency is responsible for neurocognitive impairments . Lidballe et al observations on 839 old people have shown that score of cognitive functions as measured by mini-mental state examination and low wellness have significant corralations with markers of cobalamin insufficiency such as low serum cobalamin, high methylmalonic acid, high homocysteine but their causatives are uncertain  of the elderly with cobalamin <300 pg/ml and folate <6.5 ng/ml and HHcy had dementia. In these patients correlation between dementia and cobalamin, folate deficiency and HHcy was statically insignificant, but elderly with dementia had higher homocystein serum levels (17.91±8.18 µmol/l) than elderly without dementia. Correlation between Serum Hcy, cobalamin , folate and MMSE test et al  in 10 European Country found low but significant relation between MMSE score and cobalamin and also Homocysteine  is positive in mental disorders. Normal score is >20. In our study serum Hcy, cobalamin and folate levels in two groups of positive and negative MMSE test patients had no significant differences, but the elderly with positive MMSE test had higher Hcy level than negative group. This was not in contrast with the findings of Ellison et al . Kado etcysteine . Correlation between Serum Hcy, cobalamin, folate and geriatric depression scale (GDS)Our results showed that serum Hcy, cobalamin and folate average in the 2 groups with positive (score>5) and negative (score≤5) geriatric depression scale test have not significant differences. The same insignificance was observed with Motivation, Insomnia and easily crying.Correlation between Serum Hcy, cobalamin, folate anddeep tendon reflex(DTR), Romberg testWe investigate relations between Hcy ,cobalamin, folate, and Neurocognitive Markers by deep tendon reflex test and Romberg test. In two tests, relations were insignificant but the elderly with positive and high scores in DTR test had higher homocystein (18.41 µmol/L) than negative. The same insignificant differences were observed with Vibration and Pain perception.Consistent to results in some studies, we showed that there are not significant relations between neurocognitive-psycholigical impairments and hyperhomocysteinemia or cobalamin and folate deficienciy in the elderly.Hcy and MMA  levels without any improvement on cognitive performance and they only delay progression of the disease. This may be due to an insufficient dose and duration of supplements, irreversible or vitamin-independent neurocognitive impairments (Correlations between laboratory and clinical markers for these disorders in various studies are heterogenous and it is reasonable to assume that there are multi factors resulting in these variations including: different etiologies, stage of the disease, degree of manifestation of symptoms, sampling representative the entire population, diversity of tests and measurement techniques. Normal values for serum cobalamin and its metabolites is not established and various criteria, tests, and cutoff points have been used to define cobalamin deficiency . Also miairments , 37, 38.In this study we carefully collected participant data by biochemical tests and questionnaires and this was probably more accurate than neurocognitive and psychological examinations. It seems these clinical or cognitive tests are not adequate or sensitive enough to detect mild dysfunctions in these disorders.et-al explaining that since the Nerocognitive disorders are the first symptoms of cobalamin and folate deficiency, their diagnosis need much qualified instruments and their investigations require enough subjects and appropriate composition of population and correct sampling (et al  refer to the sensitivity and specificity of metabolite measurements for milder deficiency status that are uncertain ( These problems are consistent to Bjorkegren sampling . Christoncertain .Due to prevalence of neurocognitive and psychological impairments, hyperhomocysteinemia ,cobalamin and folate deficiency in the elderly, finding crucial relationships between mentioned impairments and deficiencies, which seem to be controversial, is essential. By confirming aforementioned relationship, diagnostic and preventative operations could be initiated to improve neurocognitive- psychological impairments for desired peoples. So primarily, methods for assessment of neurocognitive and psychological status and validity and reliability of clinical and laboratory tests should be revised and secondly, longitudinal studies and clinical trials should be performed in old population to determine whether lowering of homocysctein or increasing cobalamin and folate levels will improve neurocognitive psychological impairments or not.


These lessons of ancient and modern viruses have implications for modern human pandemics from viral reservoirs and for human interventions that may come with unintended consequences.Paleovirology is the study of ancient viruses. The existence of a paleovirus can sometimes be detected by virtue of its accidental insertion into the germline of different animal species, which allows one to date when the virus actually existed. However, the ancient and the modern often connect, as modern viruses have unexpected origins that can be traced to ancient infections. The genomes of two species of mongooses and an egg-laying mammal called an echidna show that a virus currently present in poultry, the reticuloendotheliosis virus (REV), is actually of ancient exotic mammalian origin. REV apparently spread to poultry through a circuitous route involving the isolation of malaria parasites from a pheasant from Borneo housed at the Bronx Zoo that was contaminated with REV. Repeated passage of this virus in poultry adapted the virus to its new host. At some point, the virus got inserted into another virus, called fowlpox virus, which has spread back into the wild. Although REV may still exist somewhere in a mammalian host, its modern form links an 8 million-year-old infection of the ancestor of a mongoose to a virus that now is circulating in wild birds through malaria studies in the mid-20 Outbreaks of “new” viruses are real causes of concern for human and animal health. But where do they come from? The vast majority of emerging viruses in the human population originate from other animals Trypanosoma brucei (sleeping sickness) in the 1920s Whether or not a virus will “emerge” in new species depends on a complex set of requirements including contact, mode of transmission, and adaptation to the new species Ultimately, though, where did these viruses come from? Fossils of animals and plants have been used to understand the age of groups of species. Although no physical fossils exist for viruses, clues about virus history can be gleaned from the remnants of viral sequences  that have been accidently inherited in the genome of the host they infected in the past. That is, although viruses typically spread between hosts by transmission from one individual to another , on rare occasions viral nucleic acids can get integrated into the host germline and become endogenous viral elements (EVEs) Among retroviruses, a group of viruses related to the reticuloendotheliosis virus (REV) has especially interesting ancestral origins. REV currently circulates in chickens, turkeys, pheasants, ducks, and geese, where it sometimes causes disease. However, its genome is not related to other avian retroviruses; rather, it is most closely related to some mammalian retroviruses PLOS Biology, Niewiadomska and Gifford elucidate the complex evolutionary history of REV and related viral strains by using paleovirology and a historical review of the literature to infer the viral origin, the successive inter-species transmissions, and the adaptive events that led to the circulation of REVs in wild birds In this issue of The link between this avian retrovirus and endogenous elements in mammals was first documented in a screen of available animal genomes in which the most closely related sequence to REV was from a fragment of the genome of an echidna, an egg-laying mammal from Australasia Plasmodium parasites from a pheasant, originally from Borneo, that was housed at the Bronx Zoo. For experimental vaccine and drug research purposes, the Plasmodium stock was passaged over time in poultry until the 1980s and was