Cryptococcus neoformans were increasingly recognized as a significant threat to thehealth of immune compromised populations throughout the world. Until recently, the closely relatedspecies C. gattii was considered to be a low-level endemic pathogen that was confined totropical regions such as Australia. Since 1999, C. gattii has emerged in the Pacific Northwestregion of North America and has been responsible for a large disease epidemic among generallyhealthy individuals. The changing epidemiology of C. gattii infection is likely to be a consequence of alterations in fungal ecology and biology and illustrates its potential to cause serious human disease.This review summarizes selected biological and clinical aspects of C. gattii that areparticularly relevant to the recent North American outbreak and compares these to the Australian and SouthAmerican experience.During the latter half of the twentieth century, fungal pathogens such as Candida remain the most common cause of invasive yeast infection; however,opportunistic filamentous fungi such as Aspergillus spp, Fusarium spp, Scedosporium spp, Penicillium spp, and the zygomycota are becoming more prevalent in oncology and transplant centers [Cryptococcus neoformans increasedsignificantly since the onset of the HIV epidemic and continues to be commonamong individuals that do not have access to effective antiretroviral therapy [C. gattii, a closely related speciesthat was traditionally associated with tropical and subtropical climates, isnow gaining prominence as a cause of human and veterinary disease in NorthAmerica. While most clinical cases have occurred among generally healthyindividuals that reside in the Pacific Northwest, a few tourists or visitors tothe region have also been affected. The purpose of this review is to summarizeimportant biological and clinical characteristics of C. gattii that are relevant to the understanding of human diseasecaused by this emerging fungal pathogen.Although less than 500 of the estimated 1.5 million species of fungipose a threat to humans and animals , the pre centers , 4. Hum therapy . C. gattC. neoformans was first isolated in 1894 from fermented peach juice by the ItalianFrancesco Sanfelice [anfelice . Since tanfelice . Cryptoanfelice . Infectianfelice , 13.C. gattii was first isolated from a leukemic patient in 1970 and described asa variant of C. neoformans [C. gattii is closely related to C. neoformans, although its distribution is not global. C. gattii is typically restricted to tropical and subtropicalgeographical regions such as Australia, Brazil, and southern California [C. gattii belongs to serotypes B and C, while C. neoformans belongs to serotypes A and D [C. neoformans, C. gattii typically causes pneumonia andmeningitis [C. gattii appears to have a greater propensity to infect immune competent humans [oformans .C. gattlifornia , 16. In A and D . Like C.ningitis . Howevert humans , 19.C. gattii has acquired the ability to colonize new biogeoclimaticregions and is responsible for a recent outbreak of infection among humans andanimals in the temperate climate of Vancouver Island, British Columbia (BC),Canada [C. gattii infection were identified, including 8 fatalities [C. gattii infection on Vancouver Island peaked at 36 cases/millionpeople/year, a number that was significantly higher than the 0.94 cases/millionpeople/year observed in endemic regions of Australia [C. gattii infection on Vancouver Island was pneumonia [C. gattii infection on the Pacific coast of North America, it will be important forclinicians and laboratory scientists to remain vigilant for diseases that maybe caused by this fungal pathogen.For reasons that are not yet fully understood, ,Canada . Betweentalities . Betweenustralia , 23. Theneumonia . Althougneumonia , 24, 25.Cryptococcus is a largely polyphyletic genus that consists of at least 37different species and belongs to the kingdom Fungi, phylum Basidiomycota,class Tremellomycetes, and order Tremellales [C. neoformans species complex was firstclassified according to structural variations of the extracellularpolysaccharide capsule that are distinguished by agglutination assays withantigen-specific antibodies [C. neoformans wasclassified into four serotypes, A through D, in the 1950s and 1960s [C. neoformans species complex, and rarehybrid serotypes between C. neoformans and C. gattii such as BD and AB havealso been observed [mellales –28. Themellales , 27, 29.tibodies , 31. Usnd 1960s , 32. Theobserved –37.C. gattii was initially classified as a variety of C. neoformans, bearing the name C. neoformans var. gattii or var. bacillispora [C. gattii and C. neoformans were shown to havesubstantial differences in their biochemistry, ecology, epidemiology, andclinical manifestations  and C. gattii (serotypes B and C). The sexual state of C. gattii is known as Filobasidiella bacillispora [llispora , 38. Sue review ) as a ree review , 39. Fo complex , 41,cryC. neoformans may be classified into VNI/VNII genotypes corresponding toAFLP1/1A/1B (serotype A), VNIII/AFLP3 (serotype AD), and VNIV/AFLP2 (serotypeD), while C. gattii may bedistinguished as VGI/AFLP4 (serotype B), VGII/AFLP6 (serotype B), VGIII/AFLP5(serotype B or C), and VGIV/AFLP7 (serotype B or C). Interspecific hybridserotypes BD and AB correspond to AFLP8 and AFLP9, respectively [C. gattii isolates have aVGII genotype that is further subtyped into VGIIa (major) and VGIIb (minor) [C. gattii. Based on thesefindings, it was suggested that these lineages should be considered as separatetaxa, similar to the two monophyletic lineages within C. neoformans that correspond to varieties grubii and neoformans [C. gattii strains havecolonized a novel environment, they are not phylogenetically unique [The most recentclassification of cryptococci was established by genetic typing using PCRfingerprinting, random amplification of polymorphic DNA (RAPD), amplifiedfragment length polymorphism (AFLP) analysis and multilocus sequence typing(MLST) , 42–45. ectively , 37. The (minor) . A receny unique .C. gattii is endemic in tropical and subtropical regions such as Australiawhere it is most commonly associated with eucalyptus trees, particularly Eucalyptus camaldulensis and Eucalyptus tereticornis [Prunus dulcis), golden shower (Cassia fistula), and Douglas fir (Pseudotsuga menziesii) in Colombia, Brazil, and Vancouver Island,respectively [C. gattii has also been isolated frominsect frass in Australia and a wasp nest in Uruguay [Cryptococcus amylolentus, Tsuchiyaeawingfieldii, and Bullera dendrophila,have also been isolated from insect frass [C. gattii in the Pacific Northwest isintriguing, since this temperate climate is characterized by mild and wetwinters and warm and dry summers. On VancouverIsland, C. gattii has mainly beenfound on trees in the coastal Douglas fir (CDF) biogeoclimatic zone such asfir, cedar, and maple [ticornis , 47–49. ectively , 47. C.  Uruguay , 51. Otct frass –54. In tnd maple , 47. Thend maple , 23, 47.C. gattii. A survey conducted in 16countries revealed that the molecular types mostcommonly found in the clinic and environment were VGI and VGII [C. gattii clinicalinfection is rare in this continent, represented by only six of 535 serotyped Cryptococcus isolates [C. gattii isolatesidentified were not determined. In Australia, the C. gattii VGI genotype is the most common clinical or environmentalisolate, while the VGII genotype is an infrequent cause of human or animalinfection [C. gattii infection. Interestingly, most human cases in this region are due to VGII,suggesting that C. gattii hascolonized a new environmental niche [Ecological andgeographical differences exist among the different molecular types of and VGII . Genotypand VGII , 55–58. isolates . In thisnfection , 50. A nal niche , 61. Thial niche .C. gattii isolates in Australia's Northern Territory and on Vancouver Islandare of the VGII genotype, a notable difference between the two regions is theremarkably limited diversity of the latter. Specifically, ninety five percentof the environmental and clinical samples on Vancouver Island are of the VGIIgenotype, and within this group 90% are of the major genotype VGIIa [C. gattii strains: the 1975 Seattle human isolate NIH 444  and the 1992 San Francisco environmental isolate CBS 7750 isolatedfrom E. camaldulensis [C. gattii clinicaland environmental isolates from South America have shown that VGII predominatesover the other molecular types in both Colombia and Brazil as observed onVancouver Island [α mating-type cells within the Brazilian C. gattii VGII population and the fact that all Vancouver Island C. gattii isolates found to date havealso been of the α mating type [C. gattii VGII isolates (serotype B) in Colombia are mainly ofthe opposite a mating type, although large numbers are present in regions thathave a temperate climate that is similar to Vancouver Island [While the majority of pe VGIIa . MLST stdulensis . These sdulensis . These dulensis . Notablydulensis . Thereadulensis . The VGIdulensis , 46. Rer Island , 64, 65.ing type , 65, 66.C. gattii and C. neoformans share many attributes that increase their ability toinvade and survive in a host organism [C. gattii to date include an outer polysaccharide capsule, melanin,mannitol, extracellular proteinase, products of the laccase pathway, superoxidedismutase, phospholipases, urease, and the STE12α transcription factor (ahomologue of Saccharomyces cerevisiae STE12) that is present only in the α mating type  to mice suggests thaturease may aid in the dissemination of the fungus from the lung to the centralnervous system, although the exact mechanism is unknown [C. gattii was observed to share an endophytic relationship with decaying wood of botheucalypt and noneucalypt trees, where the laccase enzyme system appears toplay a role in digestion of lignin [A carbohydrate-richouter capsule that is composed primarily of glucuronoxylomannan (GXM) withsmaller proportions of galactoxylomannan  and mannoproteins is the majorvirulence factor for both oformans . The capoformans , 95. Kiognition . Speciesglobulin –82. Thismination . Similar. gattii . Intratr unknown . It is af lignin , 50.C. neoformans and C. gattii appear to share many of the same virulence factors, it is intriguingthat C. gattii most commonly infectsimmune competent individuals while C. neoformans primarily infects the immune compromised host. One explanationfor this observation is that immune compromised individuals simply have moreenvironmental exposure to C. neoformans compared to C. gattii [C. gattii disease [C. neoformans with comprehensive profiling ofindividual mutants in an animal model [C. gattiiand may in turn stimulate the development of novel therapeutic strategies.Giventhat . gattii . Further disease . Modern al model . A similC. gattii. The term phenotypicswitching refers to an adaptive mechanism characterized by structuralmodifications of the extracellular capsule and cell wall [C. neoformans [C. gattii strain NP1 has also been identified[C. gattii NP1 phenotypic variant demonstrated increased virulenceof the mucoid form. In this study, both mucoid and smooth variants were foundin the lung homogenates while only the smooth variant was found in the brainhomogenates. In terms of the immune response, infection with the smooth variantelicited a greater inflammatory response as characterized by lymphocyte andmonocyte infiltration and gave rise to smaller cryptococcomas than the mucoidvariant [Phenotypic switching andsame-sex mating are two interesting attributes that contribute to the virulenceof ell wall . This phoformans , 100. Rentified. Evaluatvariant .C. gattii isolates, it wasdemonstrated that all Vancouver Island isolates belong to the α mating-type andare unusually fertile [C. gattii undergoes same-sex mating, a process that has beenstudied in C. neoformans [α haploid cells can undergo sexualrecombination via fruiting, a process by which haploid cells fuse, chromosomalreassortment and recombination occur, followed by meiosis and sporulation [α cells (rather than an