Detailed patterns of primary virus acquisition and subsequent dispersal in wild vertebrate populations are virtually absent. We show that nestlings of a songbird acquire polyomavirus infections from larval blowflies, common nest ectoparasites of cavity-nesting birds, while breeding adults acquire and renew the same viral infections via cloacal shedding from their offspring. Infections by these DNA viruses, known potential pathogens producing disease in some bird species, therefore follow an ‘upwards vertical’ route of an environmental nature mimicking horizontal transmission within families, as evidenced by patterns of viral infection in adults and young of experimental, cross-fostered offspring. This previously undescribed route of viral transmission from ectoparasites to offspring to parent hosts may be a common mechanism of virus dispersal in many taxa that display parental care. Protocalliphora azurea ) may have on the biology of a forest passerine migrant species, the European pied flycatcher ) Arthropods are well-characterized vectors of many viruses of plants and animals before they jump across individual hosts.Polyomaviruses are a group of small, double-stranded DNA viruses best known from mammals and birds Protocalliphora have ever been recorded parasitizing individuals other than nestling birds. In pied flycatchers, eggs laid in the nest by the adult fly will hatch into larvae that develop to the pupal stage (our sampling unit) by hiding in the nest material. During this period, the larvae will take intermittent blood meals from nestlings, and will then burrow into the nest cup to pupate The modes of polyomavirus acquisition in this study system are clearly limited by the known biology of its rather specialized, putative vector, as no larvae of Holarctic species of P. azurea, to assess its potential as vector of the virus. After dissecting the patterns of APV prevalence in adult and young birds in relation to ectoparasite prevalence, we performed an experiment to discard alternative routes of virus transmission across host generations.In this study, we evaluated whether the presence of an avian polyomavirus (APV hereafter) infecting nestlings of the pied flycatcher is associated with the presence of the nest ectoparasitic blowfly n = 568 and 48.5%, n = 641 in 2005 and 2006, respectively) and breeding females  and lower in breeding males  while a sex difference in virus prevalence did not exist at fledging age . The abundance scores of another nest ectoparasite  and all mites were negative for APV (n = 120).Prevalences of infection of APV in pied flycatcher blood were similarly high in nestlings in both study years . Broods also resembled their parents, females in particular, as to prevalence of APV .The striking association found in both study years suggests that the transmission of the virus occurs from blowflies to offspring, and subsequently from offspring to parents, and not in the opposite direction, as virtually no nestlings from uninfested nests were infected with the virus , rather than their genetic parents . This result indicates that APV is not passed to offspring from the mother, as occurs in mice The correspondence between the prevalences of blowflies and APV, on the one hand, and between APV infections in nestlings and their parents, on the other, points to genetic and/or environmental sources of resemblance between parents and offspring, e.g. in susceptibility or common exposure to APV infections, as potential sources of confusion in the interpretation of the routes of transmission of APV in this system. We therefore performed a cross-fostering experiment of whole clutches to test the hypotheses of parent-offspring similarity in patterns of APV infection due to vertical transmission of APV through the egg P. azurea. Analyses of 40 live blowfly larvae whose oral region was dissected and screened for APV presence supported the inferred pattern of virus transmission to nestlings, with 100% of foregut samples of the larvae exhibiting positive evidence for APV presence, most probably due to the likely installation of the virus in the salivary glands of its vector. APV was more rarely detected in the hindgut samples of the same blowfly individuals .Therefore, both natural patterns of infection in parents and offspring and experimental evidence with cross-fostered broods point to a common environmental primary source of infection, most likely the larvae of n = 40 nestlings from 26 nests) while APV was not isolated in feces from nestlings which had no detectable APV presence in blood . This excludes the possibility of nestlings showing APV infection in the digestive tract with APV passing unnoticed in blood. Both sexes remove fecal sacs in the pied flycatcher but, as in other bird species Our results point to the most plausible route of APV infection from nestlings to both their male and female parents as being through the parents' nest sanitation tasks. This is clearly supported by the finding that most APV-positive nestlings from nests infested by blowflies were actively shedding the virus in the sampled feces , brooding females could also be directly infected by attacks from blowfly larvae, as seemingly apparent from the increase in APV prevalence in females with the greater number of blowfly larvae counted in their nests. However, neither our long-term hands-on experience (23 years) with flycatchers nor the literature support the possibility that blowfly larvae ever attach to adult birds. Furthermore, excluding common infections as nestlings, a similar but weaker relationship in male flycatchers cannot be explained by the same mechanisms as in females, because males do not incubate or brood nestlings To our knowledge, larvae of only when their nest was infested by blowflies. However, this circle on the ‘true’ source of virus infection could be addressed at different levels of explanation. One hypothesis is that the non-parasitic, adult female flies may transmit the APV via vertical transmission through their eggs , and then throughout the larval  stages to the adults  as known for some arboviruses P. azurea. We think this issue might be most profitably addressed through phylogenetic analysis of APV variants within the family Calliphoridae and, ideally, also within its sister group, flies in the family Sarcophagidae The design and results of this study only allow us to speculate on where the APV infection originates, as all blowflies were positive for APV and the virus was only detectable in their primary  host, the nestlings, when and In conclusion, nest ectoparasites of birds transmit polyomaviruses to nestlings, which in turn pass them on to their parents. To our knowledge, this is the first known natural example of a primary, rather than sporadic, route of upward transmission of a potential pathogen from offspring at an early ontogenetic stage to adult individuals. This route of infection may reveal itself as a common mechanism of virus transmission in the many taxa that exert parental care and/or feed and preen their offspring and thus might be a hitherto unnoticed n = 273) and trapped almost all breeding males and females within their nests while they fed nestlings aged 8–11 days. Nestlings were sampled at 13 days of age. A drop of blood was extracted from the brachial vein of all individuals and stored frozen in EDTA for molecular sex determination of nestlings The study was conducted in 2005 and 2006 in an intensively studied population of pied flycatchers breeding in nest boxes in central Spain n = 94 nests, i.e. 47 dyads) were at least 1 km apart. Cross-fostered eggs were replaced by the same number of rubber, blue-painted canary egg dummies mimicking size and color of pied flycatcher eggs to avoid desertion by the females during clutch exchanges. In all cases, females ‘incubated’ the egg dummies during the time needed for transportation, as indicated by direct observation or our estimated temperature of egg dummies. Eggs were kept safe and warm in water-heated containers  and transported by car in about 20 to 30 min. As a result of our procedure, all pairs of exchanged nests contained broods reared by totally unrelated adult birds. Final sample sizes were unbalanced due to some nests being lost to predation or because we were unable to trap the parent(s).In 2006, we exchanged all eggs in the second day of incubation between matched pairs of nests of the same (±1 d) breeding date and clutch size. Experimental nest dyads  were calculated by using 10 increment copies, so 0 is 0 copies detected, 1 from 10 to 102 copies; 2 from 102 to 103 copies, 3 from 103 to 104 copies; 4 from 104 to 105 and, finally, 5 from 105 to 106 copies. Detection of APV in nestling feces, blow fly larvae and Dermanyssus mites was conducted by using a classical PCR assay Blood samples from adult and nestling pied flycatchers were tested for the presence of APV by means of a sensitive and specific real-time quantitative PCR (RQ-PCR) assay. Following previous work with a murine polyomavirus


Tick-borne encephalitis virus (TBEV) causes human epidemics across Eurasia. Clinical manifestations range from inapparent infections and fevers to fatal encephalitis but the factors that determine disease severity are currently undefined. TBEV is characteristically a hemagglutinating (HA) virus; the ability to agglutinate erythrocytes tentatively reflects virion receptor/fusion activity. However, for the past few years many atypical HA-deficient strains have been isolated from patients and also from the natural European host tick, Ixodes persulcatus. By analysing the sequences of HA-deficient strains we have identified 3 unique amino acid substitutions  in the envelope protein, each of which increases the net charge and hydrophobicity of the virion surface. Therefore, we genetically engineered virus mutants each containing one of these 3 substitutions; they all exhibited HA-deficiency. Unexpectedly, each genetically modified non-HA virus demonstrated increased TBEV reproduction in feeding Ixodes ricinus, not the recognised tick host for these strains. Moreover, virus transmission efficiency between infected and uninfected ticks co-feeding on mice was also intensified by each substitution. Retrospectively, the mutation D67G was identified in viruses isolated from patients with encephalitis. We propose that the emergence of atypical Siberian HA-deficient TBEV strains in Europe is linked to their molecular adaptation to local ticks. This process appears to be driven by the selection of single mutations that change the virion surface thus enhancing receptor/fusion function essential for TBEV entry into the unfamiliar tick species. As the consequence of this adaptive mutagenesis, some of these mutations also appear to enhance the ability of TBEV to cross the human blood-brain barrier, a likely explanation for fatal encephalitis. Future research will reveal if these emerging Siberian TBEV strains continue to disperse westwards across Europe by adaptation to the indigenous tick species and if they are associated with severe forms of TBE. Flavivirus within the family Flaviviridae. Human pathogens within the genus Flavivirus include Japanese encephalitis virus, Dengue virus and Yellow fever virus that cause annual epidemics of fever, encephalitis and hemorrhagic fever in the tropics and some sub-tropical regions Tick-borne encephalitis virus (TBEV) causes up to 14,000 human cases of tick-borne encephalitis (TBE) across Eurasia annually In its natural habitat, TBEV is maintained by transmission between infected and non-infected ticks when they