distributed to many US laboratories. However, the culture of Plasmodium lophurae was contaminated with retroviruses from the REV lineages . The authors depict a compelling scenario in which the zoological park, an environment in which exotic animal species from diverse lineages are brought together in unusually close proximity, could have favored the inter-class transmission of REVs from mammals to the pheasant from which Plasmodium was isolated. However, as inter-class transmissions often result in dead-end infections, the authors surmise that serial passages of the retrovirus-containing Plasmodium stock in poultry favored the adaptation of the mammalian virus to bird hosts. Sometime along the way, the REV virus integrated into two large DNA viruses: a poxvirus (FWPV) that contained a full-length insertion of REV and a herpesvirus (GHV-2) containing only a fragment of REV How then did this mammalian retrovirus get into birds? From here the story gets spectacular and strange (yet still very plausible). In the 1930s, the need for an experimental model system for malaria research led Lowell Coggeshall to isolate While the main text describes the origins of the reticuloendotheliosis virus (REV) and its close relatives as pathogens, the study of these retroviruses has also contributed to several areas of basic science and basic virology. The original description of REV was from a tumor taken from a turkey that contained two viruses. One, REV-A, was replication-competent, and the other, REV-T, was defective, but carried an oncogene called Rel that allowed it to transform cells Further efforts are needed to determine if REV still has a modern mammalian host; the authors of this study speculate it could be a bat species


Arenaviridae, genus Arenavirus, consists of two phylogenetically independent groups: Old World (OW) and New World (NW) complexes. The Lassa and Lujo viruses in the OW complex and the Guanarito, Junin, Machupo, Sabia, and Chapare viruses in the NW complex cause viral hemorrhagic fever (VHF) in humans, leading to serious public health concerns. These viruses are also considered potential bioterrorism agents. Therefore, it is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of arenavirus outbreaks. However, these arenaviruses are classified as BSL-4 pathogens, thus making it difficult to develop diagnostic techniques for these virus infections in institutes without BSL-4 facilities. To overcome these difficulties, antibody detection systems in the form of an enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence assay were developed using recombinant nucleoproteins (rNPs) derived from these viruses. Furthermore, several antigen-detection assays were developed. For example, novel monoclonal antibodies (mAbs) to the rNPs of Lassa and Junin viruses were generated. Sandwich antigen-capture (Ag-capture) ELISAs using these mAbs as capture antibodies were developed and confirmed to be sensitive and specific for detecting the respective arenavirus NPs. These rNP-based assays were proposed to be useful not only for an etiological diagnosis of VHFs, but also for seroepidemiological studies on VHFs. We recently developed arenavirus neutralization assays using vesicular stomatitis virus (VSV)-based pseudotypes bearing arenavirus recombinant glycoproteins. The goal of this article is to review the recent advances in developing laboratory diagnostic assays based on recombinant viral proteins for the diagnosis of VHFs and epidemiological studies on the VHFs caused by arenaviruses. The family  Arenaviridae consists of only one genus, but most viruses within this genus can be divided into two different groups: the Old World arenaviruses and the New World arenaviruses  , th, th69], For the diagnosis of many viral infections, PCR assays have been shown to have an excellent analytical sensitivity, but the established techniques are limited by their requirement for expensive equipment and technical expertise. Moreover, the high degree of genetic variability of the RNA viruses, including arenavirus and bunyavirus, poses difficulties in selecting primers for RT-PCR assays that can detect all strains of the virus. Since the sensitivity of the Ag-capture ELISA is comparable to that of RT-PCR for several virus-mediated infectious diseases, including Lassa fever and filovirus hemorrhagic fever ,71,72,73The virus neutralization assay is accepted as the “gold standard” serodiagnostic assay to quantify the antibody response to infection and vaccination of a wide variety of viruses associated with human diseases ,84,85,86The VSV-based vector has already been used to generate replication-competent recombinant VSVs to study of the role of GPs of various viruses ,91,92. RWe have recently developed a VSV-based pseudotype bearing Lassa virus GP (VSV-LAS-GP) for the detection of neutralizing antibodies in the sera obtained from a Lassa fever patient. An example of the LASV neutralization assay using the VSV pseudotype is shown . In the The Lujo virus is a new member of the hemorrhagic fever-associated arenavirus family from Zambia and southern Africa, and the virus is classified as a BSL-4 pathogen . The genHemorrhagic fever outbreaks caused by pathogenic arenaviruses result in high fatality rates. A rapid and accurate diagnosis is a critical first step in any outbreak. Serologic diagnostic methods for VHFs most often employ an ELISA, IFA, and/or virus neutralization assay. Diagnostic methods using recombinant viral proteins have been developed and their utilities for diagnosing of VHF have been reviewed. IgG- and IgM- ELISAs and IFAs using rNPs as antigens are useful for the detection of antibodies induced in the patients’ sera. These methods are also useful for seroepidemiological surveys for HFVs. Ag-capture ELISAs using MAbs to the arenavirus rNPs are specific for the virus species or can be broadly reactive for New World arenaviruses, depending on the MAb used. Furthermore, the VSV-based pseudotype system provides a safe and rapid tool for measuring virus neutralizing antibody titers, as well as a model to analyze the entry of the respective arenavirus in susceptible cells without using live arenaviruses. Recent discoveries of novel arenavirus species ,26,110 a


Herpes simplex virus type 2 (HSV-2) is sexually transmitted, leading to blisters and ulcers in the genito-anal region. After primary infection the virus is present in a latent state in neurons in sensory ganglia. Reactivation and production of new viral particles can cause asymptomatic viral shedding or new lesions. Establishment of latency, maintenance and reactivation involve silencing of genes, continuous suppression of gene activities and finally gene activation and synthesis of viral DNA. The purpose of the present work was to study the genetic stability of the virus during these events.HSV-2 was collected from 5 patients with true primary and recurrent infections, and the genes encoding glycoproteins B,G,E and I were sequenced.No nucleotide substitution was observed in any patient, indicating genetic stability. However, since the total number of nucleotides in these genes is only a small part of the total genome, we cannot rule out variation in other regions.Although infections of cell cultures and animal models are useful for studies of herpes simplex virus, it is important to know how the virus behaves in the natural host. We observed that several glycoprotein gene sequences are stable from primary to recurrent infection. However, the virus isolates from the different patients were genetically different. Herpes simplex viruses (HSV) are widely distributed pathogens transmitted by close contact. Infection by type 1 (HSV-1) starts in childhood and increases during the following years so that more than 70% of the population is seropositive at an age of 40 years ,3. EncepHerpes simplex type 2 (HSV-2) is sexually transmitted, leading to blisters and ulcers in the anogenital region . Prevalphaherpesvirinae subfamily of herpesviridae. They are all neurotropic, and like many other members of the herpesviridae family cause latent infections. After primary infection in the skin/mucosal membranes the virus enters axons of innervating neurons, migrates retrograde and establishes latent infections in neuronal cells in sensory ganglia. Reactivation from