α and an a cell) may confera survival advantage and could explain why all Vancouver isolates are of the αmating type [C. neoformans have been observed in the laboratory but not innature [In contrast to mostAustralian  fertile , 66. Theoformans . Upon nurulation . Same-sing type . Both trnnature , 102.C. gattii infection have been reported in literature. One grouphas investigated the survival of A/J inbred mice following intranasal infectionwith different Vancouver Island C. gattii molecular types. In this report, the major genotype VGIIa appeared mostvirulent , the minor genotype VGIIb wasavirulent , and the VGI genotype wassimilar in virulence to VGIIa [A limited number of animal model studies of to VGIIa . These fto VGIIa , 24, 103C. gattii isolates that werenot obtained from Vancouver Island. One study compared mouse and humanpulmonary inflammatory responses to intratracheal infection with 10 differentAustralian isolates of C. gattii [C. gattii yeast, anddestruction of lung tissue. In humans, variablepulmonary pathology has been detected including poorly formed granulomascontaining intracellular C. gattii,lymphocytic interstitial pneumonitis, tissue necrosis, and fibrosis. In bothspecies the local lymphocyte pool consisted largely of T-cells with a 2:1 ratioof CD4 to CD8 cells in humans [Furtherinsights into disease pathogenesis have been derived through animal studiesusing . gattii . In BALBn humans .C. gattii 9714  under differentexperimental conditions [C. gattii disease,contrasting with the apparent predilection of immune competent humans to thisinfection. At high doses of C. gattii,immune suppressed mice were more likely to develop severe disease compared toimmune competent mice. SCID mice that lack T- and B-lymphocytes were moresusceptible to infection relative to wild-type BALB/c, suggesting a protectiverole for these cell types [C. neoformans 9759  has increased virulence compared to C. gattii 9714 after systemic infection of BALB/c mice, regardlessof the immune state of the animal. Specifically, C. neoformans 9759 infected the brain and lungs of BALB/c mice,while C. gattii 9714 affected thelungs and skin. Ulcerative lesions on the tail due to intravenous C. gattii infection were more common inimmune competent mice, while rectal prolapse was more common in immunesuppressed mice. This study also demonstrated that infection by either C. neoformans 9759 or C. gattii 9714 may causegastrointestinal pathology, although its incidence was rare [A second study examined the pathology seen in BALB/c mice followingsystemic infection with the clinical isolate nditions , 106. Itll types . Comparawas rare .Cryptococcus isolates administered to BALB/c mice via theintraperitoneal route [C. gattii GR52 and GR56 (both serotype B), from immune competent goats thatdied of pneumonia in Spain; C. gattii B4506(serotype B), an Australian environmental isolate known to be highly pathogenicin mice; C. gattii I-682 (serotypeC), from a Colombian native almond tree; and C. neoformans GR297 (serotype D), from an AIDS patient in Spainwith meningitis. Two of five mice infected with C. neoformans GR297 developed liver and peritoneal abscesses andone mouse died after four weeks. Fungal cultures of spleen, liver, kidney,testes, lung, and brain were done for each group and were positive in at leastone organ for GR52, GR297, GR56, B4506, and I-682 in 80%, 77%, 70%, 70%, and33% of the cases, respectively [Cryptococcus (91%), followed by the liver (75%), kidney (75%),testes (71%), lung (62%), and brain (29%). C. gattii GR52, the cause of a pneumonia outbreak in goats, was also isolatedfrom the lungs of all BALB/c mice studied, suggesting that it has a particulartropism for lung tissue. Pathologic findings associated with infection of thebrain included areas of spongiosis in the white matter with an abundance ofencapsulated yeasts in the basal ganglia while infected lungs showedencapsulated yeasts within alveoli, peribronchial vessels, and interalveolarspaces. Interestingly, inflammation was not observed in either organ amonginfected mice [A third study examined the pathogenesis of five al route . The isoectively . Based oted mice .C. gattii administration through thetail vein, and significant involvement of the visceral organs and peritoneum inthe third animal study may have resulted from intraperitoneal inoculation ofthe fungus. Nonetheless, the tissue tropism observed in humans is similar tothat seen in these mouse models [C. gattii isolate, infectious dose, and immune status of the host also play a role inthe virulence of the fungus and the severity of infection [These studies show that the route of experimental cryptococcalinfection is a major determinant of the site of disease. In general,intravenous infection directly induces systemic disease, whereas inhalation ofthe fungus via the airway mimics the natural route of infection and leads toprimary disease in the lung . For exae models , 15, 21.C. gattii has the potential to infect awide range of animals throughout the world, including wild, farm, domestic, andaquatic animals, along with a variety of birds. This broad host range wasobserved during the recent outbreak in British Columbia [C. gattii infection in domestic animals in southwestern BC,involving 78 feline and 51 canine cases [C. gattii infection,and that all cases in dogs and 50% of cases in cats were due to C. gattii serotype B [C. gattii disease differs slightly in cats and dogs. Among canines, theaffected organs included the respiratory system (52%), central nervous system(42%), and the skin or gastrointestinal tract (6%), while in felines 56% wererespiratory, 26% involved the central nervous system, and 19% were dermal withno cases of gastrointestinal involvement [Columbia , 110. InColumbia , 111–113ne cases . It was rotype B . The tisolvement .C. gattii. Of the 91 animalsthat underwent swabbing of the nasal cavity, two eastern grey squirrels werefound to be positive for C. gattii. This number is similar to the incidence of infection in asymptomatic companionanimals; specifically 1.1% of dogs, 4.3% of cats and 1.5% of horses werepositive for C. gattii [C. gattii infection in dogs and cats does not necessarily progress to clinical disease. In some cases it may be cleared while in other cases it may persist or developinto more severe disease [C. gattii from Vancouver Island to geographic regions that do not harbor theorganism and that veterinary cases may also be a reliable sentinel of humandisease [C. gattii infection was identified inanimals prior to the human outbreak that began on Vancouver Island in 1999 andthat nearly twice as many animals appeared to be infected with C. gattii compared to humans between1999 and 2003 [C. gattii has not been reported to date,transmission of C. neoformans frompet birds to humans has been documented, and possible human-to-humantransmission has also been described [C. gattii involving goats in Spain [Another study wascarried out on Vancouver Island to determine whether wild animals were infectedwith . gattii . Subclin disease . It has disease . The latand 2003 . These oescribed –119. Inin Spain  and psitin Spain  have alsC. gattii may cause mild to severe clinical disease in the apparently healthyas well as immune compromised host. Like other cryptococcal species, C. gattii enters the human host throughthe inhalation of airborne propagules and targets the lung as a primary site ofinfection [C. gattii infectioninvolve the lungs in the tropical climate of Australia (66% pulmonary) as wellas the temperate climate of Vancouver Island and its surroundings (75%pulmonary) [nfection , 108. Innfection , 15, 82.lmonary) , 60.C. gattii infection on VancouverIsland from 1999 to 2006 [C. gattii infection were pulmonary (75%), neurological (8%), combined (9%),and unknown (8%). Of those patients with combined disease, the main sitesinvolved the lung and CNS, and less commonly the skin and lung, or skin andCNS. Death was a rare outcome of C. gattii infection on Vancouver Island with 8 fatalities from 1999 to 2006,or approximately one death per year. Of the deceased patients, five presentedwith both pulmonary and CNS pathology, three had other underlyingcomorbidities, and one had an adverse reaction to therapy. The mean age of thedeceased patients was 61 years (range from 26 to 87) [A study performedby the British Columbia (BC) Cryptococcal Working Group at the University ofBritish Columbia reviewed the clinical aspects of 171 individual human cases of  to 2006 . It was  to 2006 . The cli6 to 87) .C. gattii on Vancouver Island or wereexposed many years earlier. This suggests that another source of C. gattii may exist outside of VancouverIsland, or that the fungus may have been transiently present in othergeographic regions due to dispersal mechanisms [C. gattii may remain latent in the bodyand reactivate at a later time [A summary of themost recent case reports associated with the Pacific west coast is presented inchanisms , 121. Ater time .C. gattii infection may bequite subtle. For example, patients 3 and 9 were asymptomatic while patient 6experienced only a nonspecific cough [In the BCCryptococcal Working Group study, the pulmonary symptoms most commonly observedin the 171 cases were cough and dyspnea, while the main symptom involving theCNS was headache . Similaric cough , 24, 114ic cough . The speic cough . Meanwhic cough , 25, 114C. gattii cases from the BC CryptococcalWorking Group study were caused by C. gattii serotype B [C. gattii serotypeC in the environment had not been described prior to 1998 [C. gattii majorgenotype VGIIa is highly associated with human disease: 82% of the cases wereVGIIa, while the others were VGIIb (9%) and VGI (6%) [C. gattii disease were smoking (50%), oral steroid use (30%), invasive cancer(24%), and chronic lung disease (13%). HIV infection (4%) and receipt of anorgan transplant (3%) were considered less common risk factors for thedevelopment of C. gattii disease inthis study population [All 171 rotype B . This fi to 1998 , 122. ItVGI (6%) . Similarpulation . ExaminaC. gattii on Vancouver Island has been estimated to be 6-7 months with a range from 2 to 11months [C. gattii infection. It is likely that the clinical incubation period depends on several variablessuch as differences in host immunity and the intensity of C. gattii exposure.Thus far, themedian incubation time of 1months . One exc1months . The potC. gattii infection and generally requires analysis of tissue or fluids from infectedsites such as cerebrospinal fluid, bronchial washings, blood, and urine [C. neoformans and C. gattii readilygrow as round cream-colored mucoid colonies on Sabouraud dextrose agar, aselective medium that is widely used for the isolation of yeast [C. neoformans or C. gattii fromother fungi [C. gattii colours the medium blue while C. neoformans does not (medium remains yellow) [Laboratory testingis essential to diagnose nd urine . Light mnd urine , 20, 91.nd urine . In the of yeast . Alternaof yeast , 47. Dier fungi . Furthe yellow) . Though  yellow) . Finally yellow) , 25.C. gattii to antifungaltherapy has been attributed to serotype-specific differences in antifungalactivity though there are no consistent or predictable data to support thishypothesis [C. neoformans and 42 C. gattii isolates tovarious antifungal drugs including amphotericin B, flucytosine, fluconazole,posaconazole, voriconazole, and isavuconazole [C. gattii serotypesB or C compared to other serotypes. In vitro activity of antifungal agentsmay be helpful in guiding the clinical management of severe cryptococcalinfection including meningoencephalitis . A slowepothesis , 128. Toconazole . The majC. gattii on Vancouver Island, andpossibly other adjacent regions, indicates that this fungus has the ability toadapt to new environmental conditions. It has been suggested by Kidd et al. that the temperate climate ofVancouver Island may provide a favorable niche for the survival and dispersalof C. gattii [C. gattii colonization of new geographicregions [C. gattii havebeen studied on Vancouver