co-feed on small forest animals Conventionally, each TBEV subtype has been associated with distinct geographic ranges within the Old World region of the northern hemisphere, hence the groupings Far East, Siberia and Western Europe TBEV virions are spherical particles with an ∼11 kb-RNA genome embedded in a capsid that is surrounded by a lipid envelope mainly containing a virus envelope (E) glycoprotein. This E protein plays a key role in many stages of the virus life cycle; it mediates virus binding to receptors on the cell surface (adsorption) which triggers receptor-mediated endocytosis. Exposure of the endocytosed virus to the acid pH converts the native E protein dimers into fusogenic trimers Ixodes ricinus ticks reflecting altered, E protein-mediated, receptor/fusion functions. We also illustrate how this might result in continued westward dispersal and emergence of new highly pathogenic virus variants  strains, in contrast to the more common antigenically competent (AC) strains; the term “AD” is derived from the strict correlation between loss of HA and immunoprecipitating activities ants see .Yar-strains of TBEV i.e. Yar71, Yar 114, Yar 46-2, and Yar 48 were isolated in the European part of Russia (Yaroslavl' region) between 1999–2001 and details for their isolation are listed in Phylogenetic analyses based on a 1110-bp fragment of the E gene (positions 1114–2224 in Vs virus L40361) showed that the Yar-viruses belong to the SIB subtype of TBEV To identify amino acid(s) responsible for the loss of HA-activity, we sequenced Yar viruses and aligned them with 290 available TBEV E sequences . One amiI. ricinus ticks H. marginatum ticks The loss of HA activity in TBEV has only previously been reported in relation to selective adaptation of TBEV to ticks. One substitution, E87K was generated during the propagation of a WE strain to Whilst the molecular details of interactions between virions and erythrocytes remain unknown it has been suggested that HA activity might be mediated by the trimeric E protein in its post-fusion conformation rather than by native E dimers Thus, we hypothesised that each of these three amino acid substitutions could individually abolish HA deficiency in TBEV. To test this hypothesis experimentally we used a TBEV infectious clone to engineer three mutant viruses IC-D67G, IC-E122G and IC-D277A See  that simAll three engineered mutants demonstrated delayed growth in PS cells, most visible during the first 24 hours . NeverthAll 3 natural HA-negative isolates  displayed small-plaque phenotype 1 mm, , in compVs virus is different from many other laboratory-maintained TBEV strains; in cell culture it develops cytopathic effect (cpe) relatively slowly After ip inoculation, the control virus pGGVs produced a relatively high morbidity rate (65%) in correspondence with previous results nd, 7th, 14th and 21st day following infection. The reproduction characteristics of IC-E122G and IC-D277A in fasting ticks were similar to those of control pGGVs virus whereas the titres of IC-D67G were significantly lower  residues that were replaced by hydrophobic and/or neutral amino acids (i.e. glycine) and localized on the virion envelope protein surface . In totaNotably, the substitution D67G was detected in 7 viruses isolated only from mammalian hosts and human patients. No other correlation between isolation source, geography or virus subtype specificity was observed. A significant number of TBEV E proteins have been sequenced partially (227 viruses of 290 available from GenBank), therefore it could not be excluded that TBEV strains with increased surface charge/hydrophobicity are quite common in nature. Indeed among 63 completely sequenced E proteins those with increased charge/hydrophobicity on the virion surface comprise ∼20% (including Yar viruses).I. ricinus (WE-strains) with those isolated from I. persulcatus (FE- and SIB-strains), to identify group amino acids that might be involved in TBEV adaptation to these two different tick species (I. ricinus–transmitted WE-strains in contrast with FE-and SIB-strains (I. ricinus or I. persulcatus) D277E amino acid substitution overlapped with the D277A substitution of Yar 48  or on the membrane-oriented side (S267A), i.e. under the virion surface. Four substitutions  were located in the transmembrane domain of the E protein, with one (T427A) more hydrophobic for “ricinus” strains. Louping ill virus (LIV), a TBEV-related virus which is transmitted by I. ricinus in the UK, also showed the same more hydrophobic pattern and localisation of “tick-specific” amino acids as the I. ricinus-transmitted WE TBEV strains  and three non-EU Member States  Here, we have investigated the molecular mechanisms and epidemiological implications of the emergence of unusual TBEV strains originally identified as HA- or AD-deficient I. ricinus ticks can be routinely maintained in laboratories, SIB TBEV is normally associated with transmission by I. persulcatus, which is not as readily available for laboratory experiments. Nevertheless, WE TBEV strains show ∼100% transmission efficiency in their natural tick vector I. ricinus (manuscript in preparation). Thus, logically, Yar viruses (i.e. SIB TBEV strains) should be transmitted efficiently in their natural vector I. persulcatus. Geographically, I. ricinus and I. persulcatus overlap in Europe and numerous reports describe the isolation of Siberian strains from I. ricinus which is now recognised as a second vector for SIB TBEVs I. ricinus ticks. This hypothesis is also supported by our analysis of E protein comparative alignments between FE, SIB and WE subtypes that revealed more hydrophobic “ricinus” amino acid patterns, compared with “persulcatus”, in correspondence with tick preference of WE or FE/SIB strains respectively. Louping ill virus, which is transmitted by I. ricinus in the UK, shares this “more hydrophobic” pattern with the WE TBEVs.Although For many viruses, HA-activity is recognised as a reflection of receptor I. persulcatus or SIB-strains into I. ricinus. Clearly these barriers to infection are not absolute since Vs virus (SIB TBEV) has a limited capacity to replicate in I. ricinus  were used to produce TBEV stocks, to recover mutant viruses, for plaque assay and studies of cytopathogenicity. Yar-strains of TBEV i.e. Yar71, Yar 114, Yar 46-2, and Yar 48 were isolated in the European part of Russia (Yaroslavl' region) between 1999–2001  and storThe RNA of each Yar virus was extracted from 200 µl of 10% infected mouse brain suspension or infected PS cell supernatant using Total RNA Isolation System (PROMEGA). The RT-PCR was used to amplify the 5′-C-prM-E gene region of Yar viruses as described Nucleotide and deduced amino acid sequences were aligned using program package BioEdit http://www.pymol.org).The unique amino acid substitutions in the E protein of each Yar virus were mapped onto the crystal structure of the native dimeric The routine protocols for cpe, plaque assay and growth curve experiments were described in detail previously 6 PFU/ml were concentrated 100 times using 7% polyethylene glycol (PEG) in the presence of 2.4% NaCl overnight at 4°C and precipitated by centrifugation at 8000 rpm for 3 h. The resulting pellet was resuspended in 500 µl of PBS. For routine hemagglutination test 8 PFU/ml) were placed in the first well of 96-well plates and diluted two-fold in borate buffer (pH 9.0). Then 100 µl of a suspension of 0.5% newborn chick erythrocytes was added to each well. PS cells were infected at a moi of 0.1 PFU/cell in 500 ml culture flasks and infectious supernatant medium was collected at day 5pi. The TBEV virions with estimated initial virus titers of 2−8×10in vitro of two overlapping plasmids, pGGVs660–1982H and pGGVs660–1982del produces full-length infectious clone of TBEV strain Vs 660–1982H  DNA strand originating from template bacterial dsDNA and the other one (nicked) from newly amplified PCR product. Following 15 cycles, the accumulation of amplified linear complementary ssDNA strands resulted in the formation of annealed circular (twice nicked) molecules. To facilitate screening, the dam-methylated bacterial (template) DNA was digested with 40 U of DpnI (New England Biolabs) at 37°C for 1 h. Following this, PCR products were electroporated into AbleK bacterial cells (Stratagene) and selected clones were completely sequenced.The ligation 60–1982H . For the660–1982H, were ligated with pGGVs660–1982del to restore the full-length cDNA of TBEV as described previously I and used for SP6 transcription to produce full-length RNA. The SP6-transcribed RNA was transfected into PS cells using Lipofectin reagent (Invitrogen) according to the manufacturer's protocols. Infectious supernatant medium was collected on day 5 pi. The presence of virus in infected cells was confirmed by immunofluorescence microscopy using monoclonal antibodies specific for flavivirus E proteins To recover engineered viruses, the mutated plasmids, constructed on the basis of pGGVsI. ricinus ticks were inoculated with virus under a stereo zoom microscope  into the coaxial plate of the second pair of legs using a digital microinjector TM system . Clean nitrogen served as a gas source to produce an injection pressure of 20 psi ( = app. 1.38bar). The injection interval was set to 1.0 sec. Hollow glass needles with a microscopically fine tip were prepared using a P-30 Micropipette puller .Unfed adult females of To investigate virus reproduction in fasting ticks, groups of 45 female ticks were infected with 500 PFU/tick of one TBEV strain. Infected ticks were incubated at room temperature (24±4°C) and 85–90% RH in a desiccator for 21 days. At 2, 7, 14 and 21 days pi salivary glands of 6 ticks were dissected, individually homogenized and the concentration of infectious virus was estimated by plaque titration.I. ricinus nymphs that were attached in close proximity (1–1.5 cm) to the feeding donor females. Surviving nymphs and salivary glands of donor females were used to determine the titres of infectious virus using plaque assays. The co-feeding transmission rate was estimated as the proportion of nymphs (%) that became infected.Virus tick-to-tick transmission experiments were carried out essentially as described previously Experimental animal procedures were performed in accordance with the guidelines for care and maintenance of animals . All animal experiments were approved by the State Veterinary and Food Administration of the Slovak Republic (permission numbers 12284/03-220 and 2362/06-221). The ethical permission to carry out the work with mice was obtained from the Ethical Review Committee of the Institute of Virology, Slovak Academy of Sciences”.Ten adult ICR mice were inoculated intraperitoneally (ip) with 2000 PFU/mouse. Mice were observed for 21 days and morbidity rate was estimated as the proportion of animals that showed clinical symptoms that included hind-leg paralysis. Sick and healthy mice were tested for the presence of anti-TBEV antibodies by HA-inhibition test Statistical analysis was performed on the data obtained from virus replication studies in PS cells and ticks, neutralization and neuroinvasiveness test using EXCEL and SigmaPlot 11 software . Standard errors of mean (SEM) were estimated for each dataset. Between-groups comparisons were performed using unpaired, two-tailed Student's t-test. Values of p<0.05 were considered as significant.Figure S1Cartoon illustrating the molecular mechanisms of emergence of new strains of TBEV with possible pathogenic characteristics for humans.(10.14 MB TIF)Click here for additional data file.