latency is a commonly occurring event during which the virus migrates anterograde in the axon, leading to new infection in the periphery or to asymptomatic shedding of the virus. About 90% of the persons with primary HSV-2 infection have recurrence during the first year afterwards, and one fifth of them more than 10 times during this period [Together with varicella zoster virus (VZV) and pseudorabies virus (PRV), HSV-1 and HSV-2 belong to the s period -20. Reacs period .The molecular processes involved during the various steps of primary and recurrent infection have been studied extensively and reviewed in recent reports ,22. NeitMajor regulatory steps in these events are silencing of viral genes, continuous suppression of gene activities and finally stimulation of such activities followed by DNA synthesis. Several aspects of these processes have been studied in cell culture and in animal models, but with little focus on the genetic stability of the virus. The purpose of the present work was to study this stability, in the natural host, during these different series of events. We isolated HSV-2 from true primary and recurrent infections and sequenced a set of selected genes. The isolates were genetically different, but no sequence differences were observed between the initial infection and the reactivation.HSV-2 isolates were collected from patients with genital lesions attending a clinic for sexually transmitted diseases in Bergen, Norway. Blood samples were analysed for the presence of HSV-2 antibodies as described previously . BrieflyThe study was approved by the Regional Committee for Medical and Health Research Ethics, Western-Norway, University of Bergen. All patients were informed about the various aspects of the project, including isolation of virus from the samples, analysis of the viruses and publication of the results. The samples and results were coded to by anonymous. Consent was obtained from all patients.Virus DNA was isolated using the Qiagen DNeasy Tissue Kit, as described by the manufacturer. The concentration varied from 25 to ≥ 1200 ng per μl. Three regions of the HSV-2 genome were amplified prior to sequencing. The UL27 gene  was amplified as a 2942-bp fragment spanning the region from 78 bp upstream of the start codon to 149 bp downstream of the termination codon (the positions refer to strain HG52). Several additional primers were used for sequencing (Table PCR and gel electrophoresis of the amplified fragments were performed as described previously . BrieflySets of overlapping primers as shown in Table http://www.ebi.ac.uk/cgi-bin/readseq.cgi) and alignment performed using Clustal W2 . Both programs are connected to the EMBL-EBI database.The sequences were analysed using the SeqScape software , using JN561323.1 in the NCBI GenBank as a reference sequence. Sequences for the reference strain and for the clinical isolates were converted to FASTA , glycoprotein G (gG), glycoprotein I (gI) and glycoprotein E (gE), respectively. The nucleotide diversity of these genes can be studied by comparing the sequences of clinical isolates of HSV-2 with the reference strain HSV-2 HG52. In the NCBI GenBank each of these genes have been sequenced in a number of isolates, varying from 48 to 69. The results of this comparison are shown in Table A consistent feature was that whenever a substitution was observed at a given position, it was identical in all isolates affected, regardless of the number of isolates subjected to change. This was the situation for all 4 genes. Except for deletion of 9 nucleotides in UL27 (gB), and of three nucleotides at two different positions in US4 (gG), the remaining changes were single nucleotide substitutions. Reiteration of sequences may cause alternative alignment, as observed both for the gB and the gG gene. Thus, alternative positions for substitutions may be indicated by the computer. From positions 62 to 88 in the former gene the sequence CGG CGG CCC is repeated three times. Similarly, CGG is repeated three times from position 1277 to 1285 in the gG gene. Identical variations in the same position in two or more clinical isolates indicate recombination. This was observed at numerous sites in all genes and is consistent with the fact that the isolates are from studies demonstrating different clades of virus and recombination ,33. HoweNucleotide diversity affecting one isolate only varied from 1.14% (UL27/gB) to 2.24% (US4/gG). Diversity affecting two isolates or more was lower, but still highest for the US4 gene. These results indicate that the 4 selected genes might be suitable candidates for detection of potential genetic variation among viruses included in the present study.All viral isolates were from females between 19 and 35 years old , it is important to know how the virus behaves in the natural host. We have studied lesional virus isolates from humans with genital HSV-2 infection and observed that several glycoprotein gene sequences are stable from primary to recurrent infection. However, isolates from the different patients were genetically different.The authors declare that they have no competing interests.AN collected blood samples and lesional material from the patients. LH was responsible for growing virus. Sequencing was a collaboration between PK and LH. NL was involved in design of the study and further discussions, and in the preparation of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/63/prepub


Arthropod-borne viruses, that is, arboviruses, belong to different virus families and genera and are maintained through transmission between vertebrate hosts by virus-infected blood feeding arthropods . Several factors have contributed to the emergence and reemergence of arthropod-to-human infections in the last decade, also in nonendemic areas: climate changes which are believed to have contributed to increased dispersion of the virus vectors; the international trading system that transports an enormous range of goods, animals, and human beings all over the world, inadvertently increasing the possibility for vectors and viruses to spread; and the global increase of human population density, particularly in areas densely populated by arthropods with transmission capability for several different pathogenic viruses. As a consequence, the incidence of arboviral infections and diseases is increasing worldwide and therefore placing a heavier burden on public health agencies and requiring the need to ensure timely recognition and treatment of virus infections.This special issue is aimed at the presentation of recent results obtained by prominent colleagues in the field of human infections caused by vector-borne viruses. Faced with a limited number of manuscripts presented, the high quality of these papers will contribute significantly to our knowledge of these important issues.Flavivirus infection, that is, dengue fever, in the United States of America where this infection is currently generating huge concerns. In particular, the authors emphasize how, despite the current economic difficulties and restrictions, strict mosquito control policies and activities must be implemented and maintained in localities that have the potential to become the route of entry for viruses and the focus of epidemics.Pathogens, such as dengue virus, Japanese encephalitis virus, and alphaviruses are addressed by the authors. G. Añez and M. Rios described the epidemiology of what is likely to be the most widespread mosquito transmitted in vitro infection of mononuclear cells from diabetes mellitus patients induces higher levels of both IL-4 and the anti-inflammatory Th2-cytokine IL-10, in addition to increased levels of GM-CSF, and they speculated that these findings might result from a counterbalance to the comparatively highly activated proinflammatory cytokines/chemokines in the diabetic hosts.A second paper by C.