Island. Some trees in the region show intermittentpositivity, suggesting the movement of fungal spores [C. gattii levelsexist in public areas such as park entrances, parking lots, and beaches despitethe fact that these areas are characterized by low tree density [C. gattii dispersal appears to be mediated by human activity, including contact withfootwear and car wheel wells, deforestation, and gardening [The colonization of . gattii . Global regions . Studiesregions , 130. Thl spores . High C. density , 121. Aardening , may alsC. gattii colonized other regions on thePacific west coast? It is known that C. gattii (serotype C) was isolated from a eucalyptus tree in San Francisco in1992, but since then C. gattii hasnot been found in that environment. In order to determine whether C. gattii has spread to the UnitedStates, Fraser et al. investigatedpossible colonization on the San Juan Islands, the closest U.S. region toVancouver Island [Basidiomycota,namely Cryptococcus laurentii and Cryptococcus cellulolyticus, were isolated, C. gattii was not detected. This suggests that C. gattii may not have yet colonized the San Juan Islands, ordispersal of the fungus is impeded by a geographical barrier that is the Juande Fuca strait [C. gattii wasisolated from environments outside of Vancouver Island including the BritishColumbia mainland, the Gulf islands, and Washington state, but no positiveenvironmental samples were found in Oregon [C. gattii has beenisolated in Seattle (1975),Vancouver Island, and the British Columbia mainland, and most recently inOregon, Washington, and Alberta [Has r Island . The Sana strait . In a ren Oregon . Inthe  Alberta –25, 63.C. gattii infection in the Pacific Northwest highlights the fact that this fungusis an important emerging pathogen that can adapt to new potential environmentalniches. C. gattii isolates found onVancouver Island appear to be hypervirulent, as the number of infectedindividuals per million people per year between 2002 and 2005 was more than 36times higher on Vancouver Island compared to endemic regions such as Australia [C. gattii disease in order to discover effective prevention and treatment strategies,including measures to limit human exposure to this pathogenic fungus. Employing C. neoformans as a model organism tounderstand the disease-causing potential of C. gattii is no longer sufficient since fundamental disparities such asdifferences in host tropism and the expression of certain virulence factorsexist between the two species.The recent outbreak of ustralia , 23. It


KI and WU Polyomaviruses in Children, France To the Editor: Two new members of the Polyomaviridae family, provisionally named Karolinska Institutet virus (KIPyV) and Washington University virus (WUPyV), have been recently discovered ; influenza virus types A and B; parainfluenza virus types 1, 2, and 3; and adenoviruses (AdVs) by direct immunofluorescence assay. The aspirates were also tested for human metapneumovirus (HMPV) by an enzyme immunoassay  and for the human bocavirus (HBoV) by PCR  children. The viruses found were RSVs in 175 (32.6%), HBoVs in 54 (10.0%), HMPVs in 50 (9.3%), rhinoviruses/enteroviruses in 11 (2%), influenza A viruses in 8 (1.5%), human AdVs in 6 (1.1%), and parainfluenza type 3 viruses in 4 (0.7%) samples. Aspirates were not tested for coronaviruses; detection of rhinoviruses/enteroviruses was likely low because cell culture is less sensitive than molecular assays.A total of 13 (2.4%) samples were positive for WUPyV; of these 4 (30.8%) were co-infected with another virus. The 13 children with samples positive for WUPyV had a median age of 11.2 (2–48) months and the male/female sex ratio was 2.2. KIPyV DNA was detected in samples from 3 (0.6%) boys ; 1 of those samples was co-infected with RSV and HMPV.Sequences of WUPyV and KIPyV isolates varied little from each other and from other GenBank sequences, which suggests that these polyomaviruses are genetically conserved viruses. Clinical characteristics of children infected with WUPyV and KIPyV are retrospectively recorded . All chiOur data are in agreement with the 2 original reports that show that the new KI and WU polyomaviruses may be detected in respiratory secretions from patients with respiratory diseases (KIPyV and WUPyV were mainly detected in samples from children with lower respiratory tract disease, such as bronchiolitis or atypical pneumonia, and in samples from children with exacerbated asthma. These preliminary data on the likely role of these viruses as respiratory pathogens need to be interpreted with caution. Aspirates were obtained only from those with observed symptoms; no asymptomatic controls were tested. Detection of WUPyV and KIPyV in respiratory samples may simply reflect a respiratory transmission route as previously suggested for BK virus and JC virus (As with other polyomaviruses, WUPyV and KIPyV could establish persistent and latent infections with likely asymptomatic reactivations (


Drosophila melanogaster is widely used to decipher the innate immune system in response to various pathogens. The innate immune response towards persistent virus infections is among the least studied in this model system. We recently discovered a picorna-like virus, the Nora virus which gives rise to persistent and essentially symptom-free infections in Drosophila melanogaster. Here, we have used this virus to study the interaction with its host and with some of the known Drosophila antiviral immune pathways. First, we find a striking variability in the course of the infection, even between flies of the same inbred stock. Some flies are able to clear the Nora virus but not others. This phenomenon seems to be threshold-dependent; flies with a high-titer infection establish stable persistent infections, whereas flies with a lower level of infection are able to clear the virus. Surprisingly, we find that both the clearance of low-level Nora virus infections and the stability of persistent infections are unaffected by mutations in the RNAi pathways. Nora virus infections are also unaffected by mutations in the Toll and Jak-Stat pathways. In these respects, the Nora virus differs from other studied Drosophila RNA viruses. The ultimate goal for viruses is to survive and propagate within its host. They do that either by infecting and propagating in a consecutive series of individual hosts, usually causing overt disease or death, or by infecting and persisting in a single host for a longer period of time. In the latter case, viruses need to infect the cells, manipulate the host replication machinery to replicate their genomes and get released from the infected cells without killing the host or triggering the immune system. Whereas some DNA- and retroviruses are able to persist by integrating their DNA into the host genome, it is still unclear how persistence is maintained by RNA viruses and by what mechanisms they evade the immune defense of the host.Drosophila melanogaster, which lacks an adaptive immune system, depends entirely on the innate immune response. The absence of an adaptive immune response and the tractable genetics available in Drosophila melanogaster makes it a valuable model system to study the role of innate immunity in controlling viral infections.In mammals, the innate and adaptive immune mechanisms act together to protect the host from viral infections. By contrast, the fruit fly Drosophila controls viral infections. The Jak-Stat pathway has been shown to affect the course of Drosophila C virus infection Drosophila X virus It is still not fully understood how DrosophilaThe RNAi machinery uses small RNAs as guides to target complementary RNA and initiate the silencing of its expression. These small RNAs are divided into three groups: small interfering RNAs (siRNA), microRNAs (miRNA) and the piwi-associated interfering RNAs (piRNA) Drosophila two different RNase III enzymes, Dicer 1 and Dicer 2, process the double-stranded RNA to produce the miRNA and siRNA duplexes respectively Drosophila C virus, flock house virus, cricket paralysis virus and the Sindbis virus Drosophila X virus, a double-stranded RNA virus. Moreover the virus appeared to replicate faster in cells deprived of AGO2 than in wild-type cell Vago, which participates in the control of viral titers in the fat body In Drosophila viruses are all pathogenic, causing acute lethality or increased mortality in the infected stocks Drosophila melanogaster, causes persistent infections that are virtually asymptomatic Drosophila. We observed a large variability in the ability of individual flies to clear the virus, depending on the viral titer in the infected fly. Surprisingly, we found that none of the known antiviral responses in Drosophila seems to play any role against the Nora virus.The best-characterized 51BAGO2414AGO2S165fsXr2d2Q1147XDcr-1 and L811fsxDcr-21piwi and 2piwiTumhop2pllAll flies used were 3–5 days old and were reared at 25°C on standard yeast/agar media. Oregon R and Canton S flies were used as wild-type flies. The 2 and approximately 0.1 µl of the viral suspension was injected in the thorax. Flies were allowed to lay eggs during 7–10 days after the infection, and the viral titers were quantified in the offspring to confirm that we had established a permanently infected stock.Flies were infected by injection as described in 5′-TTTCACTTTACTGTTGGTCTCC-3′; reverse, 5′-ATTCCATTTGTGACTGAT-TTTATTTC-3′ and a FAM/TAMRA probe: 5′-FAM-AGAGTTAGTGGACAA-GTTAGAGACTGGCAT-TAMRA-3′. cDNA synthesis was performed at 50°C for 10 min and 95°C for 3 minutes, followed by 40 cycles of amplification at 95°C for 10 seconds and 55°C for 30 seconds. The probe-based protocol works at an efficiency of 85%, and this value was used to calculate the relative concentration of viral RNA.Total RNA was prepared using the Aurum total RNA kit (BioRad), according to the manufacturer. Quantitative RT-PCR was performed in duplicate, using the probe detection system in an I-cycle iQ Thermal Cycler (BioRad). The probe-based quantitative PCR was done with the following primers: forward, To follow the viral production in single flies, we collected the feces of flies kept individually in 1.5 ml centrifuge tubes containing fly food. After 24 hours the flies were transferred to a new tube with fresh fly food. The same procedure was repeated daily during at least 19 days. We isolated viral RNA from the feces deposited in the tubes during days 1, 2, 3, 4, 7, 14 and 19. Total RNA was prepared using the Aurum total RNA kit (BioRad), after vortexing the feces deposited on the wall of the centrifuge tubes, with RNA extraction buffer (BioRad). For the clearance experiments, five days old single flies were kept in bottles and transferred twice per day into fresh bottles.6–107 arbitrary units, whereas a second population showed low or undetectable titers . Only few flies had intermediate titers. Similar results were seen with Oregon R (OR) and Canton S (CS) flies.We observed a large variability in the Nora virus titers between individuals of the same infected stock. The titers vary by three orders of magnitude We hypothesized that these individual variations in viral titers could be related to the replication cycle of the virus, causing bursts of viral production in the flies. To test this possibility, we next followed the virus production in single flies over time, by determining the virus titers in their feces. We transferred individual flies to fresh tubes with fly food every 24 hrs, and then quantified the viral RNA deposited in the tube during that time period . This exThe clearance of the virus in individuals of an infected stock, allowed us to study whether any of the known innate immune pathways are important in controlling the Nora virus. To study this question, we established Nora virus-infected stocks with mutations that affect these pathways. Such infected mutant flies were transferred to fresh food twice per day during four days, and then the viral titers in the flies was assayed to see the effect of the mutations on the clearance of the virus in low-titer