Diabetic retinopathy is one of the most common microvascular complications of diabetes and one of the major causes of adult visual impairment in national surveys in Taiwan. This study aimed to identify the healthcare costs of Taiwan's National Health Insurance program on behalf of diabetic patients with stable or progressive retinopathy.A retrospective cohort study was conducted with 4,988 medication-using diabetic retinopathy subjects ≥ 40 years of age under National Health Insurance Program coverage between 2000 and 2004. Study cohort subjects were recorded as having diabetic retinopathy according to ICD-9-CM codes. States of diabetic retinopathy were strategically divided into stable and progressive categories according to subjects' conditions at follow-up in 2004. Expenditures were calculated and compared for the years 2000 and 2004.During the 4-year follow-up (2000 through 2004), 4,116 subjects (82.5%) of 4,988 diabetic subjects were in the stable category, and 872 (17.5%) were in the progressive category. Average costs of those in the normal category increased by US $48 from US $1921 in 2000 to US $1969 in 2004 (p = 0.594), whereas costs for those progressing from normal to non-proliferative diabetic retinopathy (NPDR) or proliferative diabetic retinopathy (PDR) increased by US $1760, from US $1566 in 2000 to US $3326 in 2004 (p < 0.001). The PDR category had the highest average costs at US $3632 in 2000. The NPDR-to-PDR category experienced the greatest increase in costs at US $3482, from US $2723 in 2000 to US $6204 in 2004 (p = 0.042), and the greatest percentage of increase at 2.3% .This large-scale longitudinal study provides evidence that increased healthcare costs are associated with progressive diabetic retinopathy among diabetic NHI enrollees in Taiwan. Diabetes mellitus is a common, chronic disease among adults, with a worldwide prevalence estimated at 4% in 1995 and projected to rise to 5.4% by the year 2025 [Healthcare costs for diabetics are more than double the costs of those without diabetes in the United States  and the Increased healthcare costs attract the attention of policy makers seeking strategies to reduce medical costs. Despite the enormity of the problem, nationwide data about the costs of treating diabetic patients with diabetic retinopathy are still limited. A common source of administrative data is health insurance claims data associated with private or public health insurance programs. In Taiwan, 96% of all residents have been enrolled in the National Health Insurance (NHI) program since 1996. In this study, we used these administrative claims data to identify the costs of the NHI program paid on behalf of diabetic patients with either stable or progressive retinopathy.In Taiwan, the National Health Insurance (NHI) Program began on March 1, 1995. As of 2007, 22.6 million of Taiwan's 23 million persons were enrolled in this program. Therefore, the NHI Program has accumulated 23.8 million administrative and claims records. In order to respond to current and emerging issues rapidly and effectively, the National Health Research Institutes (NHRI) cooperated with the NHI Bureau (NHIB) to establish and maintain an NHI research database. The NHRI safeguards the privacy and confidentiality of subjects and routinely transfers insurance health data from the NHIB to health researchers for analysis, with the aim of improving the health of Taiwan's citizens. Data analysed in this study were also retrieved from the National Health Insurance Research Database. Access to research data has been approved by the Review Committee of the NHRI. The NHRI provides a unique validated database of medical claims from 200,432 random subjects, representing about 1% of the population, for use in health insurance studies. These data consisted of inpatient and ambulatory care records, as well as registration files, and resulted in the establishment of an academic, retrospective cohort group. This academic cohort database has been tested and no notable differences were found in age, sex, or medical costs for all enrollees . LikewisSince 1999, the NHRI database has used the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) for outpatient and inpatient diagnostic coding. By searching these electronic records and pharmacy prescriptions in similar fashion to a previous study , individAll estimates of medical costs, including copayments, were calculated in New Taiwan dollars (NT$). Fee-for-service costs included outpatient and inpatient care, prescription drugs, and laboratory fees. Costs represented healthcare expenditures for an individual for the whole year, not focusing exclusively on those associated with diabetes or diabetic retinopathy. Outpatient services included outpatient visits, emergency department visits, end-stage renal dialysis, preventive services, dental care, home healthcare, and traditional Chinese medicine treatments. Inpatient care costs were calculated over a one-year period in both 2000 and 2004. The costs in this study did not include services that might have been provided but were not covered by NHI. Indirect costs of diabetes such as lost productivity due to disability or premature death were not estimated.Excess costs associated with changes in diabetic retinopathy were measured and compared as average annual costs for 2000 and 2004. Changes in annual costs associated with diabetic retinopathy were estimated by comparing mean medical costs in 2000 and 2004 for the two main categories of stable and progressive retinal status. These included two subcategories in the stable group , and three subcategories in the progressive group .t test because claim years for the same individual were assumed to be inherently related.The statistical significance of differences identified in mean costs was evaluated using the paired In our study population, the number of medication-using diabetic subjects ≥ 40 years of age was 4,988 under NHI coverage in 2000. The expenditures of all subjects were calculated and compared for the years 2000 and 2004. The characteristics of these subjects are presented in Table Table Table The normal category in both 2000 and 2004 had the lowest costs, and smaller relative increases in costs from 2000 to 2004. The PDR category had the highest average costs of NT$117,630 (US $3632) in 2000, but the NPDR-to-PDR category experienced the greatest increase in costs , from NT$88,182 (US $2723) in 2000 to NT$200,961 (US $6204) in 2004. Regarding the percentage of increased costs between 2000 and 2004, the normal-to-NPDR-or-PDR category was 2.1%  and the NPDR-to-PDR category was 2.3% (adjusted to 2.2%), which were relatively higher than those in the stable group (Table Our current study is a longitudinal study that aimed to provide cost estimates associated with different severities of diabetic retinopathy in Taiwan. The number of medication-using diabetic subjects ≥ 40 years of age was 4,988 under NHI coverage in 2000. Of these 4,988 diabetic subjects, 82.5% were in the stable category, and 17.5% were in the progressive category during the 4-year follow-up. The normal category had the lowest costs and smaller relative increases in costs. The PDR category had the highest average costs in 2000, but the NPDR-to-PDR category experienced the greatest increase in costs from 2000 to 2004.Any analysis of claims data is subject to significant limitations that have been well defined in the literature ,17. HoweIn Taiwan, the direct costs of healthcare for diabetic patients was 11.5% of the total national healthcare costs and was 4.3 times higher than the average cost of care for non-diabetic individuals from 1997 to 1998 .In our study, diabetic subjects with diabetic retinopathy in 2000 had higher 2000 costs than those diabetic subjects with a normal retinal status in a cross-sectional view, but the normal-to-normal category had smaller relative increases in costs from 2000 to 2004 in a longitudinal view. One cross-sectional study conducted in Germany in 2002 also revealed that costs associated with diabetic retinopathy tend to increase as diabetic retinopathy progresses, being highest in patients with proliferative diabetic retinopathy and lowest in patients with mild, non-proliferative diabetic retinopathy . One stuIn our study, subjects with proliferative diabetic retinopathy had the highest average costs in both 2000 and 2004, and subjects who progressed from NPDR to PDR experienced the greatest increases in costs. This evidence implies that strategies aimed at preventing and mitigating diabetic complications such as diabetic retinopathy are likely to be relative to long-term reductions in medical costs.We acknowledge several limitations in our study. First, our analysis only included costs of medical care incurred by the NHI program in Taiwan. Second, we did not adjust for co-morbid diseases, and just focused on the relationship between overall medical costs and severities of diabetic retinopathy. Third, technological changes and economic inflation constitute a dynamic process. Diagnosis and treatment in 2000 may be somewhat different than that in 2004 as well as into the future, and cost escalation due to inflation may also be a factor. Both the results of absolute excess costs and relative percentage of increased costs should be taken into consideration. Fourth, misclassification bias in both the diagnosis and the grading of diabetic retinopathy should be considered when using claims data.In conclusion, we provide nationwide, large-scale, and longitudinal evidence that increased healthcare costs are associated with progressive diabetic retinopathy among diabetic NHI enrollees in Taiwan. It implies that strategies aimed at preventing and mitigating diabetic complications such as diabetic retinopathy are likely to be relative to long-term reductions in medical costs.The authors declare that they have no competing interests.PC and WHT participated in the design of the study and performed the statistical analysis. LCW and CYT have made substantial contributions to the study concept and design, or acquisition of data, or analysis and interpretation of data. HKC, MTT and STS conceived of the study, and helped to draft the manuscript or revise it critically for important intellectual content. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/10/136/prepub


Adult CBA mice thymectomized, treated with antilymphocytic globulin (ALG) and inoculated with human leprosy organisms were accidentally infected with polyoma virus and all developed tumours. After cessation of ALG administration, some animals were given spleen cells from syngeneic donors immunized with polyoma virus; none developed tumours. Similar results were obtained in mice deliberately infected with polyoma virus but not with leprosy organisms. Passive transfer of antibody before but not after virus inoculation prevented tumour formation in immunosuppressed recipients. Virus infection in thymectomized, lethally irradiated and bone marrow reconstituted mice resulted in only a very low incidence of tumours. These results emphasize the role of immunological surveillance in preventing polyoma tumour formation under natural conditions.