-F. Liu and coworkers reported on the complementary role of clinical practice-based laboratory data in facilitating suspicion and diagnosis of dengue in clinical settings, thus ameliorating the overall diagnostic capabilities for this infection. Although the authors state that further studies are needed to confirm the validity of their approach, we believe that the study is important to define which procedures will have the most significant impact on clinical practice in tropical countries, where medical resources are limited. R. Rodriguez-Roche and E. A. Gould contributed a comprehensive review about the complex epidemiology and pathogenesis of the dengue viruses. They underlined the need for more effort to understand and accelerate the development of suitable vaccines and/or antiviral therapies for the control of this “scourge” of the tropical and subtropical world. Another paper by M. L. Muñoz and coworkers is dedicated to the investigation of the interplay between dengue virus proteins and the receptor polypeptides located on the surface of mosquito cells, since this interaction is the fundamental basis for viral entry into susceptible cells, followed by development and transmission in the vectors. The paper by I.-K. Lee and coworkers provides an insight into the immunopathogenesis of dengue virus in patients with type 2 diabetes mellitus and reports on the ability of mononuclear cells from these patients to be infected by dengue virus. The aim of the paper is to fill a gap in our knowlwdge that concerns epidemiological data and immunological findings of frequent dengue haemorrhagic fever and Th2 cytokines in patients with diabetes mellitus. The authors demonstrated that Two papers describe different aspects of Japanese encephalitis virus (JEV) related diseases. The paper by G. Kakoti and coworkers reports on the clinical profile and the outcome of Japanese encephalitis virus infection in children up to 12 years old. This is a prospective study based on serological assays and it shows some points of interest from a clinical and epidemiological point of view as it investigates the presence of clinical or technical factors likely to be significantly correlated with increased mortality by JEV in children with acute encephalitis syndrome in North East India. B. Bandyopadhyay and collaborators reported the epidemiology of JEV in West Bengal (India) showing that 22.76% and 5% of the acute encephalitis syndrome cases were positive for JE IgM in 2011 and 2012, respectively, and that JE is mainly prevalent in children and adolescents below 20 years of age with no gender predilection. This is a significant observation from an epidemiological point of view, which shows how active surveillance of JE cases is still warranted. It also emphasizes the need for vigilance to identify the introduction of new genotypes in the endemic areas or to detect evidence of dispersion into newer geographical districts.This series of papers is completed by a paper by I. Assunção-Miranda that describes the molecular mechanisms involved in the pathogenesis of alphavirus-induced arthritis, the paper by A. Burgueño and coworkers that investigated the seroprevalence of Saint Louis encephalitis related antibodies among horses in Uruguay, where no sharp epidemiological data about this virus were available so far, and the article by G. Sautto and collaborators that speculated about the possible use of candidate monoclonal antibodies to be possibly used in a future passive immunotherapy for arthropod-borne infections. This approach seems of particular importance, given the current absence of specific antiviral drugs as well as effective vaccines for the most diffused arbovirus infections.


The hybridization of nucleic acid targets with surface-immobilized probes is a widely used assay for the parallel detection of multiple targets in medical and biological research. Despite its widespread application, DNA microarray technology still suffers from several biases and lack of reproducibility, stemming in part from an incomplete understanding of the processes governing surface hybridization. In particular, non-random spatial variations within individual microarray hybridizations are often observed, but the mechanisms underpinning this positional bias remain incompletely explained.This study identifies and rationalizes a systematic spatial bias in the intensity of surface hybridization, characterized by markedly increased signal intensity of spots located at the boundaries of the spotted areas of the microarray slide. Combining observations from a simplified single-probe block array format with predictions from a mathematical model, the mechanism responsible for this bias is found to be a position-dependent variation in lateral diffusion of target molecules. Numerical simulations reveal a strong influence of microarray well geometry on the spatial bias.Reciprocal adjustment of the size of the microarray hybridization chamber to the area of surface-bound probes is a simple and effective measure to minimize or eliminate the diffusion-based bias, resulting in increased uniformity and accuracy of quantitative DNA microarray hybridization. DNA microarrays, developed in the mid 1990s Generally, at the single spot scale it is thought that after an initial period where hybridization kinetics are reaction-limited, probe depletion near the spot renders the process diffusion-limited In this study we identify a widespread spatial bias in microarray hybridization experiments that depends on probe spot position, and we characterize this bias experimentally using a simplified array design on standard 1”×3” glass slides. We demonstrate the underlying mechanism through a simple physical model, which reproduces the spatial bias and captures the salient features of the experimental observations. Finally, we explore the influence of hybridization conditions on the positional bias using numerical simulations. This analysis sheds light on the importance of array design and hybridization chamber geometry and suggests design modifications to minimize the spatial bias.5′-GAA CCC AAC GGC CCG ACA-3′) A published 16S rRNA-targeted oligonucleotide probe, DVHO831  was amplified using the PCR primer set 616V-630R in vitro transcription labeling of PCR products. The PCR cycling program consisted of 95°C for 3 min followed by 30 cycles of 95°C for 30 s, 52°C for 30 s and 72°C for 1 min, and concluded with 72 °C for 3 min. The target was labeled via an in vitro transcription reaction and subsequently template DNA was digested 2 at 60°C for 30 min and was then stored in the dark at −20°C.The target gene was prepared and labeled fluorescently as described previously Labeled RNA was added to hybridization buffer  buffer, 10% Denhardt's reagent, 0.1% sodium dodecyl sulfate (SDS), 15% formamide) and incubated at 65°C for 5–15 min. Microarrays were sealed with the HybriWell Hybridization Sealing System , which have a well depth of 0.25 mm. Static microarray hybridizations, i.e. without active mixing of the hybridization solution, were performed in a custom-made hybridization chamber Slides were scanned using a GenePix Personal 4100A Array Scanner  with a 3 line average and 10 µm resolution. Photomultiplier tube gain was adjusted to just below saturation intensity of the brightest signals and identical scanning settings were used for each experimental series. Scanned images were analyzed with GenePix Pro 6.0 software (Axon Instruments) and signal intensity surface plots of microarrays were generated using ImageJ with the Interactive 3D Surface Plot plug-in P. amoebophila whole genome array with oligonucleotide probes was produced in the same fashion as the simplified array and hybridized using fragmented labeled genomic DNA as a target n-lauryl sarcosine, 0.1% blocking reagent, 50 µg ml−1 salmon sperm DNA), and denatured at 95°C for 10 min. Slides were pre-hybridized with blocking reagent  for at least 2 hours at 42°C, and were then washed with double-distilled water and with isopropanol and finally dried by centrifugation. Slides were hybridized using sealed coverslips with a 0.25 mm well depth  under constant shaking at 400 rpm  for 16 h at 42°C. Slides were washed at 42°C with three serial washes of increasing stringency , rinsed with double-distilled water, and air-dried. Images were recorded by scanning the slides with a GenePix Personal 4100A array scanner . Hybridization images were used directly for creation of signal intensity surface plots with the ImageJ software.A www.ncbi.nlm.nih.gov/geo/) under accession number GSE26275.The microarray data have been deposited in the NIH National Center for Biotechnology Information's Gene Expression Omnibus database  at the spot surface. To show this explicitly, we present a formal scaling analysis that demonstrates the smallness of a dimensionless parameter, P, measuring the ratio of the hybridization timescale to the diffusion timescale.Extensions of Langmuir-based models to diffusion-reaction models are necessary to capture spatial variations because they incorporate transport processes C, isD is the diffusion coefficient of the target, r and z are the radial and vertical coordinates, k is the hybridization rate constant, and B is the concentration of probe. In equation (1), we have not included the dissociation of bound target strands because an assumption of irreversible hybridization kinetics is justified for perfectly complementary hybridization The diffusion-reaction equation for the concentration of target, R, the chamber height h, a reference probe concentration 0B, an (arbitrary) reference target concentration 0C, and the diffusion timescale 2/DR. Equation (1) can be written in terms of the dimensionless variables as,R<h  yields the condition, C = 0).The value of R = 50 µm, D/,R-2.)