infected flies, and on the persistence in the flies with a high-titer infection.AGO2, Dicer-2, and r2d2 genes, which are all essential for the siRNA pathway and known to affect other Drosophila RNA viruses. Surprisingly, we observed no difference in the ability of these mutants to clear the Nora virus after an infection compared to the wild-type stocks (We first tested homozygous null mutants of the e stocks . For allpiwi, which is involved in the control of retrotransposon RNA precursors via the piRNA pathway. Again, we saw no difference between piwi mutants and wildtype flies in their ability to clear the virus (Dicer-1 gene, which is important in the miRNA machinery that controls endogenous mRNA. Dicer-1 is required for normal development, and the Q1147XDcr-1 mutant is therefore homozygous lethal, but at least in the heterozygous condition it does not affect the viral titers (We also tested two different null mutants in he virus . Finallyl titers .Drosophila, we finally investigated if these pathways affect the Nora virus. For the Toll pathway we tested a strong loss-of-function allele in the pelle gene, 2pll, which blocks Toll signaling when homozygous. However, that did not affect the ability of the mutant flies to clear the virus (Tumhop, which has a continuously activated Jak-Stat pathway. However, Tumhop flies have normal Nora virus titers. That experiment does not exclude the possibility that Jak-Stat signaling might play a role, but it is clearly not sufficient to affect the Nora virus.Since the Toll and Jak-Stat pathways have also been implicated in controlling viral infections in he virus . Thus, wOur data show that the Nora virus is able to establish a persistent infection in the individual fly for a long period of time. This persistence seems to be determined by the virus titer in a threshold-dependent manner. A high-titer infection tends to stabilize at a high level, resulting in a persistent infection. On the other hand, flies with a lower titer seem to be more efficient in fighting the infection and reducing the initial viral load. Our experimental approach allowed us to test for two possible levels of Nora virus infection control. First, a mutation that inactivates the antiviral defense should result in flies that are unable to clear a low-titer infection. As a consequence, we expect all flies in the population to become persistently infected. Second, if the defense also limits viral replication in the persistently infected flies, we could expect increased viral titers and perhaps lethality. Surprisingly, none of these effects were observed for any of the mutants we tested. A fraction of the flies in all tested stocks were able to clear the virus, fully or in part, and another fraction reached a similar level of persistent infection as in the wild-type controls.Drosophila C virus The lack of effect of the siRNA system was unexpected. This system is generally active against foreign replicating RNA and it is known to be essential in the defense against several RNA viruses in different organisms. It is possible that the Nora virus produces a suppressor of the siRNA-dependent RNAi machinery, or that it uses a stealth approach to avoid triggering the antiviral response. Viral suppressors of RNAi have been described from several viral systems 8 viral genomes per hour, viral replication is never so high that the survival of the host is endangered Drosophila against the Nora virus. If the virus carries its own mechanisms to limit replication, such a balance could be reached even in the complete absence of effective defenses. This situation would be ideal for the virus, where it would be able to evade the immune system and control its titers to levels that would maintain its presence by transmission to the next generation via the fecal-oral route. However, the clearance of the Nora virus from the low-titer flies is on the other hand a strong indication that defense mechanisms do exist in Drosophila, as it is unlikely that the virus encodes a mechanism for its own elimination.Although persistently infected flies produce Nora virus at a very high and stable rate, on the order of 10Drosophila viruses. Further genetic studies in Drosophila and in the Nora virus are likely to resolve these issues.The existence of both, a high variability in viral titers within the infected fly populations and a threshold titer that determines the persistence of the virus, makes us think that the immune system does interact with the Nora virus and influences the outcome of the infection. It remains to be determined by which mechanisms the flies clear the virus. To our knowledge this is the first example of an RNA virus that is completely unaffected by the inactivation of the RNAi machinery. It is also intriguing that the virus is not sensitive to any of the other immune pathways that have been shown to be important during an infection with other


Arabidopsis plants expressing amiRNAs designed to target the genome of two unrelated viruses were resistant, in a highly specific manner, to the appropriate virus. Here, we pursued two different goals. First, we confirmed that the 21-nt target site of viral RNAs is both necessary and sufficient for resistance. Second, we studied the evolutionary stability of amiRNA-mediated resistance against a genetically plastic RNA virus, TuMV. To dissociate selective pressures acting upon protein function from those acting at the RNA level, we constructed a chimeric TuMV harboring a 21-nt, amiRNA target site in a non-essential region. In the first set of experiments designed to assess the likelihood of resistance breakdown, we explored the effect of single nucleotide mutation within the target 21-nt on the ability of mutant viruses to successfully infect amiRNA-expressing plants. We found non-equivalency of the target nucleotides, which can be divided into three categories depending on their impact in virus pathogenicity. In the second set of experiments, we investigated the evolution of the virus mutants in amiRNA-expressing plants. The most common outcome was the deletion of the target. However, when the 21-nt target was retained, viruses accumulated additional substitutions on it, further reducing the binding/cleavage ability of the amiRNA. The pattern of substitutions within the viral target was largely dominated by G to A and C to U transitions.Plant microRNAs (miRNA) guide cleavage of target mRNAs by DICER-like proteins, thereby reducing mRNA abundance. Native precursor miRNAs can be redesigned to target RNAs of interest, and one application of such artificial microRNA (amiRNA) technology is to generate plants resistant to pathogenic viruses. Transgenic  RNA viruses are well-known for their tremendous capacity to evolve, a characteristic that threatens the development of effective antiviral strategies. A new antiviral strategy was recently proposed to control plant RNA viruses that relied on the expression in plants of artificial microRNAs (amiRNAs) targeting short sequences of 21-nt in the viral genome. Here, we have evaluated the likelihood that changes in the 21-nt target sequence would result in resistance breakdown. We found that changes at different sites in the target had different consequences on the ability of the virus to evade amiRNA surveillance. Then, we evolved viruses with a single substitution within the target under the selective pressure imposed by amiRNAs but without any selective pressure at the protein level. We found extra mutations accumulated in the target that further reduced base pairing with the amiRNA. These results showed that when allowed to replicate, RNA viruses would readily generate genetic variability that would facilitate evasion from the engineered miRNA-mediated virus resistance. The amiR-P69 was designed to target sequences encoding the P69 suppressor of TYMV whilst amiR159-HC-Pro would target sequences for the HC-Pro silencing suppressor of TuMV. Transgenic lines carrying both 35S-pre-amiR159-P69 and 35S-pre-amiR159-HC-Pro transgenes can express the appropriate amiRNA at high levels and showed specific resistance to either TYMV and TuMV, depending on the expression of the cognate amiRNA 159-TuCP directed against the TuMV coat protein (CP) gene Plant miRNAs regulate the abundance of target mRNAs by guiding their cleavage at the sequence complementary region. Previous reports have shown that changes of several nucleotides within a miRNA 21-nt sequence do not affect its biogenesis and maturation In animal systems, RNA interference (RNAi), a gene-silencing mechanism similar to that of miRNA, has been used in clinical trials as antiviral therapeutics to inhibit replication of several human pathogenic viruses  have used an P69 gene targeted by amiR159-P69. This heterologous-virus system allows us to modify any nucleotide within the 21-nt target site without altering virus coding sequences and thus without affecting replication and activity. In other words, this heterologous system allows separating the selective pressure imposed by protein functionality from the selective pressure imposed at the sequence level by RNA silencing. The 21 scanning mutant viruses were inoculated on amiR159-P69 plants and the proportion of transgenic plants that became infected was used to determine the importance of the mutated nucleotide position within the amiRNA target site.Here, we first investigated whether the 21-nt of an amiRNA target site is both necessary and sufficient for amiRNA-mediated specific resistance. Second, we were interested in identifying critical positions within the target site for this resistance. Third, we have explored the patterns of sequence polymorphism of viral sequences that evolve under the only selective pressure of amiRNA-mediated silencing. To address these issues, we established a heterologous-virus resistance system using a TuMV-GFP viral vector to carry a non-essential 21-nt sequence of the green fluorescence protein (GFP) gene carrying a 21-nt sequence (5′-CCACAAGACAAUCGAGACUUU-3′) of the TYMV P69 gene at its 3′-end and inserted the 21ntGFP-P69 fusion gene in between the NIb and CP genes to generate a TuMV-GP69 chimeric virus (CUGAGAGACUUU-3′5′-CCACAAGAC) to give 21ntmGFP-P69, which was inserted in the same position of the viral genome to generate the TuMV-GP69m chimeric virus  from the TuMV-GFP viral polyprotein (The presence of two TuMV NIa protease cleavage sites (CVYHQA) at both the N- and C-termini of the GFP-P69yprotein . Virus iArabidopsis thaliana plants expressing amiR-P69 can specifically target the P69 gene of TYMV and displayed specific resistance to TYMV P69 gene into TuMV-GFP, which was targeted by amiR159-P69, rendered these amiR159-P69 plants resistant to TuMV-GP69 . Progeny plants of these lines were challenged with TuMV-GFP, TuMV-GP69 or TuMV-GP69m.Next, we established the heterologous virus resistance system in N. benthamiana plants expressing amiR159-P69 were resistant to TuMV-GP69 but susceptible to TuMV-GFP and TuMV-GP69m  . As expeMV-GP69m . Plants MV-GP69m . These r159-P69 target sequence is non-essential to the TuMV-GP69 chimeric virus.As the 4 nt mutation on the central positions of the target sequence (position 8 to 12) compromised specific resistance, we decided to further investigate nucleotide positions within this 21-nt sequence that are critical for amiRNA-mediated resistance. Note that the sequence can be systematically altered without affecting essential viral gene functions because the amiRP69 in the background of TuMV-GP69 . WT plants were 100% susceptible to all 21 scanning mutant viruses and symptoms appeared at 5 dpi (data not shown), indicating that the single nt substitutions on the target 21-nt sequence did not affect mutant virus replication nor movement. On the other hand, these mutant viruses showed variable pathogenicity after inoculation on amiR9 plants . For theIn good agreement with the pathogenicity data, symptoms elicited by these mutants were generally delayed in comparison with TuMV-GFP . For the159-P69 plants and analyzed the 21-nt target sequence on the