Near term infants are a main part of preterms. They are at higher risk for mortality and morbidity than term infants and could show a quite different development of tone and reflexes from them. The aim of the present study was to describe longitudinally, in a large sample of healthy near term infants, the development of the forward parachute reaction (FPR) and its correlation with the age of acquisition of independent walking.The assessment of FPR  was performed at 3, 6, 9, 12 months of corrected age in 484 infants, with a gestational age between 35.0 and 36.9 weeks. The age of acquisition of independent walking was monitored until its appearance. A correlation analysis was done between the age of walking and the acquisition of a complete or incomplete FPR, using the Spearman Rank correlation. The Mann-Withney U test was used to identify significant gestational age differences for the age of FPR appearance.Most of infants had a two-step development pattern. In fact, they showed at first an incomplete and then a complete FPR, which was observed more frequently at 9 months. An incomplete FPR only, without a successive maturation to a complete FPR, was present in the 21% of the whole sample. Infants with a complete FPR walked at a median age of 13 months, whereas those with an incomplete FPR only walked at a median age of 14 months.We identified two groups within our sample of near term infants. The first group showed a progressive maturation of FPR, whereas the second one was characterised by the inability to get a complete pattern, within the one year observation's period. Furthermore, we observed a trend toward a delayed acquisition of independent walking in the latter group of infants. During the last two decades, the population of near term infants (34 weeks through 36 weeks of gestation) is increased progressively and it is actually the 70% of the whole population of preterms -3. ImproThe forward parachute reaction (FPR) is a milestone "key" for the neuromotor development of infants. In fact, several authors showed that infants need a previous acquisition of this reaction before standing and walking ,8. In clth percentile), major congenital malformations, severe postnatal infectious diseases, metabolic or haematological complications and variable follow-up program. The study protocol was previously approved by the Ethics Committee of our Institution and a signed informed consent was obtained from parents.The infants described in this study are a part of a large cohort admitted to the Neonatal Unit of the Department of Paediatrics of the University of Catania between January 2001 – December 2005. They were consecutively enrolled in a follow-up research program until the age of 2 years. Although the near term well babies are not likely to stay at hospital longer than a few days, our follow-up research program included one assessment for cranial ultrasound (US) during the first week, one after 14 days and one at term age. The last two US scans were done after discharge from the hospital. As a part of the follow up program, neurological development was systematically followed with a structured neurological assessment, performed at 3, 6, 9 and 12 months of corrected age (CA) , and a nThe FPR was assessed at 3, 6, 9, 12 months CA in all infants included in the present study.FPR was evoked by holding the infant by the hips in a prone position  and by dropping him suddenly downwards at 45 degrees with the head directed towards a surface with the eyes open. The manoeuvre was applied when the infant was in the most appropriate behavioural state, corresponding to state 4 of Brazelton  . It was The age of onset of independent walking was defined as the time when the infant took the first five successive steps without support . The parValues are reported as median, minimum-maximum and 25°–75° percentiles. For analysis purposes, infants were divided in two groups. A first group (group A) was composed by infants who gained a complete FPR within the observation's period; in this group are enclosed those infants showing an incomplete FPR followed by a complete FPR too. A second group (group B) was represented by those who had an incomplete FPR only. A third group of infants contemplate those infants with no FPR (both incomplete or complete) within the first year of life; because of the limited number of this group, it was not enclosed in the statistical analysis. The number of infants who had a complete or incomplete FPR at 3, 6, 9, 12 months were calculated. A correlation analysis was done between the age of walking and the acquisition of a complete or incomplete FPR, using the Spearman Rank correlation. The Mann-Withney U test was used to identify significant GA differences for the age of FPR appearance. The level of significance was set at p < 0.05.During the study period, 648 consecutive newborns with a GA between 35,0 and 36,9 weeks were discharged from our Neonatal Unit; from this population 164 babies were excluded because of major lesions at US scanning (n = 86) or an incomplete follow-up program (n = 78); 484 infants fulfilled the inclusion criteria; 228 were born at 35 weeks and 256 at 36 weeks GA. A Normal cranial US was found in 415 infants, whereas transient flares or IVH 1 were respectively observed in 59 and 10 infants. The 51% of this sample was male. No significant differences (p > 0.05) between infants born at 35 and those born at 36 weeks were found for the age of acquisition of FPR. Infants with minor lesions on US showed similar findings to those observed in the group with normal US. The results of neuromotor assessments performed during the first year of life are reported in a previous paper [In Fig. Fig. All infants showed an independent walking within 17 months of age, with a median of 13 months. Infants with a complete FPR at 3 months developed precociously an independent walking at a median age of 10 months, whereas those who had a complete FPR at 6, 9 and 12 months walked respectively at a median age of 12, 13 and 14 months .The correlation analysis (r) between the age of acquisition of an independent walking and the age of a complete FPR by 12 month was 0.32 (p < 0.0001) whereas for those with an incomplete FPR only, Our study investigated the development of FPR in a large sample of near term infants, during the first year of life. We found that most of infants showed a complete FPR as previously shown both in term  and pretThe possible physiological background of FPR was proposed by Touwen , who sugThe incomplete FPR is characterised by an incomplete extension and/or by an asymmetric extension of the upper limbs and/or of closed hands ,10. Its An interesting result of our study is the presence of a small subgroup of infants who develop an early incomplete reaction (at 3 months) showing an independent walking around 12 months. These infants were not able to gain a complete FPR within the first year of life and, in the same line of evidence reported by other authors ,8, it seThe systematic observation of the sensory-motor development of infants has shown that it occurs according to a timed calendar. In fact, a normal infant acquires the sitting position at about 6 months, on all-fours at 9 months, supported walking at 12 months and independent walking between 12–15 months ,8,17,18.Then, our study shows for the first time not only the features and the development of FPR in near term infants, but also the temporal relationships between the development of an incomplete FPR and an independent walking.A peculiar characteristic of our sample is represented by the high number of infants who were recruited for the study. In fact, it seems to be sufficiently consistent, from a statistical point of view, to follow the development of FPR during the first year of age. Since, the infants enrolled for this investigation were near term babies only, it is not possible to extend our observations to healthy infants born at term age. However, we can conclude by indicating that, in our near term sample, a milestone "key", as a complete FPR, follows a development quite overlapping to that of term infants and similarly correlated to the acquisition of independent walking. On the other hand, we should consider two main limitations of this study. Firstly, the fact that our observation's period was limited to the first year of age and secondly that the interval between each evaluation was of 3 months. Consequently, we cannot indicate the exact time of acquisition and maturation of FPR.In conclusion, by this study we point out the role played by the FPR in the process of near term infant's maturation and its correlation with the acquisition of functional abilities as independent walking. The knowledge of the maturation of these patterns could be useful for clinicians to identify those infants with motor delay. Future studies could be needed to enrol large samples of infants at low and high risk to identify the predictive value of FPR maturation in relation to the development of posture and movement.FPR: Forward Parachute Reaction; US: Ultrasound; CA: Corrected Age; CAT-CLAMS: Clinical Adaptive Test/Clinical, Linguistic and Auditory Milestone Scale; GA: Gestational Age; IVH 1: Germinal layer haemorrhages grade 1; r: Correlation analysis.The authors declare that they have no competing interests.DMMR participated in the design of the study, performed the clinical neurological assessments and helped to draft the manuscript. MC participated in the design of the study and drafted the manuscript. MS performed the clinical neurological assessments. FP performed the statistical analysis. AP and AS participated in the design of the study. MGR conceived the study, participated in its design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The role of genetic factors involved in the determination of risk of cutaneous malignant melanoma (CMM) in humans remains unclear owing to genetic heterogeneity and reliance on simplistic models of inheritance. Here, we report a statistical genetic analysis of cutaneous malignant melanoma in Sinclair swine (SSCM), a unique animal model for human CMM. Using complex segregation analysis a two-locus model involving an unknown major locus and a second locus that lies within or close to the swine leukocyte antigen (SLA) complex jointly determine risk of SSCM in pedigreed animals. These loci also influence severity of affection, accounting for approximately 20% of the phenotypic variation in quantitative tumour burden.