-−1. = 250 s, thus obtaining P = 5×,10-−5.<<1. The smallness of this value supports the conclusion – obtained by Gadgil et al. C = 0.For example, for the case considered by Gadgil et al. R = 75 µm. We used a diffusion coefficient of D = 1×,10-−9., m-2., s-−1., based on published estimates for single-stranded RNA k according to the Wetmur-Davidson equation 4., M-−1., s-−1. Although k is known to be sensitive to several factors 0B, we followed the method of Cheung et al. R and has a concentration BS, then dries to a disk of the same radius and height hD, while conserving the total amount of probe. Assuming Dh = 2 µm S,B = 50 µM from our experiments, this yields, 0B = (2/3),SB = 1.25 mM. Using these values, we find a reaction timescale P = 2.8×,10-−3.≪1. It is thus justified to assume that the spot acts as a perfect sink, where C = 0.The same result applies to the system under consideration here. We performed a careful analysis of the parameters involved by resorting to the relevant literature  understanding the dominant process responsible for hybridization heterogeneity, and (ii) exploring the role of geometrical parameters in determining the magnitude of this heterogeneity. For this purpose, we solved the three-dimensional unsteady diffusion equationL and height H, containing a square block of 30×30 spots that covers an area of M×M mm2. Each spot had a diameter of 150 µm and the inter-spot distance is 150 µm. Hence, the 30×30 spots covered 8.5×8.5 mm (M = 8.5 mm). Both the height of the chamber and the relation between M and L will be seen to be important in determining hybridization heterogeneity.Equation (4) was solved numerically using a finite-element code (COMSOL Multiphysics) for the dimensions of the simplified microarray , with thC = 1 in the entire chamber, i.e. a constant concentration of target (well-mixed). Because the diffusion equation is linear in the concentration, the solution is independent of the particular initial target concentration , unless the concentration is so high that individual spots become saturated (a case we do not consider here).Each simulation was carried out over 24 hours, with a diffusion coefficient of C = 0), consistent with reaction being considerably faster than diffusion, as detailed above. The flux was thus computed based on Fick's law as –DA(∂C/∂z), where ∂C/∂z is the gradient of target concentration normal to the spot and A is the surface area of the spot. This yielded the total amount of target that had been bound to the spot over the simulation time. At all other surfaces , which were impermeable to the target, no-flux boundary conditions were imposed.For each spot, the flux of target strands to the spot was calculated at each instant in time and subsequently integrated over the total time (24 h). For this, the boundary condition imposed at the spot was one of perfect absorption , differing from the real one only in the chamber height . The second models a large and deep hybridization chamber , to explore the effect of chamber height. The third case is that of a small and shallow chamber , to understand the effect of the amount of surface area not covered with spots. In all cases, the number of spots and the area covered by the spots are the same.Three different cases were considered. The first is the closest to the simplified array described above, with a large and shallow hybridization chamber . Increasing the target concentration reduced the boundary effect and could even quench it at high levels . The conThe variation of lateral mass transfer with position in the array raises the possibility that ratio signal intensities calculated from competitive two-color hybridization experiments, which for example are performed to measure relative differences in gene expression, may be sensitive to probe location when the competing target nucleic acids are present at different concentrations. To test this, we mixed identical targets labeled with either Cy3 or Cy5 at a 3∶1 ratio (900 ng/300 ng) and competitively hybridized them under standard conditions (18 h hybridization) . We obseBecause transport within the chamber of our simplified microarray occurs only by molecular diffusion and spots do not cover the chamber surface area uniformly , we hypothesized that the boundary bias results from the outer spots in the array having a larger supply of target strands coming from the outer spotless area. We thus developed a mathematical model based on Fickian diffusion to predict hybridization behavior for the simplified array design and found that it reproduces the fundamental features of the observed behavior. Inner spots get less of their mean share of the target strands from the outer, spotless area, because they are in the shadow of outer spots. This, then, leads to the predictions that (i) the amount of spotless area is an important determinant of the bias; therefore in the limiting case of a chamber that is entirely covered by spots, we expect no bias, and (ii) deeper chambers cause less bias, because greater depths alleviate the shadowing effect and favor a more equal partitioning of the target strands from the outer, spotless area to all spots.To test these predictions concerning the design of the hybridization slide and chamber geometry, we compared mathematical modeling results for three configurations : a largeAnother option for minimizing the boundary effect is to increase hybridization time to equilibrium, though this may be prohibitively long in cases where the target concentration is very low. Alternatively, hybridization systems with active mixing generally enhance target delivery and hold the potential to reduce diffusion limitations and thereby minimize the boundary effect. However, the mechanism by which active mixing is ensured differs greatly among the different hybridization systems (such as pressure-driven flow-through or surface acoustic wave micro-agitation systems) We observed and explained systematic spatial gradients in nucleic acid surface hybridizations, both experimentally and theoretically, and found that heterogeneity in lateral diffusive fluxes generate a consistent position-dependent bias in target signal intensity in single-color hybridizations as well as intensity ratios in competitive two-color hybridizations. A simple diffusion model supported the conclusions drawn from the experimental data and revealed that the geometry of the hybridization chamber and the probe spot area play an important role in determining the intensity of the spatial bias. The benefits realized from optimal adaptation of probe spot area to hybridization chamber size are a shift from diffusion-limited to reaction-limited conditions and increased microarray hybridization uniformity. Many commercially available hybridization systems with active mixing  are expensive and lack proof of a homogenous distribution of target molecules, which is required to effectively reducing diffusion limitation problems during hybridization. Our proposed strategy, adjusting the geometry of the hybridization chamber to the surface area of spotted probes or vice versa, is a relatively simple and cost-effective solution to diminish biases resulting from diffusion limitation and enhance accuracy of microarray-based quantification even without active mixing.