viruses by RT-PCR and sequencing. Several possibilities could account for the resistance breakdown. (1) The scanning mutation could affect amiR159-P69-mediated cleavage to different degrees in amiR159-P69 plants. (2) The scanning mutant virus could undergo rapid evolution accumulating additional mutation(s) within the target site to further increase the number of mismatches and consequently the ability to replicate in presence of the amiR159-P69. (3) Since the 21-nt sequence and the GFP gene are non-essential for virus survival, the surviving mutant virus could undergo in-frame deletions in this region that would render the virus unrecognizable by the amiR159-P69. To discriminate amongst these possibilities, we designed two primers (PTuNIb-8671 and MTuCP-8982) to amplify an 1136 bp DNA fragment including a partial NIb gene, GFP gene, the target 21-nt sequence, and a partial CP gene (GFP-21nt sequence. The recovery of lower molecular mass PCR fragment(s) would indicate deletion of this region. In addition, we designed another primer set (PXFP-532 and MTuCP-8982) to amplify a 482-bp fragment that included a partial GFP gene and a partial CP gene  and TuMV-GP69m3 (lanes 10 and 13). In addition, several viruses, such as TuMV-GP69m5-13, -15, and -19 contained partially-deleted 21-nt sequence . This bias amongst transitions is expected if the viral RNA was edited by cytidine deaminase enzymes.Interestingly, we found that 40 out of 55 observed additional mutations were transitions. Over 50% of the additional mutations in positions 3, 4, 6, 8, 11, and 15 were transition mutations. For example, there were 100% U→C or C→U transitions in position 3 and 4. In position 15, the G→A transition represented 88.89% of all observed mutations at this particular site. Moreover, there were 50 to 66.67% of G→A transitions at positions 6, 8, and 11. This result is not surprising, since it is well known that virus coding regions show an excess of transitions over transversions 159-P69 transgenic plant were resistant to TYMV, but not to TuMV (a heterologous-virus), because there was no sequence homology with amiR159-P69 on the TuMV viral genome P69 gene cannot infect amiR159-P69 plants because of amiR159-P69-mediated cleavage. By contrast, the TuMV-GP69m virus with mutations on the central region within this sequence is sufficient to prevent amiRNA-mediated cleavage on the viral RNA and compromise specific virus resistance. These results indicated that the 21-nt target site is portable and is necessary and sufficient to confer virus resistance.Here, we have developed a heterologous-virus resistance system to study and identify critical positions of amiRNA target site for amiRNA-mediated resistance. The amiRNIb and CP genes. The encoded GFP protein contains two NIa proteinase cleavage sites (CVYHQ/A) at the N- and the C-terminus such that GFP can be released from the viral polyprotein by proteolytic processing. In addition, the additional 21-nt target site that encodes seven amino acids is also nonessential for TuMV. Therefore, any modification on the GFP gene and the 21-nt target site would not affect the chimeric TuMV as evidenced by its ability to infect plants and stably replicate.Because of the genome organization and proteolytic processing strategy of potyvirus, TuMV can express GFP when a cDNA for this protein is inserted in-frame between the in vivo assay we identified critical positions on the 21-nt target sequence for RISC-amiRNA-mediated cleavage. Scanning mutations on the 21-nt target site of the challenging chimeric virus showed different degree of resistance breakdown on amiR159-P69 transgenic plants. Based on the proportion of amiR159-P69 plants that become susceptible, we defined critical, moderately critical and non-critical positions on the 21-nt sequence. Positions 3–6, 9, and 12, are found to be critical for resistance because chimeric virus with mutations at these sites were pathogenic, on average, on ∼82% of amiR159-P69 plants. Positions 2, 10, 11, 13, 15, and 18 are classified to be moderately critical mutations giving average pathogenicity of ∼36% in transgenic plants. The remaining positions are classified as non-critical for resistance since mutants at these sites were only pathogenic in less than 7% of inoculated plants. In summary, most critical positions are localized on sequences complementary to the 5′ portion of the amiRNA whereas the moderate critical positions are mainly localized on the central region of the target site. The exception being position 18, which is complementary to the 18th nucleotide on 3′ portion of the amiRNA, and was also moderately important for amiRNA-mediated resistance. These results are consistent with those obtained with in vitro miRNA-mediated cleavage using a wheat germ system Using an Plum pox virus (PPV) carrying an endogenous miRNA target site can also overcome the resistance by deletion and mutation on the 21-nt target sequence Representative results showed that several virus sequences recovered from symptomatic amiRNA plants contained deletions and additional mutations within the 21-nt target site. This observation is consistent with the hypothesis that mutations in certain critical positions within the target site reduced amiRNA-mediated cleavage efficiency . The redGFP gene, along with small portions of the NIb gene C-terminus or the CP gene N-terminus, has been deleted from the viral genome (data not shown). These results suggest that TuMV can tolerate small deletions in the NIb or CP gene. In addition, deletions in between the GFP gene and the target site  were analyzed for transgene and miRNA levels and 4 independent lines  with high amiR159-P69 expression levels were selected for virus challenge experiments. Seeds were surface-sterilized and chilled at 4°C for 2 d before being placed on Murashige and Skoog (MS) medium with/without antibiotics or sowed directly on Florobella potting compost/sand mix (3∶1). Plants were maintained in a growth room .Two amiRNA transgenic 32P-γ-ATP and T4 polynucleotide kinase (New England Biolabs) to generate high specific activity probes. Hybridization was carried out using the ULTRAHyb-Oligo solution according to the manufacturer's directions (Ambion) and signals were detected by autoradiography. In each case, the probe contained the exact antisense sequence of the expected miRNA to be detected.Ten µg of total RNA was resolved in a 15% polyacryamide/1× TBE /8M urea gel and blotted to a Hybond-N+ membrane (Amersham). DNA oligonucleotides with the exact reverse-complementary sequence to miRNAs were end-labeled with GFP gene was inserted between NIb and CP genes. This chimeric TuMV-GFP virus was used as a surrogate wild type virus and as a backbone for construction of various chimeric recombinant viruses described here.The TuMV infectious clone (p35STuMV-GFP) comprises of a 35S promoter and the full-length cDNA of TuMV-GFP. The 5′-CCACAAGACAAUCGAGACUUU-3′) of the TYMV P69 gene targeted by amiR159-P69 to the 3′ end of the GFP gene. The GFP-P69 fusion sequence was then inserted in between the NIb and CP genes to generate the p35STuMV-GP69 infectious clone. As a control, the central 4 nts (underlined) of the 21-nt target sequence (CUGAGAGACUUU-3′5′-CCACAAGAC) was mutated to give GFP-P69m which was also inserted in the same position of the virus to generate p35STuMV-GP69m.We fused the 21-nt sequence , local lesions were obtained on inoculated leaves. Viruses were then isolated from single lesions and transferred to N. benthamiana for amplification. Four dpi leaves of N. benthamiana with viral infection symptoms were used as the source of inoculum to challenge WT and amiR159-P69 N. benthamiana plants for evaluation of virus pathogenicity . Twenty amiR159-P69 plants were used for each experiment, and the experiments were repeated 3 times. Resistance efficiency of amiR159-P69 plants challenged with recombinant viruses were compared with those obtained with control viruses, including TuMV-GFP, TuMV-GP69 and TuMV-GP69m.To evaluate the efficiency of amiRNA-mediated specific resistance toward wild type and mutant viruses, we have established a standard protocol for virus challenge inoculation and quantitative evaluation of pathogenicity. Our overall aim was to reduce the time for virus maintenance and propagation in host plants so as to minimize possible virus evolution. All recombinant viruses were propagated from DNA infectious clones. Aliquots of 20 µL, containing 1 µg of DNA in sterilized water, were mechanically applied onto carborundum-dusted leaves of Pathogenicity was evaluated between two and four times for each one of the 21 TuMV-GP69mX recombinant viruses. Count data from experiments that were statistically homogeneous were pooled into a single set, whereas experiments that behaved as outliers were removed from the dataset.5′-GAACCAGCTCAAGAGGATCT-3′) and MTuCP-8982 (5′-GCCACTCTCTGCTCGTATCTTGGCACGCGC-3′) for amplification of the viral region between the partial NIb and the CP genes . One µg total RNA was used for reverse-transcriptional polymerase-chain reaction (RT-PCR) with PTuNIb-8671  was used as a measure of the goodness-of-fit and the model with the lowest BIC was considered to be the best one TuMV-GP69mX recombinant viruses were classified into groups of similar pathogenicity using the two-step cluster analysis


Measles outbreaks in 1999 in Queensland and Victoria, Australia, were caused by a novel strain of clade G virus (proposed name g3). Epidemiologic and molecular evidence supports independent circulation of this virus in Queensland, northern Australia, in addition to importation of the virus by East Timor refugees seeking safe haven in Australia. Measles virus (MeV) circulation in industrialized countries, such as aggressive vaccination campaigns targeting children, have proven successful Strategies to reduce In Victoria, measles is a reportable disease, and an enhanced surveillance strategy has been operational since 1997. A registered nurse visits the homes of patients with reported cases to collect specimens for laboratory confirmation, by polymerase chain reaction or detection of MeV-specific immunoglobulin (Ig) M, of the clinical diagnosis and subsequent identification of the measles genotype >2.5% nt divergence in the 450-bp carboxyl-terminal end of the N gene and 2% in the H gene Variable regions in the MeV genome include the hemagglutinin (H) and nucleoprotein (N) genes. The most variable region is the carboxyl-terminal end (450 nucleotides [nt]) of the N gene. A uniform nomenclature approved by World Health Organization (WHO) has existed since 1998 and is used in classifying and naming measles viruses. Currently, 20 genotypes and 1 proposed new genotype exist, encompassing eight clades designated A–H. Each clade contains MeV genotypes that are related by Until recently, MeV strains belonging to clade G had not been detected for >15 years, and the lineage was considered to be either extinct or inactive In June 1999, the novel genotype was first identified in Victoria in a 24-year-old Australian man who had symptoms of MeV infection shortly after visiting the northern state of Queensland. In the next 2 months, four more cases of infection with the same genotype were identified in Queensland. Epidemiologic links between these five cases could not be established.In the second week of September 1999, refugees from the newly independent country of East Timor arrived in Darwin, Northern Territory, where they underwent preliminary medical examinations before being moved to a safe-haven refugee camp in regional Victoria. Several days after arriving at the safe haven, a 4-year-old girl had symptoms that matched the clinical case definition of measles infection (rash and cough with fever at onset of rash) The commonality of the virus strains circulating in both Australia and East Timor was confirmed through analysis of MeV RNA The N gene sequences of measles viruses identified from June to November 1999 were most closely related to the Amsterdam prototype G2 strain identified by de Swart et