Wolbachia pipientis has been shown to increase host resistance to viral infection in native Drosophila hosts and in the normally Wolbachia-free heterologous host Aedes aegypti when infected by Wolbachia from Drosophila melanogaster or Aedes albopictus. Wolbachia infection has not yet been demonstrated to increase viral resistance in a native Wolbachia-mosquito host system.The bacterial endosymbiont Wolbachia-induced resistance to West Nile virus  by measuring infection susceptibility in Wolbachia-infected and Wolbachia-free D. melanogaster and Culex quinquefasciatus, a natural mosquito vector of WNV. Wolbachia infection of D. melanogaster induces strong resistance to WNV infection. Wolbachia-infected flies had a 500-fold higher ID50 for WNV and produced 100,000-fold lower virus titers compared to flies lacking Wolbachia. The resistance phenotype was transmitted as a maternal, cytoplasmic factor and was fully reverted in flies cured of Wolbachia. Wolbachia infection had much less effect on the susceptibility of D. melanogaster to Chikungunya (Togaviridae) and La Crosse (Bunyaviridae) viruses. Wolbachia also induces resistance to WNV infection in Cx. quinquefasciatus. While Wolbachia had no effect on the overall rate of peroral infection by WNV, Wolbachia-infected mosquitoes produced lower virus titers and had 2 to 3-fold lower rates of virus transmission compared to mosquitoes lacking Wolbachia.In this study, we investigated Wolbachia can increase resistance to arbovirus infection resulting in decreased virus transmission in a native Wolbachia-mosquito system. The results suggest that Wolbachia reduces vector competence in Cx. quinquefasciatus, and potentially in other Wolbachia-infected mosquito vectors.This is the first demonstration that  Wolbachia pipientis is an intracellular, α-proteobacterial symbiont that infects a wide variety of invertebrates, including insects, spiders, mites, isopod crustaceans, and filarial nematodes Culex pipiens and has been most studied for the broad range of reproductive phenotypes that it induces in its various hosts, including cytoplasmic incompatibility, feminization, parthenogenesis, and male killing Wolbachia a highly successful symbiont, infecting up to 66% of arthropod species Wolbachia has been shown to infect at least 19 species of fruit flies of the genus DrosophilaD. simulans, Wolbachia causes robust and complex patterns of cytoplasmic incompatibility, while in other species, like D. melanogaster, reproductive phenotypes are generally weak or absent Wolbachia infection of D. melanogaster is nonetheless widespread Wolbachia-infected D. melanogaster, thereby explaining the observed persistence of Wolbachia infection. A variety of non-reproductive phenotypes have additionally been identified in Wolbachia-infected D. melanogaster, including effects on behavior, viability, insulin signaling, and iron homeostasis Wolbachia-dependent phenotypes is generally modest and frequently variable, some of these could, nonetheless, provide Wolbachia-infected flies with a selective fitness advantage Wolbachia infection of D. melanogaster has also been shown to increase the fly's resistance to some viral infections, resulting in infections with lower virus titers and less associated pathology D. melanogaster by Drosophila C virus or cricket paralysis virus  is strongly suppressed by Wolbachia infection with titers of DCV being reduced up to 10,000-fold Nodaviridae) is not inhibited by Wolbachia at the level of virus replication, yet the pathology associated with FHV infection is strongly reduced D. melanogaster that cause pathology, such as DCV, resistance to viral infection would clearly confer Wolbachia-infected D. melanogaster a significant fitness advantage in the face of viral infection.Wolbachia from D. melanogaster can also confer resistance to viral infection in a heterologous host. Wolbachia strain wMelPop, which normally infects D. melanogaster, lacks normal replication control, resulting in significant pathology and a shortened lifespan in the fly Wolbachia-free mosquito Aedes aegypti with a mosquito-adapted strain of wMelPop produces mosquitoes with both a shortened lifespan and increased resistance to viral infection Ae. aegypti with Wolbachia from Aedes albopictus also increases viral resistance Wolbachia can increase viral resistance when infecting a heterologous mosquito host. It is less clear, however, whether Wolbachia ever increases viral resistance when infecting their native mosquito hosts. To date, this question has only been addressed in Ae. albopictus, and no increase in susceptibility to dengue virus  or Chikungunya virus  infection was observed in Ae. albopictus mosquitoes cured of their normal Wolbachia symbionts, suggesting that Wolbachia infection does not increase viral resistance in this native Wolbachia-mosquito system Wolbachia-induced increases in resistance to infection by West Nile virus  in D. melanogaster and in the southern house mosquito Culex quinquefasciatus, a natural vector of WNV. In both cases, we looked for viral resistance induced by the Wolbachia strain that naturally infects each species. We demonstrate that Wolbachia infection of D. melanogaster increases resistance to infection by WNV and that this protective effect is relatively specific to WNV compared to other arboviruses. We further demonstrate that Wolbachia also increases resistance to WNV infection in the mosquito Cx. quinquefasciatus. While more modest than the level of resistance observed in flies, the resistance phenotype in Cx. quinquefasciatus was sufficient to significantly reduce the proportion of infected mosquitoes that transmitted virus during feeding.To investigate this question further, we looked for D. melanogaster increase susceptibility of flies to infection by WNV 414Ago2 had the opposite phenotype, being highly resistant to WNV infection  BER1 flies were found to be resistant to infection, with an ID50 for WNV of 4190 plaque forming units (pfu) and low titers of virus in infected flies (Wolbachia(-) BER1-T flies were susceptible to infection, with an ID50 for WNV of 1.5 pfu and consistently high titers of virus in all infected flies , which are not infected with Wolbachia . Thus, tetracycline treatment itself does not cause increased viral susceptibility, supporting the conclusion that it is the loss of Wolbachia that is responsible for the observed increase in WNV susceptibility.To address the possibility that tetracycline treatment itself caused the observed increase in WNV susceptibility independent of the loss of olbachia , with teolbachia . In contolbachia . Their IFlaviviridae (including WNV), Togaviridae, and Bunyaviridae families. To assess the relative strength of Wolbachia-induced viral resistance against representative Togaviridae and Bunyaviridae viruses, we compared the susceptibility of BER1 and BER1-T flies to infection with Chikungunya virus  and La Crosse virus . CHIKV proliferates robustly after inoculation into D. melanogaster (50 for CHIKV in Wolbachia(+) BER1 flies was 30 pfu, and the average titer of virus in infected animals for the various concentrations of CHIKV inoculated ranged from 6.4 to 9.1 log10 pfu/animal (Wolbachia(−) BER1-T flies were more susceptible to CHIKV infection than were the BER1 flies. The ID50 for the BERT1-T flies was 3.6 pfu, 8-fold lower than that for BER1 flies, and the average titer of virus in infected BER1-T flies was higher at every concentration of virus tested, averaging 4.2-fold higher when calculated across all inoculation doses combined . Thus, CHIKV infection of BER1 flies is inhibited by Wolbachia, but the effect is weaker than observed for WNV; specifically, the ID50 decreased by 8-fold (CHIKV) versus 2793-fold (WNV) and the virus titer increased by 4.2-fold (CHIKV) versus 918-fold (WNV) when averaged across all infected animals  BER1 flies was 30 pfu, and the average titer of virus in infected animals ranged from 1.5 to 3 log10 pfu/animal for the various concentrations of LACV that were inoculated (Wolbachia(+) BER1 and Wolbachia(−) BER1-T flies. The ID50 for the latter was 32 pfu, and the average titer of virus in infected animals for the various concentrations of LACV tested was not significantly different from the titer determined in Wolbachia(+) BER1 flies  strain and males from the original Wolbachia(+) strain were fully infertile, while the reciprocal cross and crosses between Wolbachia(−) individuals were fertile (data not shown). Finally, there was no significant difference in the wet weight or wing length of newly emerged females from the Wolbachia(+) and Wolbachia(−) strains, suggesting that Wolbachia infection does not have a significant effect on the growth of Cx. quinquefasciatus (data not shown).A laboratory strain of reatment . In addiWolbachia infection of Cx. quinquefasciatus affects susceptibility of the mosquitoes to WNV, we fed Wolbachia(+) and Wolbachia(−) mosquitoes a blood meal containing WNV, and determined the frequencies of infected mosquitoes, of virus dissemination into the legs, and of virus transmission in the saliva at 5, 7, and 14 days post blood meal  and Wolbachia(−) mosquitoes became infected (Wolbachia(+) and Wolbachia(−) mosquitoes were similar even when the overall frequency of infection was reduced by adding less virus to the blood meal or by culturing blood-fed mosquitoes at lower temperatures (data not shown). In contrast to the similarity in overall infection frequency, the frequencies of virus dissemination into the legs and of virus transmission in the saliva were higher in the Wolbachia(−) mosquitoes at all time points tested at both five and fourteen generations (Wolbachia(−) mosquitoes were higher at all time points is highly significant , and the fact that the same increases were observed both five and fourteen generations after antibiotic treatment suggests that the increased rates of WNV dissemination and transmission are a permanent phenotypic consequence of removing Wolbachia from Cx. quinquefasciatus mosquitoes rather than being a transient artifact caused by antibiotic treatment.At the relatively high titer of virus added to the blood meal, most of both the infected . The freerations . The incWolbachia(+) and Wolbachia(−) mosquitoes, it was unclear if the increased rate of virus dissemination and transmission observed in Wolbachia(−) mosquitoes was a consequence of increased susceptibility of the mosquitoes to WNV infection (Wolbachia(+) and Wolbachia(−) mosquitoes, the average titer was higher in Wolbachia(−) mosquitoes at all time points tested (Wolbachia(−) mosquitoes, however, were only significant at five generations, but not at fourteen generations after treatment . More importantly, the probability of virus dissemination and transmission was strongly correlated with virus titer in both the five generation and fourteen generation experiments . Infections producing titers beyond a threshold of about 4.5 log10 pfu/mosquito were likely to result in virus dissemination, and in infections with the highest titers, virus transmission. This correlation is particularly apparent when dissemination and transmission status is compared with virus titer for each mosquito (green and red pluses in Wolbachia(−) mosquitoes compared to Wolbachia(+) mosquitoes having the highest virus titers at each time point tested. The increase in the number of Wolbachia(−) mosquitoes with the highest virus titers correlates with, and can explain, the 2 to 3-fold higher rates of virus dissemination and transmission observed for the Wolbachia(−) mosquitoes , resulting in the overall rate of viral infection being independent of the presence or absence of Wolbachia. As the WNV infection spread, however, the virus would encounter tissues containing Wolbachia, and therefore more resistant to infection, resulting in the lower virus titers and decreased rates of virus dissemination and transmission that were observed. The degree of overlap in the tissue distribution of Wolbachia and viral pathogen is likely to be an important determinant in the extent to which Wolbachia can inhibit vector competence in any given Wolbachia-mosquito-arbovirus interaction Wolbachia-induced resistance to WNV may be weaker in Cx. quinquefasciatus than in D. melanogaster simply because levels of Wolbachia are lower in the somatic tissues of Cx. quinquefasciatus than in D. melanogaster. Preliminary comparison of Wolbachia levels in the two species is consistent with that conclusion (unpublished observations).nfection . In the Wolbachia, and until recently, all the Wolbachia strains shown to increase viral resistance have been supergroup A strains that normally infect D. melanogaster and D. simulanswAlbB from Ae. albopictus was shown to increase resistance to DENV in the heterologous host Ae. aegyptiWolbachia infection are know to