We then determined the sites undergoing selection during passage in Vero E6 cells. Four locations within the Ebola virus Kikwit genome were identified that together segregate cell culture-passaged virus and virus obtained from infected non-human primates. Three of the identified sites are located within the glycoprotein gene (GP) sequence: the poly-U (RNA editing) site at position 6925, as well as positions 6677, and 6179. One site was found in the VP24 gene at position 10833. In all cases, in vitro and in vivo, both populations (majority and minority variants) were maintained in the viral swarm, with rapid selections occurring after a few passages or infections. This analysis approach will be useful to differentiate whether filovirus stocks with unknown history have been passaged in cell culture and may support filovirus stock standardization for medical countermeasure development.To identify polymorphic sites that could be used as biomarkers of Ebola virus passage history, we repeatedly amplified Ebola virus (Kikwit variant)  Filoviridae) are etiological agents of severe viral hemorrhagic fever in humans and non-human primates with case-fatality rates ≈25–90%. The family Filoviridae includes two genera, Ebolavirus and Marburgvirus, to which five ebolaviruses  and two marburgviruses (Marburg and Ravn) are assigned, respectively Filoviruses  gene GP gene, which consists of a single open reading frame (ORF) and expresses a single glycoprotein , the ebolavirus GP gene expresses three products . This is achieved by the alternative use of three overlapping ORFs. In particular, the expression ratio of the three proteins is regulated via a stretch of seven uridylyls (7U) commonly referred to as the mRNA editing site. During replication in the host cell, a soluble glycoprotein of unknown function (sGP) is the primary expression product of the GP gene. GP1,2 is expressed only when an extra (eighth) adenylyl is inserted into the nascent mRNA via stuttering of the EBOV RNA-dependent RNA polymerase over the editing site. Likewise, ssGP, another soluble glycoprotein of unknown function, is produced when the polymerase adds two nontemplate adenylyls (or ignores a template U) 1,2 is a structural protein that forms trimers and localizes to the Ebola virion membrane. It is primarily responsible for attachment of the virion to its host cell-surface receptor and subsequent fusion et al. demonstrated that serial passage of a recombinant EBOV  containing a wild-type (7U) editing site in Vero E6 cells resulted in a viral population that predominantly contained an 8U editing site. However, when 8U virus was injected into guinea pigs, the population became predominantly 7U 1,2 and/or sGP. This study was targeted exclusively to the GP gene mRNA editing site and was performed using traditional clonal analysis.Recently, studies from Volchkova et al. are variant- and/or animal-specific and whether hotspots other than the mRNA editing site exist in EBOV subpopulations. This is an area of particular interest with the increasing use of animal data for the approval of therapeutics for human use when human trials are not possible. We focused the study on the interface between in vivo infection and in vitro propagation of viral stocks.For our study, we used deep sequencing to characterize the entire EBOV-Kik genome during viral passage in cell culture and after infection of NHP, one of the standard animal models for filovirus infection and currently the most highly regarded model for MCM research and development. The aim of the study was to determine whether the results of Volchkova Zaire ebolavirus, genus Ebolavirus, family Filoviridae, order Mononegavirales) was performed at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) at Fort Detrick, Frederick, MD, USA within maximum (biosafety level 4) containment. We used the only Kikwit isolate of Ebola virus (EBOV-Kik) available at USAMRIID, isolate 9510621 – this isolate had been used for all major USAMRIID NHP studies. EBOV-Kik 9510621 was originally isolated at the CDC Special Pathogens Unit from a sample taken from a female patient who died during the 1995 Ebola virus disease outbreak in Kikwit, Zaire (now Democratic Republic of the Congo). According to available records, the first and second passages of the virus were performed in grivet (Chlorocebus aethiops) kidney epithelial Vero E6 cells (ATCC #CRL-1586) to produce an initial virus stock, designated here as #135 (passage 2). After an additional passage in Vero E6 cells, the virus, designated #134 (passage 3), was sequenced at USAMRIID using classical methods by Chain et al. and Ichou et al. Near-complete genomic sequence (lacking 3′ and 5′ UTRs) was deposited in GenBank (accession # AY354458). Importantly, classical sequencing revealed an 8U mRNA editing site. We used three independent lineages of virus adaptation after passaging in Vero E6 cells from the original passage 2 stock, 135. Macaca fascicularis). Samples were harvested on days 5, 8, or 10 from animals challenged with viral stock 16502 (passage 3). These samples are referred to as M1D8, M2D10, M3D5, M3D8, M4D8, and M4D10. All viral samples were obtained from mock-treated animals; only samples with viral burden above ∼100,000 gc/ml were included.All work with infectious Ebola virus  and used for cDNA synthesis by sequence-independent single primer amplification (SISPA) 5 reads. Amplification (SISPA) primer removal, quality trimming, and trim-to-mer were performed on reads with a minimum similarity of 93% (four base mismatch). Single-nucleotide polymorphisms (SNPs) with fewer than 200 read depth were removed from the analysis. Only SNPs present in the population above the 2% threshold are presented in this report. A consensus change is defined here as a change relative to the published sequence for EBOV-Kik (GenBank accession # AY354458) present in ≥50% of the population. Below that threshold, SNPs are considered subclonal substitutions and part of a minority subpopulation of the virus. Because the sequence is derived by random hexamer amplification using the SISPA protocol, these results do not distinguish between genomic and messenger RNA.Viral assemblies were completed in DNAStar Lasergene nGen  with ≈4×10Near-complete genome reconstruction was achieved for all EBOV-Kik passages and for viruses in NHP serum samples. The assemblies generated were at a depth of ≈1,000 and coverage of >99% for all specimens . For puret al. We observed that, indeed, the EBOV-Kik 8U variant was the preferred state in vitro (passages from Vero E6 cells), whereas the 7U variant was the preferred state in vivo (virus obtained from infected macaques) , there