al. (4). Although phylogenetic analysis indicated that these sequences belonged within the clade G viruses, they differed from the G2 prototype strain by 12 (2.6%) nt and by deduction 6 (4%) amino acids . N gene Full or partial H gene sequences were derived from the measles viruses of four samples obtained from patients 8, 13, 15, and 16. Phylogenetic analysis of the H gene of the first 842 nt of these samples confirmed their assignment to the clade G viruses (results not shown). The four sequences analyzed showed up to 1.1% intranucleotide divergence. The MeV from patient 8 varied by 2.1% nt sequence compared with the prototype G2 strain. Comparison of nucleotide and amino acid sequence of the H gene of the one sample (from patient 13) with full H gene sequence showed differences from the prototype G2 strain of 35 (1.9%) nt and 13 (2.1%) amino acids. This sequence also contained amino acids in the H protein not previously found in other reference prototypes, namely 212Q, 225H, 238D, and 495N. Phylogenetic analysis of the full H gene sequence showed similar patterns of relatedness to those obtained for the N gene (results not shown).Together, phylogenetic analysis of the N and H genes and the appearance of novel amino acids in the H protein provided strong evidence that these measles virus were sufficiently unrelated to the prototype G2 strain to enable their classification as a new genotype within clade G. We propose that measles virus/Vic.AU/24.99 be the reference sequence for a new genotype, g3, pending isolation of a reference strain The novel g3 MeV was not the only strain circulating in Australia during this time. Coinciding with the genotype g3 outbreak, four cases of genotype D8 were identified in Victoria. Three months earlier, small clusters of measles cases were identified in two other Australian states, genotype D5 in the Northern Territory and genotype D3 in Western Australia. Soon after the g3 outbreak, genotype D7 circulated in Victoria, Queensland, and the Northern Territory.In recent years, vaccination campaigns have been undertaken in East Timor under the guidance of United Nations Children's Fund (UNICEF) and WHO. In October 1999, UNICEF conducted a major immunization campaign in Dili, East Timor, reaching approximately 4,000 children <5 years of age; in March 2000, >45,000 children were vaccinated In an industrialized country like Australia, where no apparent circulation of an indigenous MeV strain occurs, the use of epidemiologic surveillance and molecular characterization is important in tracing the source and transmission pathways of MeV imported from areas where the disease is endemic. Apart from expanding our global knowledge of MeV genotypes, molecular characterization is also useful in clarifying epidemiologic links and distinguishing between vaccine-associated and wild-type infection.Despite the novelty of the circulating MeV genotypes, our findings highlight the need for continued vigilance if the virus is to be eradicated. The humanitarian movement of refugees from a country where MeV infection is uncontrolled to countries with relatively high MeV herd immunity is now a common occurrence. Thus, the potential for transmission of this highly infectious virus to residual susceptibles in the wider community remains a distinct possibility, as demonstrated by this outbreak. Measures, such as vaccination of military personnel and support staff working directly with displaced persons; use of appropriate infection-control procedures when attending sick refugees; and screening of newly arrived refugees from measles-endemic areas, are likely to decrease transmission of MeV in industrialized countries.


As part of an investigation of an encephalitis outbreak in New York City, we sampled 430 birds, representing 18 species in four orders, during September 13-23, 1999, in Queens and surrounding counties. Overall, 33% were positive for West Nile (WN) virus-neutralizing antibodies, and 0.5% were positive for St. Louis encephalitis virus-neutralizing antibodies. By county, Queens had the most seropositive birds for WN virus (50%); species with the greatest seropositivity for WN virus (sample sizes were at least six) were Domestic Goose, Domestic Chicken, House Sparrow, Canada Goose, and Rock Dove. One sampled bird, a captive adult Domestic Goose, showed signs of illness; WN virus infection was confirmed. Our results support the concept that chickens and House Sparrows are good arbovirus sentinels. This study also implicates the House Sparrow as an important vertebrate reservoir host.


Epstein-Barr (EB) virus was discovered in 1964 . In the decades since then an immense body of information has been accumulated on the virus and a great deal is now known about its general biological behaviour, its epidemiology, its molecular biology, the humoral and cellular immunological responses which it evokes, and about its relationship to human cancers. The fact that EB virus was thought from the outset to be a human tumour virus was no doubt responsible for the large number of laboratories in which it has been studied. Viruses causing tumours in animals have been known since early in the present century and affect frogs, fowl, rodents, rabbits, cats, cattle, monkeys and even fish . It was obvious that man could not be different in this respect and the finding of EB virus therefore promised to bring human tumours into line with those of other species.


Risk for infection was low among those who handled neotomine or sigmodontine rodents on the job. Rodents are the principal hosts of Sin Nombre virus, 4 other hantaviruses known to cause hantavirus pulmonary syndrome in North America, and the 3 North American arenaviruses. Serum samples from 757 persons who had worked with rodents in North America and handled neotomine or sigmodontine rodents were tested for antibodies against Sin Nombre virus, Whitewater Arroyo virus, Guanarito virus, and lymphocytic choriomeningitis virus. Antibodies against Sin Nombre virus were found in 4 persons, against Whitewater Arroyo virus or Guanarito virus in 2 persons, and against lymphocytic choriomeningitis virus in none. These results suggest that risk for infection with hantaviruses or arenaviruses usually is low in persons whose occupations entail close physical contact with neotomine or sigmodontine rodents in North America. Bunyaviridae, genus Hantavirus (Sin Nombre virus (SNV), New York virus (NYV), Black Creek Canalvirus (BCCV), Bayouvirus (BAYV), and Choclo virus (CHOV) in North America (–Andes virus (ANDV) and Laguna Negra virus (LANV) in South America  is a frequently fatal rodentborne viral zoonosis. Seven species in the virus family Arenaviridae, genus Arenavirus, includes 3 North American species and 14 South American species (Bear Canyon virus (BCNV), Tamiami virus (TAMV), and Whitewater Arroyo virus (WWAV). The South American species include Guanarito virus (GTOV), Junin virus (JUNV), Machupo virus (MACV), and Sabiá virus (SABV). These 4 South American species have been causally associated with severe human disease in Venezuela, Argentina, Bolivia, and Brazil, respectively (The virus family Peromyscus maniculatus) in Canada and the western United States, SNV  in the northeastern United States, NYV (Sigmodon hispidus) in Florida, BCCV and TAMV  in the southeastern United States, BAYV (–Oligoryzomys fulvescens) in Panama, CHOV  in California, BCNV  in New Mexico, WWAV . The primary objective of the survey was to assess the risk for hantavirus infections in persons whose occupations expose them to rodents. Participation in the survey was voluntary and entailed completion of a self-administered questionnaire and donation of a small volume of venous blood. Most of the 995 participants were enrolled at 1 of the following: American Society of Mammalogists meeting , Wildlife Disease Association meeting , Southwestern Association of Naturalists meeting , Wildlife Society meeting , 16th Vertebrate Pest Conference , and Colorado Pest Control Meeting . The other participants mailed their completed questionnaires and serum samples directly to CDC.The questionnaire included detailed questions about previous exposure to rodents, use of personal protective equipment to minimize exposure to rodent excretions and secretions, and any previous occurrence of a severe febrile illness that included shortness of breath. The lifetime number of rodents handled by a person was measured categorically: I (1–99), II (100–499), III (500–999), IV , V , and VI . Use of gloves, protective masks equipped with high efficiency particulate air (HEPA) filters, and protective eyewear also was measured categorically: always (>90% of the time), usually (50%–90% of the time), sometimes (10%–49% of the time), seldom (<10% of the time), or never.Neotoma spp.), other neotomine rodents, cotton rats (Sigmodon spp.), oryzomyine rodents (Oryzomys spp. or Oligoryzomys spp.), or other sigmodontine rodents. Of the persons included in the study, 699 had worked with rodents only in North America. The 58 others had worked with rodents in North America and in South America. The geographic distribution of exposure to rodents in North America was Canada (n = 36), Alaska (n = 8), the contiguous United States or District of Columbia . The total number of rodents handled by any 1 person ranged from category I (1–99) to category VI ; the median was IV . Of the 757 persons in the study, 751 (99.2%) had handled deer mice, white-footed mice, cotton rats, oryzomymine rodents, California mice, or woodrats .www.cdc.gov/ncidod/diseases/hanta/hps/noframes/epislides/episl7.htm). Of these, 259 (57.2%) were reported from 6 states in the southwestern United States: Colorado (n = 51), New Mexico (n = 71), Utah (n = 25), Arizona (n = 49), Nevada (n = 18), and California (n = 45). Sin Nombre virus is the only virus known to cause HPS in these 6 states. In this study, 387 (51.1%) persons had worked with rodents in Colorado (n = 124), New Mexico (n = 111), Utah (n = 65), Arizona (n = 90), Nevada (n = 33), or California (n = 169) and had handled deer mice. The total number of deer mice handled by persons in this group ranged from I (1–99) to VI ; the median was II (100–499).HPS was first recognized as a clinical entity in 1993 in the southwestern United States  includes Arizona, California, Colorado, New Mexico, Oklahoma, Utah, and Texas . The total number of California mice handled by persons in this group ranged from I (1–99) to III (500–999); the median was I (1–99). As indicated previously, 14 persons had worked with rodents in Florida and handled cotton rats. Three hundred and thirty-three persons had worked with rodents in Arizona (n = 87), California (n = 130), Colorado (n = 76), New Mexico (n = 101), Oklahoma (n = 40), Utah (n = 59), or Texas (n = 101) and handled woodrats. The total number of woodrats handled by persons in this group ranged from I (1–99) to V ; the median was I (1–99).BCNV virus has been found only in California (Lymphocytic choriomeningitis virus (LCMV) is the only Old World arenavirus species that is enzootic in North America. The house mouse (Mus musculus) is a member of the subfamily Murinae, family Muridae ; the median was I (1–99).Of the 757 persons in this study, 735 (97.1%) had worked with rodents before the discovery of HPS in 1993; during that time, 504 (68.6%) of them never or infrequently wore personal protective equipment  when handling rodents. In contrast, only 267 (36.3%) of these 735 persons never or infrequently wore personal protective equipment when handling rodents after the discovery of HPS. Use of personal protective equipment by the other persons in the study both before and after the discovery of HPS depended on the type of equipment.All unique identifying information was removed from the serum samples before they were tested for antibodies. Furthermore, all unique identifying information was removed from the computer (electronic) records before analysis of the demographic and serologic data.,Escherichia coli–expressed recombinant SNV nucleocapsid protein that is highly