differ between heterologous and native hosts. For example, wAlbB induces the innate immune response in heterologous host Ae. aegypti but not in native host Ae. albopictuswPip strain of Wolbachia infecting Cx. quinquefasciatus is a supergroup B strain of Wolbachia, and the results reported here are the first demonstration of viral resistance induced by a supergroup B strain of Wolbachia in its native host wNo infecting D. simulans does not increase resistance against infection by either DCV or FHV wNo were significantly lower than the levels of three supergroup A strains that also naturally infect D. simulans and that all increase viral resistance Wolbachia-dependent increase in viral resistance in Cx. quinquefasciatus demonstrates that supergroup B strains of Wolbachia are capable of increasing resistance to viral infection in their native hosts, and argues that the absence of Wolbachia-induce resistance in wNo-infected D. simulans is more likely due to low Wolbachia density than to an intrinsic inability of supergroup B strains to confer a resistance phenotype on their native hosts.There are at least eleven major supergroups of D. melanogaster-derived wMelPop and Ae. albopictus-derived wAlbB strains of Wolbachia when transferred into Ae. aegypti have been shown to significantly increase resistance of Ae. aegypti to DENV infection, with virus titers being suppressed more than 10,000-fold in both cases D. melanogaster , the strains of Wolbachia (wMel versus mosquito-adapted wMelPop), and the strains of virus (CHIKV versus CHIKVE1-A226V). Wolbachia-induced phenotypes, including viral resistance, are known to be sensitive to differences in both host species and Wolbachia strain Recently, both nogaster , s5. They strong [39]. TheWolbachia-induced suppression of viral infection in D. melanogaster varies widely amongst those viruses that have been tested. Infections by WNV (Flaviviridae) and DCV (Dicistroviridae) are both strongly suppressed, with virus titers being reduced at least 10,000-fold for both viruses (this report)Togaviridae) and NoraV virus (picorna-like family), in contrast, are only modestly suppressed, with virus titers being reduced about 10-fold, while infections by LACV (Bunyaviridae), FHV (Nodaviridae) and IIV-6 (Iridoviridae) are unaffected (this report)Wolbachia-induced resistance. Although both are positive-sense, ssRNA viruses, other positive-sense, ssRNA viruses are less strongly affected  or are not affected at all at the level of virus replication (FHV). It is also unclear whether negative-sense, ssRNA viruses like LACV and dsDNA viruses like IIV-6 will, as a general rule, be refractory to Wolbachia-induced resistance, given that only a single representative virus of either type has thus far been tested (this report)Wolbachia-induce resistance may be limited to a subset of positive-sense, ssRNA viruses. An elucidation of virus specificity, particularly the marked sensitivity shared by relatively dissimilar viruses like WNV and DCV, will require a better understanding of the underlying mechanism, or mechanisms, by which Wolbachia infection increases viral resistance.The strength of Wolbachia infection of D. melanogaster inhibits WNV infection so dramatically without causing significant deleterious effects to the host. Other than resistance to viral infection, the particular Wolbachia-infected strains of D. melanogaster studied here are unremarkable, with no obvious reductions in viability, fertility, or fecundity (unpublished observations). Elucidating the underlying molecular mechanism by which Wolbachia inhibits viral infection in D. melanogaster could, therefore, promote the development of antiviral agents that either mimic the direct antiviral action of Wolbachia proteins or modulate in therapeutically useful ways the same host pathways important for viral infection. The complexity inherent in a biological system comprising the interaction of three disparate organisms - bacterial symbiont, insect host, and viral pathogen - presents significant challenges for future mechanistic studies. Such studies will be facilitated by the availability of extensive genetic and molecular tools developed for D. melanogaster.Finally, it is noteworthy that D. melanogaster were maintained on cornmeal-brewer's yeast-glucose medium at 23°C and 45% relative humidity. Wild-type D. melanogaster strains Oregon R and BER1 were obtained from the Bloomington Drosophila Stock Center. 414AGO2 flies were obtained from Haruhiko Siomi Wolbachia infection by growing the flies for one generation on instant food  made with 200 µg/ml tetracycline. Removal of the Wolbachia was confirmed by PCR.Cx. quinquefasciatus were maintained at 26°C, 50% RH with a 16∶8 L∶D photoperiod. Larvae were reared at 300 larvae/liter with a water depth of 1.5 cm and fed standardized volumes of ground koi pellets. Adults were maintained on 10% sucrose ad libitum and fed goose blood supplemented with 2.5% sucrose for egg laying. The Wolbachia(+) Cx. quinquefasciatus colony was established from mosquitoes obtained from Benzon Research, Inc. , who established their colony in 1995 from a preexisting colony maintained at that time at Virginia Polytechnic Institute and State University. Mosquitoes were cured of Wolbachia infection by feeding adults for one week on 1 mg/ml tetracycline (pH 7) in 10% sucrose Wolbachia(−) strain. Removal of the Wolbachia was confirmed by PCR.D. melanogaster and ten pools of two animals each pool for Cx. quinquefasciatus . The presence of Wolbachia wsp gene sequences and mitochondrial 12S rRNA gene sequences was determined in each extract by PCR. Primers 81F and 691R were used for the wsp gene, and primers 12Sai and 12Sbi were used for the 12S gene, both as described previously Total DNA was isolated from five pools of five animals each pool for The WNV stock was derived from WNV NY003356, a primary isolate from kidney tissue of an American crow collected in 2000 in Staten Island, NY D. melanogaster were inoculated essentially as described previously 8 pfu/mL. Mosquitoes were fed for 1 hourr using a Hemotek membrane feeder . Virus titer in the blood meal did not change during the course of the 1 hour feeding. Fully engorged mosquitoes were sorted into pint cartons supplied with 10% sucrose ad libitum and held at 27°C, 55% RH, and 16∶8 L∶D photoperiod before being assayed. At 5, 7, and 14 days post blood meal, mosquitoes were anesthetized with triethylamine, and their legs were removed and placed into 0.5 mL MD. Saliva was collected by placing the proboscis into a capillary tube containing 50% FBS, 50% sucrose for 30 minutes, and then the solution in the capillary was dispensed into 0.5 mL MD. The body was placed into 0.5 mL MD, and the body and legs were homogenized by mixer mill. Samples were stored at −70°C until the proportion of mosquitoes with infected bodies (infected), infected legs (disseminated), and infected saliva (transmitted) was determined by plaque assay. Results obtained for the Wolbachia(+) and Wolbachia(−) strains of Cx. quinquefasciatus were compared using binomial probability analysis.Vector competence assays were performed essentially as described previously D. melanogaster were compared using Student's t-tests, and ID50 values were calculated using program ID50 5.0 Wolbachia(+) and Wolbachia(−) Cx. quinquefasciatus were compared using ANOVA analysis after confirming the data were normally distributed using the Anderson-Darling test statistic. Correlation of the probability of virus dissemination into the legs and of virus transmission into the saliva with virus titer measured in the bodies of Wolbachia(+) and Wolbachia(−) Cx. quinquefasciatus was done using χ2 analysis.Vero cell plaque assays were used to determine titers of WNV, CHIKV, and LACV essentially as described previously Text S1Supplemental data for (0.04 MB DOC)Click here for additional data file.Figure S1D. melanogaster strains Oregon R and 414Ago2 differ in their susceptibility to WNV infection. The indicated pfu of WNV was injected into D. melanogaster strains wild-type Oregon R (OR) and 414Ago2 (414). Seven days after inoculation, the titer of WNV in each fly was measured by plaque assay. (A) The fraction of flies that became infected for each genotype at each concentration of virus, and the ID50 value for each genotype as calculated from those data, are shown. (B) The titers of WNV in the infected OR (O) and 414 flies (X) are shown. The grey diagonal line indicates the amount of WNV inoculated per fly. The limit of detection of the plaque assay was 25 pfu/animal for strain OR and 2.5 pfu/animal for strain 414.(0.07 MB PDF)Click here for additional data file.Figure S2414Ago2 flies is a dominant maternal-effect phenotype. The indicated pfu of WNV was injected into progeny from the reciprocal crosses female OR x male 414 (MAT-OR) and female 414 x male OR (MAT-414). Seven days after inoculation, the titer of WNV in each fly was measured by plaque assay. (A) The fraction of flies that became infected for each genotype at each concentration of virus, and the ID50 value for each genotype as calculated from those data, are shown. (B) The titers of WNV in the infected MAT-OR (O) and MAT-414 flies (X) are shown. The grey diagonal line indicates the amount of WNV inoculated per fly. The limit of detection of the plaque assay was 25 pfu/animal for MAT-OR and 2.5 pfu/animal for MAT-414.The WNV resistance phenotype observed in (0.06 MB PDF)Click here for additional data file.Figure S3414Ago2 flies is caused by a maternal cytoplasmic factor. Twenty three pfu of WNV was injected into female progeny from each generation of five consecutive introgression backcrosses of female progeny to OR males, starting with the cross of resistant strain 414 females to susceptible OR males. As a positive control at each generation, WNV was also injected into females from the OR stock, and the inoculated females were assayed in parallel with the female progeny from the introgression backcrosses. Seven days after inoculation, the titer of WNV was measured by plaque assay in the backcross progeny flies (X) and control OR flies (O). The ratio of the number of flies infected to the number of flies inoculated for each generation is shown along the top of the graph for the OR control flies and along the bottom of the graph for the backcross progeny flies. The limit of detection of the plaque assay was 25 pfu/animal and is shown by a dashed grey line.The WNV resistance phenotype observed in (0.04 MB PDF)Click here for additional data file.Figure S4Wolbachia status of D. melanogaster strains analyzed for susceptibility to arbovirus infection. DNA was isolated from D. melanogaster strains Oregon R (OR), 414Ago2 (414) and tetracycline-treated 414Ago2 (414-T). DNA sequences corresponding to the wsp gene of Wolbachia and the 12S mitochondrial gene were identified by PCR.The (0.09 MB PDF)Click here for additional data file.Figure S5414Ago2 flies was lost after tetracycline treatment. The indicated pfu of WNV was injected into D. melanogaster strain 414Ago2 (414) and tetracycline-treated 414Ago2 (414-T). Seven days after inoculation, the titer of WNV in each fly was measured by plaque assay. (A) The fraction of flies that became infected for each genotype at each concentration of virus, and the ID50 value for each genotype as calculated from those data, are shown. (B) The titers of WNV in the infected 414 (X) and 414-T flies (O) are shown. The grey diagonal line indicates the amount of WNV inoculated per fly. The limit of detection of the plaque assay was 5 pfu/animal.The WNV resistance phenotype observed in (0.06 MB PDF)Click here for additional data file.