was an inversion of direction of change. An equally rapid reversion was observed when this passage 3 (16502) stock was utilized to infect macaques. At day 5, the earliest available time point we were able to sequence, the reversion to the 7U variant was nearly complete. These findings provide evidence that the 7U variant is not only selected for in guinea pigs, as shown by Volchkova et al., but also in NHP, suggesting that a 7U mRNA editing site is a determinant for viral fitness in mammalian hosts. This hypothesis is further substantiated by the fact that the limited human-derived ebolavirus sequences deposited in GenBank to date, with the exception of EBOV-Kik, are 7U viruses. It remains unclear why the described selection takes place. A 7U to 8U switch likely reverses the expression ratio of sGP: GP1,2 from ≈80∶20 to ≈20∶80. An obvious conclusion is that during replication in vivo there is a strong selective pressure for the virus to avoid overexpression of GP1,2 and thereby the maintenance of a 7U mRNA editing site, although it cannot be ruled out that expression of sGP, whose function is still unknown, drives the selection. GP1,2 has been described by some groups to be cytotoxic, and indeed Volchkov et al. have suggested that mRNA editing is a mechanism to control the protein's cellular concentration 1,2 antibody decoy in vivo – low plasma sGP concentrations could therefore mean that more virions are neutralized, which of course would be detrimental to the virus Our results confirm and expand the observations of Volchkova GP gene at position 6,677  yet interestingly, the direction of change was reverse in these cases . The first change was observed again in the GP gene  1,2 signal peptide (residues 1–32) and the receptor binding site of the GP gene (residues 54–201) in vitro using EBOV-May Complete EBOV-Kik genome analyses also revealed several other instances of changes that segregate between VP24 gene  and converts VP24 residue Lys163→Arg  and does not segregate between in vitro and in vivo passage Table 2.in vitroin vitro showed the same kind and type of mutations in the editing site. The rapid conversion of EBOV-May/8U to the wild-type EBOV-May/7U in guinea pigs after only one passage was interpreted as a sign that EBOV-May/8U may be incapable of efficiently replicating in the model as a result of its increased capacity of express GP1,2 and/or decreased expression of sGP. Increased cytotoxicity caused by GP1,2 overexpression; a decrease in sGP expression was also linked to a higher host clearance, because it has been postulated that sGP has a decoy function to confound the immune function. Our data confirm the general hypotheses, although the quick change to the 7U variant at day 8 after challenge might favor the cytotoxicity hypothesis rather than the decoy function.The significance of these findings is puzzling. It is highly unlikely that the observed variants arise spontaneously each time this virus changes environments, however, these viruses were not plaque purified during their historical passaging and we were unable to obtain genetic material from original clinical sample material. Thus, the selection forces behind the positive and purifying pressures are unknown. Previous work showed that the rEBOV-May/8U variant possessed a growth advantage over the rEBOV-May/7U variant and that it might enhance the rate of virion release VP24 gene. Further work to characterize this change and its effect on virus replication is needed. While these biomarkers seem to be ideal for identifying “in vitro”-grown EBOV-Kik, work would need to be completed with other circulating strains to determine if this set of SNPs is robust enough to use across several ebolaviruses and their various variants.The remaining change is located to the Our study has important implications for MCM as candidate vaccines or antivirals cannot be tested against highly lethal agents, such as filoviruses, in controlled clinical trials during a disease outbreak due to obvious ethical concerns. Therefore, the development of medical countermeasures against filoviruses relies on data accumulated from appropriate animal model studies and assessment by the Food and Drug Administration (FDA) under the 2002 “Animal Efficacy Rule” (FDA 21 CFR 601.90). Recently, the FDA has been encouraging standardization of filovirus animal experiments across agencies and institutes to ensure that results can be properly compared, making filovirus stock standardization a heavily discussed topic. Moreover, there is a large amount of interest in viral resistance development. However, the timeline for resistance development is very short during filovirus animal models (∼8–10 days). It then becomes very important to look at the viral minority population for resistance development. Understanding the selection pressures on the virus during in vitro and in vivo passaging would be critical to assess the mechanisms of selection during treatment. The results presented here confirm data presented by Volchkova et al. (22), namely that EBOV-Kik evolves into genomically different, but defined, subpopulations depending on whether they are administered to animals or cell culture. This observation raises several interesting questions: 1) Is cell-culture adaptation dependent on cell type, i.e., does 7A Ebola virus necessarily evolve to 8A Ebola virus in all cell types, or is this a Vero cell- or non-human primate cell-specific phenomenon? 2) While there are no substantial differences between animal experiments performed with EBOV-May and EBOV-Kik, our results demonstrate several differences between the putative wild-type virus (no complete genome is available) and the viral seeds. Would it be desirable to use an Ebola virus genomically as close to a wild-type, i.e.,unpassaged p0?. 3) What are the scientific explanations for the different evolution of Ebola virus subpopulations in cell culture versus animals? While the evolution to 8A Ebola viruses in animals can easily be hypothesized to be due to a yet-unidentified immunological selection process, it is less clear why a 7A Ebola virus would be preferred by individual cells in culture. 4) Can the results presented here be extrapolated to all ebolaviruses and are similar selection processes at work when marburgviruses, which do not contain an editing site, are passaged through animals and cells? Answers to these questions from scientists, in dialogue with FDA specialists, may influence how filovirus stocks will be prepared and characterized in the future . They may also emphasize the need to accumulate more p0 filovirus genome sequences, of which there are currently few, for comparison studies.