cross-reactive with other neotomine rodent-associated hantaviruses and with sigmodontine rodent-associated hantaviruses in the ELISA used in this study . The control (comparison) antigen for the SNV IgG ELISA was an E. coli–expressed recombinant protein that is antigenically unrelated to the SNV nucleocapsid protein. The arenavirus antigens were detergent lysates of Vero E6 cells infected with WWAV strain AV 9310135, GTOV strain INH-95551, or LCMV strain Armstrong. WWAV is highly cross-reactive with BCNV and TAMV in the ELISA used in this study . Collectively, WWAV, GTOV, and LCMV represent the 3 major antigen groups in the family Arenaviridae, as defined by ELISA (We tested the serum samples for immunoglobulin G (IgG) against SNV, WWAV, GTOV, and LCMV by using ELISA, as described (Antibodies against SNV were detected in 4 (0.5%) of the 757 persons in the study. Antibody titers were 1,600 in 2 persons and ≥6,400 in the other 2 persons. The total years worked with rodents and the lifetime number of rodents handled by the 4 antibody-positive persons were 9.0–30.0 (mean 21.3) and IV  to V , respectively. Two of the antibody-positive persons had worked with rodents only within the contiguous United States , 1 had worked with rodents in Arizona, Colorado, New Mexico, Utah, Texas, and Mexico, and 1 had worked with rodents in Michigan, Pennsylvania, Mexico, Costa Rica, and Argentina. All 4 antibody-positive persons had handled deer mice, white-footed mice, other neotomine rodents, cotton rats, and other sigmodontine rodents. Those who had worked in South Carolina or Argentina also had handled oryzomyine rodents. All 4 antibody-positive persons reported that they had never worn a protective mask or protective eyewear when handling rodents before the discovery of HPS. One reported having been hospitalized for an illness characterized by fever, headache, and severe shortness of breath (symptoms suggestive of HPS). This person had worked with rodents only within the contiguous United States.Antibodies against WWAV or GTOV were detected in 2 (0.3%) of the 757 persons in the study. Antibodies against WWAV  but not GTOV were detected in a person who had worked with rodents in Texas and Wisconsin and handled woodrats, other neotomine rodents, and sigmodontine rodents. Antibodies against GTOV  but not WWAV were detected in a person who had worked with rodents in Pennsylvania, Utah, and Wyoming and handled white-footed mice, other neotomine rodents, cotton rats, and other sigmodontine rodents. The lifetime number of rodents handled by the 2 antibody-positive persons were III (500–999) and IV , respectively. Both reported that they had never worn a protective mask or protective eyewear when handling rodents before the discovery of HPS. Antibodies against LCMV were found in none of the 757 persons in this study.,,Previously published studies found no antibodies against SNV in 583 persons who worked in Arizona or New Mexico in occupations that potentially exposed them to rodents or rodent droppings  of the 387 persons in this study who had worked with rodents in Colorado, New Mexico, Utah, Arizona, Nevada, or California and who had handled deer mice. Antibodies to SNV also were detected in 1 (1.2%) of the 81 persons who had worked in New York, Pennsylvania, or Rhode Island and who had handled white-footed mice. The antibodies against SNV in the 3 antibody-positive persons who had worked in the southwestern United States could be a consequence of infection with SNV. The antibodies against SNV in the person who had worked in Pennsylvania could be a result of infection with NYV.Of the 453 laboratory-confirmed HPS cases mentioned previously, 160 (35.3%) were fatal. Together, the high case-fatality ratio of HPS in North America, the lack of a vaccine against HPS, and the lack of a specific therapy for HPS should motivate persons to minimize their risk for infection while working in the field, classroom, or laboratory with rodents potentially infected with hantaviruses, especially those viruses known to cause HPS. Published guidelines for safely working with rodents potentially infected with hantaviruses include using protective gloves, respirators fitted with HEPA filters, and protective eyewear  captured in western Texas and in northern Texas , and arenaviruses antigenically closely related to WWAV strain AV 9310135 have been isolated from southern plains woodrats captured in southern Texas  captured in southern Mexico . The antibodies against GTOV in these 3 rodents are the first evidence that an arenavirus antigenically distinct from BCNV, TAMV, WWAV, and LCMV exists in North America and support the idea that the infection in the antibody-positive person in this study was a result of an arenavirus infection acquired in North America.Recently, antibodies against GTOV but not WWAV or LCMV were detected in 3 peromyscine rodents (


A key stage for all microarray analyses is the extraction of feature-intensities from an image. If this step goes wrong, then subsequent preprocessing and processing stages will stand little chance of rectifying the matter. Illumina employ random construction of their BeadArrays, making feature-intensity extraction even more important for the Illumina platform than for other technologies. In this paper we show that using raw Illumina data it is possible to identify, control, and perhaps correct for a range of spatial-related phenomena that affect feature-intensity extraction.We note that feature intensities can be unnaturally high when in the proximity of a number of phenomena relating either to the images themselves or to the layout of the beads on an array. Additionally we note that beads neighbour beads of the same type more often than one might expect, which may cause concern in some models of hybridization. We highlight issues in the identification of a bead's location, and in particular how this both affects and is affected by its intensity. Finally we show that beads can be wrongly identified in the image on either a local or array-wide scale, with obvious implications for data quality.The image processing issues identified will often pass unnoticed by an analysis of the standard data returned from an experiment. We detail some simple diagnostics that can be implemented to identify problems of this nature, and outline approaches to correcting for such problems. These approaches require access to the raw data from the arrays, not just the summarized data usually returned, making the acquisition of such raw data highly desirable. A key stage for all microarray analyses is the extraction of feature-intensities from an image. If this step goes wrong, then the experiment will certainly be compromised. Thus, much research has gone into the tasks of automatic identification of both the features on arrays and the grids on which they lie -4. IllumIllumina employ random construction of their arrays on a hexagonal grid . As a coWhile Illumina's software can report the raw bead-level data from an array (i.e. the location and intensity for every bead), more typically only summarized data are produced. We have previously shown that there are advantages to working with the bead-level data , not leaFor example, it has been shown  for micrOther theoretical aspects of the technology require investigation. The background intensity for arrays is calculated in a non-robust manner that might lead to extreme and biased values. Additionally, the high-density structure of the array may lead to situations where the intensity of a bead may influence that of its neighbours.There are three key steps in moving from an image of a BeadArray to a set of intensities. First the bead locations must be identified, second the beads must be mapped to a bead-type, and third, an intensity value extracted. Here we investigate each of these steps, showing how each can bias the final intensity, but showing also how such errors can be identified and corrected when working with bead-level data. We illustrate this using two bead-level data sets .Here we use two Illumina BeadArray platforms: The Human-WG6 V2 single-channel expression array, and the CNV370-duo two-channel genotyping and DNA copy number platform. Their differing structures are depicted in Figure et al [The copy number data we use consist of two arrays from a previously published data set, full details of which are provided by Curtis et al . The arret al .tif image file arising from the scanned array, the .txt file containing the locations of all decoded beads (decoding is discussed in the next section) as well as the associated (background-corrected) intensities, the .locs file that additionally contains the locations of the beads that were not decoded, and the .sdf file that contains details of the chip structure. To reduce download sizes the .txt and .locs file have been compressed using the package BeadDataPackR [The raw data we consider to be the .ataPackR . Instrucbeadarray package [beadarray or another BioConductor package, we make this available through an R script containing the additional functions , and Gx and Gy are the grid locations. The statistic where Gx + Δx etc.) and use the bead identities associated with these new grid positions. The shift Δx can be determined manually, or automatically by finding the shift that minimizes the mean within bead-type variance for the affected segment and maximizes correlation between replicate segments.Further, we consider the possibility that the mapping of bead identities to the beads can go wrong. This will either occur for a subset of beads, in which case we may detect it using our measure of departure from the grid, or it will occur for all beads, in which case we must remap the grid positions for each bead  within the .locs file.This process relies on being able to identify the centre of a bead to a resolution of a fraction of a pixel. Illumina report the bead locations to between 2 and 4 decimal places of a pixel in the .beadarray package.Background values are calculated from a 17 × 17 square of pixels located about the bead-centre in the non-sharpened image [Note that a 17 × 17 square could contain 12 bead-centres  is of concern.To investigate the magnitude of the bead location effect, we perform simulations of idealized beads. Each bead on the array we consider to be a sphere evenly covered in probes. This we assume results in a smoothly fluorescing feature when the bead is scanned. Then, we simulate the digitization of the image into discrete pixels by creating a pixel grid, integrating the fluorescence within each square of the grid, and rounding to an integer value. We then systematically move the grid, a fraction of a pixel at a time, and calculate the foreground intensity using the known bead-centre location.txt file to investigate the patterns of association in real data. We break a theoretical pixel into 100 × 100 bins and, for each bin, plot the mean of all log-intensities for beads whose fractional bead-centre locations coincide with the bin.We use the fractional part of the bead-centre locations and the intensities obtained from Illumina's .Where we identify an association, we investigate the nature of any causality by a) recalculating intensities with location-independent weights, and b) by averaging not the log-intensities but the residual log-intensities once the average for that bead-type has been removed. Since beads of the same type should be showing similar intensities, they should be similarly affected by the bead registration algorithm, and so this second step tests the possibility of log-intensity affecting location.locs files and mapping the neighbours of all beads. We assume that any such cluster of beads will contain a non-decoded bead with six non-decoded neighbours, and each such bead is used as a seed to identify maximal networks of neighbouring non-decoded beads (by use of an invasion process). Clusters we define as being any such network of 50 or more beads, and these are then expanded further to identify successfully decoded beads in the vicinity of the cluster.Clusters