Between 2000 and 2004, dengue virus type 1 (DENV-1) genotypes I and II from Asia were introduced into the Pacific region and co-circulated in some localities. Envelope protein gene sequences of DENV-1 from 12 patients infected on the island of New Caledonia were obtained, five of which carried genotype I viruses and six, genotype II viruses. One patient harboured a mixed infection, containing viruses assigned to both genotypes I and II, as well as a number of inter-genotypic recombinants. This is the first report of a population of dengue viruses isolated from a patient containing both parental and recombinant viruses. The envelope (E) protein gene was amplified by RT-PCR . The PCR product was analysed on 1.5 % (w/v) agarose–Tris/acetate EDTA gels and the band of cDNA of interest was excised and purified by using a High Pure PCR Purification kit (Roche) according to the manufacturer's instructions. Approximately 100 ng cDNA or 600 ng plasmid (see below) was sequenced, using 6.4 pmol oligonucleotide primer and an ABI PRISM Dye Terminator cycle sequencing ready reaction kit (Perkin Elmer) according to the manufacturer's instructions. The product was purified by using Dye-EX spin columns (Qiagen) and analysed at the Australian Genome Research Facility on a 3730XL nucleotide sequencer (Applied Biosystems). Sequences are identified as DENV serotype, country of origin, sample number (and clone number)/year sample obtained.To determine the origin of the DENV-1 circulating in New Caledonia during an outbreak in 2002 and 2003, virus was first recovered from 12 patients by culturing 100 μl serum on 25 cmpaup* package (modeltest (nexus file in JGV Online). This analysis revealed that five patients were infected with genotype I viruses, whilst six patients were infected with viruses of genotype II . However, it was not possible to obtain an unambiguous consensus nucleotide sequence, using cDNA as a template, for the E gene of a single virus isolate – D1.New Caledonia.416/03 – taken from a patient who developed dengue in New Caledonia in February 2003 . Therefore, cDNA corresponding to the E genes of viruses from this patient was derived by RT-PCR using primers D1-764F and D1-2467R or D1-938F and D1-2386R  and purified from 1.5 % (w/v) agarose–Tris/acetate EDTA gels by using a High Pure PCR purification kit (Roche) according to the manufacturer's instructions. 5′-ATP extensions were added to the cDNA  phylogenetic analysis of the complete E gene (1485 bp) by using the  package  with theodeltest , and 100bacteria . Plasmidase ml−1 , using aThe nucleotide sequences of 24 clones derived from the virus population of D1.New Caledonia.416/03  varied at 266 sites. Clones 19 and 32 had single-nucleotide deletions at E250 and E308, respectively, resulting in frame-shifts and the production of downstream stop codons. The deduced amino acid sequences of the clones varied at 110 sites (after replacement of the nucleotide deletions in the sequences of clones 19 and 32). Clone 32 also had a stop codon (UAG) at amino acid position E248, as did clone 7 at E391.rdp2 program  serum was available from patient D1.New Caledonia.416/03 after virus isolation had been performed, only seven clones of the E protein gene could be obtained from this source. The sequences of these clones served to confirm the presence of genotype 1 DENV-1 in this patient, but as none were recombinant, we were unable to distinguish whether the recombinant genomes that we detected in this virus isolate arose in the patient or during the isolation of the virus in cultures of This is only the second observation of both parental and recombinant DENV in a population within a single host .By continuing to monitor the epidemiology of the ongoing outbreak of DENV-1 virus infection in the Pacific, it may be possible to determine whether the recombination events described here have conferred any fitness benefit on these viruses, although it is likely that most recombinants are deleterious and so removed by purifying selection. This notwithstanding, it is remarkable that both parental and different recombinant viral genomes were observed in an isolate from a single patient, particularly as the recombination appeared to have involved two genotypes of DENV-1 imported into New Caledonia (


Helicobacter pylori infection, and long-term ingestion of antacids and biguanides. Management of cobalamin deficiency with cobalamin injections is currently well documented but new routes of cobalamin administration  are being studied, especially oral cobalamin therapy for food-cobalamin malabsorption.Cobalamin (vitamin B12) deficiency is particularly common in the elderly (>65 years of age) but is often unrecognized because its clinical manifestations are subtle; however, they are also potentially serious, particularly from a neuropsychiatric and hematological perspective. In the elderly, the main causes of cobalamin deficiency are pernicious anemia and food-cobalamin malabsorption. Food-cobalamin malabsorption syndrome is a disorder characterized by the inability to release cobalamin from food or its binding proteins. This syndrome is usually caused by atrophic gastritis, related or unrelated to  Cobalamin or vitamin B12 deficiency is common in elderly patients , arepotLiterature of the last ten years has providedseveral definitions of cobalamin deficiency –7. The Epidemiological studies show that in the generalpopulation of industrialized countries, cobalamin deficiency has a prevalence ofaround 2 to 20%, depending on the definition of cobalamin deficiency used , 9. Theμg ofcobalamin per day  are used during the rest of the patient's life [The classic treatment for cobalamindeficiency, particularly when the cause is not dietary deficiency, isparenteral administration—in most countriesintramuscular injection—of this vitamin , 18, 34.t's life , 19. In t's life . Hydroxot's life .μg of oralcrystalline cyanocobalamin per day) were used. These data is in accordance withthe results of the two prospective randomized-controlled studies comparingoral cobalamin with intramuscular cobalamin therapy [Vitamin B12 Cochrane Group supports theefficacy of oral cobalamin therapy, with a dose between 1000 and 2000 μg giveninitially daily and then weekly [μg daily [H. pylori infection or pancreatic exocrine failure), lifelong administration or, whenapplicable, sequential administration [Since cobalamin is absorbed byintrinsic factor-independent passive diffusion , dailyhigh-dose oral cyanocobalamin can induce and maintain remissions in patientswith megaloblastic anemia . In case therapy , 39. A sn weekly . In thin weekly . Neverthper day) . Theproper day) . Since lper day) . As a reper day) . Neverthstration .


Viruses can fall prey to their defective interfering (DI) particles. When viruses are cultured by serial passage on susceptible host cells, the presence of virus-like DI particles can cause virus populations to rise and fall, reflecting predator-prey interactions between DI and virus particles. The levels of virus and DI particles in each population passage can be determined experimentally by plaque and yield-reduction assays, respectively.To better understand DI and virus particle interactions we measured vesicular stomatitis virus and DI particle production during serial-passage culture on BHK cells. When the multiplicity of infection  was fixed, virus yields followed a pattern of progressive decline, with higher MOI driving earlier and faster drops in virus level. These patterns of virus decline were consistent with predictions from a mathematical model based on single-passage behavior of cells co-infected with virus and DI particles. By contrast, the production of virus during fixed-volume passages exhibited irregular fluctuations that could not be described by either the steady-state or regular oscillatory dynamics of the model. However, these irregularities were, to a significant degree, reproduced when measured host-cell levels were incorporated into the model, revealing a high sensitivity of virus and DI particle populations to fluctuations in available cell resources.This study shows how the development of mathematical models, when guided by quantitative experiments, can provide new insight into the dynamic behavior of virus populations. Defective interfering (DI) particles are virus-like byproducts of infections that are unable to infect cells on their own because they carry mutations, typically large deletions, in essential viral genes. However, when DI particles and standard virus particles co-infect the same cell the DI particles compete for resources that ultimately enable them to reproduce at the expense of virus particles. DI particles were discovered more than 60 years ago ,2, and tThe unique biological properties of DI particles make them interesting and important for several reasons. First, their ability to interfere with standard virus infections highlights their potential use as therapeutic agents . MoreoveA characteristic feature of DI particles is their emergence and outgrowth during high multiplicities of infection (MOI), where numerous copies of the standard virus infect each host cell. Under such conditions "predator" DI particles can productively interact with "prey" cells infected with standard virus. Ecological models of predator-prey behavior have been proposed to describe the dynamics of virus and DI particle populations -26, wher2 in Eagle's Minimum Essential media  with 5% GlutamaxTM (Gibco) with 10% fetal bovine serum ; the media was switched 2% FBS for all virus infections. All studies employed VSV-N1, a molecularly well-defined virus strain based on the San Juan isolate of the Indiana serotype of VSV [4 PFU/ml) to inoculate 8 × 106 cells in a T-75 flask , incubating 24 h at 37°C, passing through a 0.22 micron filter , and storing at -80°C. The stock titer was 1 × 109 PFU per ml, and the absence of detectable DI particle activity was confirmed by the yield-reduction assay.The maintenance and analysis of host cell and virus cultures followed established methods . Brieflye of VSV , generouThe interference of VSV infection by DI particles was quantified by the yield-reduction assay ,30. Prio6 cells per well in a six-well plate. For fixed-MOI cultures the virus level from each passage was determined by plaque assay and diluted using cell culture media with 2% FBS to set the MOI for the next passage. To infect, 100 microliters of the diluted virus solution was added to the cell monolayer. The dish was then swirled so that the virus solution would fully coat the cell monolayer. After incubation at 37°C for 1 h, the virus solution was removed, and 2 ml of cell culture media with 2% FBS was pipetted into the well. The wells were then incubated at 37°C for 23 h. After this incubation period the solution was mixed by pipetting, aliquoted, and stored at -90°C until diluted for titering or use in a subsequent infection. For fixed-volume passages, cell culture media containing 2% FBS was added to the well immediately upon removal of the media containing 10% FBS. Each passage infection was initiated by transferring 20 microliters of the mixed virus solution from the previous passage. The infection was then incubated at 37°C for 24 hours and harvested, as described above.Prior to each virus passage, fresh cells from parallel-prepared cultures were counted. Typical passage infections used 1.6 × 10As detailed in the Results section, difference equations linking inputs and outputs of infection were employed to estimate overall yields of virus and DI particles. Parameter values (p) were estimated by least squares:with the summation spanning the available data points. The corresponding confidence intervals were constructed by parametric bootstrapping percentile method using multiple resamples from the fitted model as detailed elsewhere .Over the course of 13 fixed-volume passages levels of virus production from cell monolayers dropped more than 10-fold, and passage-to-passage titers of virus fluctuated up to 100-fold Fig. , produciTo better understand how the accumulation of DI particles across fixed-MOI passages impacts virus yields we quantified the activity of interfering particles in different passage populations using the yield-reduction assay Fig. . At highz is the number of IUs per IE. The model equations are:To quantitatively assess the effects of virus-DI particle interactions during serial-passage culture we applied a mathematical model that we recently developed to quantitatively account for the input-output behavior of single passages . Brieflyth passage (VN+1) depends on virus amplification in cells initially infected with either virus alone (CV) or cells co-infected with both virus and IEs (CB). The output concentration of IEs from the Nth passage (IEN+1) depends on their average production from co-infected cells (CB) and their generation from IE-free infected cells (CV). To determine how the initial number of cells (CN) distribute among those that contain virus alone (CV) or virus and IEs (CB) we assume that particles encounter and infect cells following a Poisson distribution, as shown in equations 3 and 4. The model has nine parameters, presented in Table z, three parameters describe the productivity of virus, and five parameters describe the productivity of IEs. In the absence of DI particles a virus-infected cell produces an average of b virus particles. This productivity level is then modified by parameters p and q which describe how the virus productivity changes depending on the dose of interference. The negative sign on p reflects the reduction in virus yield from DI particle co-infection, and the positive value of