Although louping ill affects mainly sheep, a 2011 outbreak in northern Spain occurred among goats. Histopathologic lesions and molecular genetics identified a new strain of louping ill virus, 94% identical to the strain from Britain. Surveillance is needed to minimize risk to domestic and wildlife species and humans. Flaviviridae, genus Flavivirus. Louping ill virus belongs to a subgroup antigenically related to viruses known as the tick-borne encephalitis viruses (TBEVs) of Europe (Ixodes ricinus. It mainly affects sheep and red grouse (Lagopus lagopus scotica), but many other species have been reported to be susceptible, including dogs, llamas, alpacas, goats, pigs, and humans  showed the same signs and died over a 4-month period. Many ticks were found on these animals (≈10–15 ticks/goat), and Butox  was applied.Necropsy was performed on 2 adult goats; gross lesions were recorded and special attention was paid to the nervous system. Samples for histopathologic examination were taken from the brain , spinal cord, liver, kidney, adrenal glands, lungs, spleen, and gastrointestinal tract. They were fixed in 10% neutral-buffered formalin, and 4-µm hematoxylin and eosin–stained sections were produced.Flaviviridae , treated with DNase 1 , and reverse transcribed into single-stranded cDNA by using random hexamers (First Strand cDNA Synthesis Kit for reverse transcription PCR (RT-PCR) [avian myeloblastosis virus]); Roche Diagnostics, Indianapolis, IN, USA). Real-time RT-PCR primers designed to detect all viruses in the family During necropsy of the 2 goats, no gross lesions were found. No histopathologic lesions were found in any organ system except the central nervous system . The cerMolecular genetic studies enabled identification of the virus. The sequence (Genbank accession no. JQ646028) was 94% identical to the strain from Britain (EU074000) and 93% identical to the strains from Spain (EU074016) and the Negishi virus (EU074002), thus confirming identity of the Asturian strain louping ill virus. Phylogenetic analysis, conducted by using the unweighted pair-group method analysis tree  shows hoHistopathologic lesions together with molecular genetic results enabled a definitive diagnosis: tick-borne encephalitis caused by an Asturian strain of louping ill virus. These cases confirm the infection in species other than sheep in Spain and the presence of the virus in areas of northern Spain other than the Basque region. An epidemiologic survey confirmed that no clinical signs were observed for the source herd in southern Asturias. However, a few years ago, another herd, located in the same area to which the affected herd described here had been moved, showed similar signs; 8 of 20 goats died. Although a diagnosis was not confirmed for this earlier outbreak, the facts suggest that the virus might have been in this area for several years.,The concern over finding louping ill in this area lies in its zoonotic potential. Numerous cases of human infection have been described (The Bermeya goat is considered to be at high risk for extinction, and many efforts have been made by breeders and the local administration of agriculture to limit the loss of genetic variability in these goats. The cases reported here provide an example of how an infectious disease can also reduce the local genetic resources. These cases, together with the case described in the Basque region, underline the need for a specific surveillance plan in northern Spain that focuses on ticks, wildlife species, and livestock. This plan will be crucial for determining the actual effects of louping ill on hunting, animal breeding, and human health.


Previously, WNV was serially or alternately passed 20 times in vivo in chicks or mosquitoes and resulting viruses were characterized genetically. In this study, these test viruses were competed in vivo in fitness assays against an unpassed marked reference virus. Fitness was assayed in chicks and in two important WNV vectors, Culex pipiens and Culex quinquefasciatus. Chick-specialized virus displayed clear fitness gains in chicks and in Cx. pipiens but not in Cx. quinquefasciatus. Cx. pipiens-specialized virus experienced reduced fitness in chicks and little change in either mosquito species. These data suggest that when fitness is measured in birds the trade-off hypothesis is supported; but in mosquitoes it is not. Overall, these results suggest that WNV evolution is driven by alternate cycles of genetic expansion in mosquitoes, where purifying selection is weak and genetic diversity generated, and restriction in birds, where purifying selection is strong.In nature, arthropod-borne viruses (arboviruses) perpetuate through alternating replication in vertebrate and invertebrate hosts. The trade-off hypothesis proposes that these viruses maintain adequate replicative fitness in two disparate hosts in exchange for superior fitness in one host. Releasing the virus from the constraints of a two-host cycle should thus facilitate adaptation to a single host. This theory has been addressed in a variety of systems, but remains poorly understood. We sought to determine the fitness implications of alternating host replication for West Nile virus (WNV) using an  in vivo in mosquitoes and chicks against an unpassed marked reference virus to determine whether replicative fitness gains or losses had occurred. Our results demonstrate that the trade-off hypothesis accurately predicts WNV adaptation in the avian environment but not in mosquitoes. Overall, our results suggest that WNV adaptation is controlled by alternating cycles of genetic expansion in a permissive mosquito environment and restriction in avians, where purifying selection is dominant.Arthropod-borne viruses, including West Nile virus (WNV) have long been considered to be subject to a fitness “trade-off” because they must replicate in at least two taxonomically divergent hosts in order to perpetuate. Results of studies testing this trade-off hypothesis have been inconsistent and largely dependent on which virus family is studied and which experimental system used. Therefore, considerable ambiguity exists in the literature regarding how host alternation influences virus population biology. Accordingly, we allowed WNV to adapt to each of its main hosts (mosquitoes and birds) for 20 sequential passes in order to determine whether host alternation in nature imparts a fitness compromise, as predicted by the trade-off hypothesis. After host-specialization, passed viruses were competed  Flaviviridae: Flavivirus) is an arthropod-borne virus (arbovirus) that has demonstrated remarkable success since being introduced to North America in 1999. Within three years after its introduction the virus had adapted to local mosquito vectors and within 8 years had become fully endemic West Nile virus , two of largest, most medically relevant families of arboviruses. Results of these studies are inconsistent and seem to differ between virus families  and experimental systems . Among flaviviruses, the trade-off hypothesis is often only partially supported. Host specialization frequently results in fitness increases; however, that these increases carry a fitness cost in the bypassed host is less well supported. Work in cell-culture with dengue virus (genus flavivirus) has shown that single-host-specialized virus replicated faster and reached higher titers in the specialized cell-line but reciprocal fitness losses were less extreme and inconsistent flavivirus) displayed improved fitness and more rapid replication in mosquito cells with only modest and inconsistent fitness losses in chicken cells In vivo studies with flaviviruses have also been difficult to reconcile with the trade-off hypothesis. For example, chick-specialized SLEV showed increased infectivity in chicks but was unchanged in mosquitoes, while mosquito-specialized virus was unchanged in both systems in vivo serial passage on fitness of WNV within biologically relevant hosts has been difficult to resolve because the determinants of virus fitness in either host (mosquitoes or birds) have been poorly understood. Recent advances have provided a more complete mechanistic understanding of in vivo fitness determinants that may shed light on phenomena previously attributed to “trade-offs”. For example, genetic diversification of WNV is driven by, and can circumvent, mosquito immune mechanisms Nonetheless, considerable ambiguity exists in the literature concerning the impact of host alternation on arbovirus adaptation and fitness. Importantly, neither the receptor-ligand interactions most important for virus entry and tropism nor the intracellular resources that might form the basis for host specialization in a putative fitness “trade-off” are well understood. Most studies of the trade-off hypothesis have involved either flaviviruses or alphaviruses  bird-specialized WNV, (b) mosquito-specialized WNV, (c) alternately passed WNV and (d) unpassed WNV, were competed against genetically marked WNV 20 times  [17], , then adsorbed onto confluent monolayers of Vero cells at room temperature for 60 min with continual rocking. Cells were then overlaid with cell-culture medium containing 0.4% agarose and maintained at 37°C with 5% CO