of non-decoded beads are identified by finding the locations of all non-decoded beads from the .EBImage package (version 3.3.1) [readTIFF function in beadarray which allows us to interrogate the pixels surrounding a bead.To identify bright beads that might interfere with their neighbours, we use the n 3.3.1)  to segmeIdentifying beads that neighbour beads of the same type is trivial using the neighbours matrix. In an idealized array containing 1, 000, 000 beads from 50, 000 bead-types where each bead has six neighbours we would expect to have 60 such pairs. This calculation ignores some of the structure of the array , and we investigate the numbers that we would expect for our arrays under a truly random construction as follows: We maintain the neighbours matrix for an array, but permute all bead identities of beads that were successfully decoded, tally the number of neighbouring pairs of the same type, and repeat.beadarray package along with the scripts we provide here to identify affected beads and remove them prior to summarization. Analyses follow the scheme illustrated in Additional File To assess the impact of the identified phenomena we use the On investigation of the possible dependence between the fractional part of the coordinate, and the derived log-intensity, a striking association is seen Figure . That thWe conducted simulations to establish whether such a large effect of fractional location on intensity was plausible. Under our simulations, it is clear that the relative position of the pixel grid over the bead can influence the measured intensity, with the value being greatest when the fractional part of the bead-centre coordinates is close to zero. The range of values that can be achieved is small however, being of the order of 0.2 on the log intensity scale for large bright beads. This value increases as the bead image becomes smaller relative to the pixels  would also seem questionable in these circumstances.For the vast majority of beads the difference between their given and predicted locations is very small, commonly less than one pixel. However, there are often thousands of beads where the deviation is greater than a pixel, and it can commonly be as large as 4 pixels, which is a concern given the beads have a presumed radius of 2-3 pixels and between bead-centre spacings of approximately 6 pixels. Some allowance must be made for irregularities in scanning the array but, given the theoretically regular nature of the grid, this seems to be evidence that beads are being mis-registered, and there is a distinct association between the degree of departure from the grid and residual log-intensity ,  and . The consequence of mapping to the wrong bead is clear, with many outliers being created. Bead G should map to the bright bead (in the red channel) to which bead F now maps, but instead maps to a region of low intensity. As a consequence, bead G is taken to exhibit lower intensity than is normal for that bead-type, while bead F exhibits greater intensity than does its cohort. Beads N and O should map to regions of low intensity, but instead return moderately high values as a consequence of their picking out of neighbouring beads. Naturally there are cases, such as P, where the bead-centre maps to the wrong bead in the image, but the two beads exhibit similar intensities so there is little effect.With localized problems such as this, where frequently only a single bead in a neighbourhood shows a concerning discrepancy between the two channels, it is natural to call upon the redundancy built into the Illumina BeadArray platform and simply discard the beads in question. In these circumstances, correcting a few extra beads would be unlikely to reward adequately the considerable efforts required. The task then is one of identifying these beads for removal, about which we now add a note of caution.When comparing the coordinates of beads from the two channels, one will immediately be struck by a discrepancy. Since the array is not in exactly the same position in the two images, a shift of one coordinate set will be required, often by a distance of ten or more pixels. However, closer inspection will show that this shift is not constant across the image. The nature of this shift is illustrated in Additional File Not only does this have implications for the identification of beads that deviate from the expected grid, but also raises more fundamental questions about the use of a constant number of pixels to calculate foregrounds and backgrounds. Moreover, having seen that the fractional part of a bead's coordinates is influential on the derived intensity, we may ponder what might be the implications of such a changing shift between the two channels.Sometimes the image registration can go dramatically wrong, resulting in the entire grid of bead-centre locations being positioned incorrectly within the image , and exclusion of beads around such clusters would be easy to implement. The influence of this bias across an experiment is illustrated in Additional File We have observed that it is common for successfully decoded beads, in the vicinity of such clusters, to display higher intensity than is expected for their bead type , and secondly we would expect the satellite beads to show similar intensities to beads of a similar type elsewhere on the array.Figure Out of the 289 pixels that are considered for the background calculation, the number that are "true background" pixels for a given bead ranges from 117 to 237 for one expression array (4343238066_A_1), with median 149. Note that if we took a 6 × 6 square foreground mask for each bead, more than two-thirds would be considered to have no "true background" pixels within their 289 candidates. However since the outer layer of that 6 × 6 square makes a negative contribution to the foreground calculation, it is reasonable to use the 4 × 4 pixel mask. As anticipated, when there are more true background candidate pixels, the non-robust measure of background tends towards more extreme values. The effect is not large in magnitude however, due to the generally low variability in background values, and so is unlikely to be a concern  would be alternatives to consider. Figure When such pixels fall within the background calculation region for a bead, they inevitably end up as one of the five lowest pixels in the region and so contribute to the background score for that bead. The resultant background scores for such beads are lower than they would otherwise be, and the final intensity for the bead is calculated to be higher than it should. There are few such pixels on an array, and their identification and exclusion from calculations would not present a technical challenge, however a simple change to the background calculating rule might present the simplest solution. Currently the mean of the five lowest pixels is used, however using a trimmed mean of a certain number of the lowest pixels or simply using the pixel of a certain rank  across the 24 sections of the two expression chips. We can detect no significant association between the presence of a neighbour of the same bead-type and observed intensity , it will be included 'as is' , or can be included after correction (such as the approach we present here). The remaining phenomena we have described will have a lesser impact, but occur apparently more frequently (summarized in Table If beads are not providing sound estimates of the quantity that they are designed to measure, yet by chance return an intensity consistent with the true target, then they will have the effect of increasing the perceived number of beads contributing to the estimate and thus will lead to artificially low standard errors and consequently to erroneous results. Since low standard errors are inherently desirable, it is unlikely that concerns will be raised in such cases.When the intensity is inconsistent with the true target, there is a chance that the standard Illumina analysis will identify these beads as outliers. However it is sub-optimal to use such a broad tool for this purpose, when more targeted options are available. Moreover, we have shown an example where the effect of a miscreant bead is to obscure the outlying status of other beads for which we have no explanation of their abberant intensities. These are the beads for which a general outlier removal step is warranted, and we have shown that a two-pass approach of removing known problems and then applying a general outlier removal can be beneficial.We summarize our findings in Table In addition to this, we have demonstrated that in theory the relationship between the position of a bead on the array and the grid of pixels introduced by creating a digital image of the array during scanning can affect the intensity value attributed to that bead. It appears that the majority of the association seen between bead-intensity and within-pixel bead location can be attributed to the intensity of a bead affecting the location at which it is found, yet for a bead to lie away from its anticipated location on the grid we have shown to be indicative of a biased intensity. It may be that different approaches for identifying beads and computing intensities could reduce or eliminate this source of variation.Finally we have shown that there are a variety of spatial effects that may impact on the reliability of results for individual beads. Particularly bright beads appear to display a 'bleed over' effect in which neighbouring beads are swamped by the signal being emitted from the bright bead. We have also identified isolated pixels with intensities far lower than the modal background for the array. Each of these artificially inflates the intensity attributed to their neighbouring beads, an effect also seen in the neighbourhood of clusters of non-decoded beads.Yet if they can be identified as suggested in the results and in Table The identification and remedy of the problems we have described are reliant upon access to the raw, bead-level, data. Even if one plans to analyse summarized data using Illumina's software, the routine recording of bead-level data provides a safety net should problems with the arrays be identified or suspected. With such routine access to bead-level data, an automated pipeline for quality assessment could be implemented painlessly, allowing the detection of the artefacts described here, and would provide reassurance regarding an independent summary-level analysis for experimenter, reader, and reviewer alike. Given this we highly recommend the collection and storage of bead-level data.MLS conducted analyses, authored code and drafted the manuscript. MJD provided Illumina expertise and helped to draft the manuscript. ST provided statistical expertise and helped to draft the manuscript. AGL conceived the study, provided statistical expertise, conducted analyses and drafted the manuscript. All authors read and approved the manuscript.R script containing functions that are not available in beadarrayClick here for fileVignette to reproduce figures and tables in this paper.Click here for fileFigure illustrating the Illumina foreground calculation.Click here for fileFigure demonstrating the between-bead dependence of background calculations.Click here for fileFlowchart illustrating preprocessing steps.Click here for fileFigure illustrating the results of the bead simulation exercise.Click here for fileFigure illustrating the association between departure from the grid and intensity.Click here for fileFigure illustrating the inconsistent shift required to align red and green images of the same section.Click here for fileFigure illustrating an example of image mis-registration.Click here for fileFigure illustrating the influence of various biases when considered across an entire experiment.Click here for fileFigure illustrating the influence of the number of true background pixels that a bead has on its calculated background.Click here for fileThe numbers of neighbouring pairs of beads of the same type, observed and theoretical values.Click here for file