q accounts for a partial recovery of virus yield for large co-infecting doses of DI particles. The IEs are either generated de novo from virus-infected cells (CV), described by the parameter g, or amplified from co-infected cells (CB), described by the parameter d. The effects of IE co-infection on IE yield is then modified by the parameters r, s, and w. At low levels IEs enhance their own production, reflected by the positive value of r. At higher IE, where N·z/CN)   × 106 cells per well, respectively.The impact of host-cell availability for infection was tested by simulating passages using either fixed or fluctuating cell numbers. Fluctuating conditions were simulated by using cell numbers that were experimentally measured during the fixed-volume passage, at the time of infection Fig. . Both vi4 IE per ml had no effect on the productivity of virus or IE at steady state. However, transients in virus and IE productivity differed in duration, requiring up to 9 passages to achieve steady-state levels.The model was used to simulate the effects of different initial IE levels on the behavior of virus and IE levels across fixed-volume passages, as shown in Fig. q = 0, rather than q = 0.47 , as shown in Fig. infected cell. Other mechanisms may also play a role in the mismatch between model and data at low MOI. For example, DI particle populations may evolve to more potently inhibit amplification of virus in cells co-infected with both virus and DI particles, or de novo generated DI particles may emerge and displace existing DI particles [By maintaining a fixed multiplicity of infection (MOI) across serial virus passages we observed a regular pattern of population behavior: average yields of virus declined more rapidly for passages performed at higher MOIs Fig. . The eararticles ,33,34. FN+1V) and cell level(NC) shown in Eq. 1. A testable prediction of the model is that by controlling experiments to reduce fluctuations in cell levels one should observe more regular behavior of virus levels  during constant-volume passages, as reflected in Fig. What causes the chaotic population dynamics that is a hallmark of fixed-volume serial virus cultures? Mathematical models of passage behavior have previously suggested how chaotic behavior can arise during fixed-volume passages , but thede novo generation of DI particles produced interference activity that was rapidly amplified in co-infections with wild-type virus. It appears that once DI particles are available they will tend to dominate subsequent co-infections. While new or different DI particles could, in principle, be generated by de novo processes, the low productivity of such processes prevents de novo DI particles from being competitive with existing DI particles. This dominance of initial DI particles has been found experimentally by observing how parallel cultures initiated from a common stock preserve DI particles present in the stock [To run our model one must provide an initial condition that specifies not only the concentration of virus particles, but also the concentration of DI particles in the initial passage. In the laboratory, the concentration of DI particles in the initial passage will depend on the history of the stock solution. For example, if the stock is amplified from an isolated plaque at low MOI or serially passaged at high MOI it may contain negligible or high levels of DI particles, respectively. In our model this input was found to have no effect on the steady-state dynamics of the virus and DI particle populations Fig. /4h. Evenhe stock .de novo rate of DI particle generation. This rate was estimated for VSV at 0.001 IE/cell. However, when a cell is co-infected with viable virus and DI particles, then many DI particles can be made, reflecting their ability to divert resources from virus to DI particle production.Some cells can make nearly a million-fold more DI particles than other cells, depending on how they are infected. When a cell is infected with viable virus, in the absence of any DI particles, few if any DI particles will be made, reflecting the small de novo rate of DI particle generation is (0.001 IE/cell × 200 IU/IE × 1 DI particle/IU) or 1 DI particle from every five infected cells. Thus, a 500,000-fold difference in DI particle yields separates the most productive from the least productive cells. A more expanded view of productivity, which includes the production of both DI and viable particles, may be concisely expressed in terms of a particle-to-PFU ratio. Such a ratio can be experimentally determined by estimating the number of virus-sized particles in a sample by electron microscopy and dividing by the number of viable virus particles measured by the plaque assay. If we assume that only DI and viable particles are produced by a co-infected cell, then we estimate a particle-to-PFU ratio of 38  based on maximal production of DI and viable particles. This estimate is consistent with the observed range for VSV from 10 to 50 particle-to-PFU [Experiments suggest that more than 500 IE per cell can be produced under optimal conditions for DI particle production . If we ae-to-PFU .q to zero, so cells co-infected with virus and a high level of DI particles would produce only DI particles. Others have found that individual cells isolated from a co-infected cell population produced no detectable virus, suggesting a curing effect, but other cells drawn from the same population did produce virus [Although spontaneous curing of virus infections by DI particles has not been experimentally observed, the possibility has been explored in previous models ,22. Our ce virus . The absce virus ,40, as pce virus . In an iWe are seeking to develop experimental measures and theoretical methods to better understand how viruses grow and how their infections spread. Our main contribution here has been to better link quantitative experiments of virus and DI particle populations with quantitative models. This approach will be enhanced in the future by incorporating additional measures, beyond plaque counting, including electron microscopy (particle counting), quantitative PCR, and next-generation sequencing. Ultimately, our approach aims to provide a predictive capability that will enable the long-term control of virus infections.Serial-passage cultures of virus at fixed multiplicity of infection (MOI) produced regular patterns of attenuated virus production across passages. These patterns of steeper declines in virus production at higher MOI could be accounted for by a mathematical model that incorporated interactions among virus particles, defective interfering (DI) particles, and cells to predict yields of virus and DI particle production from co-infected cells. Further, analysis of fluctuating virus populations provided evidence for a high sensitivity of virus and DI particle population dynamics to small changes in the levels of cellular resources.The authors declare that they have no competing interests.KT and JY conceived of the study, and KT performed all experiments. KT and JY analyzed the data and wrote the manuscript.


We describe two cases of West Nile (WN) encephalitis in a married couple in Tel Aviv, Israel, in 1999. Reverse transcription-polymerase chain reaction performed on a brain specimen from the husband detected a WN viral strain nearly identical to avian strains recovered in Israel in 1998 (99.9% genomic sequence homology) and in New York in 1999 (99.8%). This result supports the hypothesis that the 1999 WN virus epidemic in the United States originated from the introduction of a strain that had been circulating in Israel.


We recently proposed a new strategy: 2-locus TDT for detecting two susceptibility genes through their interaction in trio families. We apply our method to two candidate genes, A and C, on the Genetic Analysis Workshop 15 (GAW15) simulated rheumatoid arthritis data and study the power to identify an interactive effect of these genes.This study was performed with full knowledge of the answers. Most multifactorial diseases result from complex interactions among multiple genes with modest effects and various environmental factors. Current strategies usually aim to detect the individual effect of each factor.We have recently proposed a new strategy: the 2-locus TDT (transmission-disequilibrium test), which tests for interaction between two candidate genes . Like thThe GAW15 Problem 3 consisted of 100 replicates of 1500 nuclear families with two affected sibs. From each replicate, we extracted 1500 trio families: one affected (the index case) and his two parents.We focus the analyses on two candidate genes in the RA susceptibility: Locus A on chromosome 16 and Locus C on chromosome 6. Locus A has an indirect effect on RA hazard. Its implication on the susceptibility to the disease depends on the HLA-DRB1 locus located on chromosome 6. Locus C is in strong linkage disequilibrium (LD) with HLA-DRB1. Both genes are involved directly or indirectly in the RA outcome through the HLA-DRB1 gene and thus do not act independently. We apply the 2-locus TDT to the SNPs indicated as functional in the two candidate genes in each replicate of 1500 trios.1, A2 and C1, C2 the SNP alleles of genes A and C, respectively. For each patient, there are nine possible genotypes that we denote AiAjCkCl, where Ai, Aj and Ck, Cl are the alleles inherited from the two parents at the loci A and C, respectively. Subscripts i, j, k and l can be either 1 or 2. A2 and C2 are considered to be the high risk alleles and A2A2C2C2 are the reference genotype.We denote AiAjCkCl) by fijkl and by εijkl the eight penetrances relatively to the reference genotype A2A2C2C2. Thus, ε2222 = 1. Let MG be the general model in which εijkl may take any value between 0 and 1. When the two genes A and B act independently, the lines and the columns of the relative penetrance matrix are proportional (multiplicative matrix) and may be written with four parameters: μ, ν, ϕ and π . In the second one, the restricted model is compared to the general one by a maximum likelihood ratio test.The 2-locus TDT is achieved in two steps. In the first step, the parameters  of the restricted model and The estimation of these parameters requires specifying the allele frequencies at each locus. This was achieved by the affected family-based controls (AFBAC) method, using the parental alleles untransmitted to the patients from which the family was ascertained . Here, tGLG and maxRLR the likelihood values for the estimated parameters of the general and restricted models, respectively. The likelihood ratio test (LRT) was calculated as follows:We denote by maxThis ratio follows, under the null hypothesis, a chi-square distribution with four degrees of freedom (df) corresponding to the difference in the number of parameters between the two models.For each replicate of 1500 trios, the LRT was computed and the significance of the test was assessed in order to evaluate the power of our test in the present situation.1A1C1C1, A1A2C1C1, and A2A2C1C1 are very rare in all replicates, we fixed the relative penetrances of these genotypes at 10-3. Consequently, under the general model, we estimate only five parameters and, under the restricted model, only three parameters ν, ϕ, and π. Thus, in the present situation, we have 2 degrees of freedom for the LRT. Figures p-values in 98 replicates (the program failed in 2 replicates), respectively. There is a good percentage of replicates (44/98) for which the test was significant at 1% level. The power of 2-locus TDT test (at a 5% level) is 66%.Because patients with genotypes Ap-values on two contrasting replicates: 9 and 27. In the Replicate 27, there is a strong evidence for interaction: LRT = 14.35 and p = 7 × 10-4. In contrast, we did not find interaction in Replicate 9 because the LRT = 2.49 and p = 0.29.We illustrate the variation of the LRT and 1A2C2C2 is half of the one expected under the restricted model.Tables In this paper, we studied the performance of the 2-locus TDT for detecting the interaction of the genes A and C in the susceptibility to RA simulated data and show that interaction may be detected with a power of 66% at a 5% level.The 2-locus TDT searches for a departure from multiplicativity in the two-locus penetrance matrix. This departure cannot be observed without an effect of the two genes. Therefore, the 2-locus TDT may also be used for detecting the effect of two candidate genes through their interaction.More generally, the 2-locus TDT may be particularly interesting in the situation of weak effect of one or both genes and strong interaction. In such a situation, traditional methods will fail because they will stop in the absence of noticeable main effect. In fact, they ignore the possibility that the interactive effect of multilocus functional genetic variants may play a larger role than the single-locus effect in the determinism of one trait .Our test applies to candidate genes already recognized as key elements of a metabolic pathway thought to be involved in the disease pathogenesis. There are difficulties in good candidate choice and the use of tagSNPs as markers for most genetic variations in the human genome has become a cornerstone of genetic research. There are, however, reasons to think that the expectations they have raised are somewhat inflated and may lead to unwarranted neglect of other strategies. In particular, we believe in particular that identification of biologically relevant pathways followed by candidate gene strategies may be quite effective at identifying new variants and their interaction in disease susceptibility .The author(s) declare that they have no competing interests.