Calcium and apatite granulations are demonstrated here to form in both human andfetal bovine serum in response to the simple addition of either calcium orphosphate, or a combination of both. These granulations are shown to representprecipitating complexes of protein and hydroxyapatite (HAP) that display markedpleomorphism, appearing as round, laminated particles, spindles, and films.These same complexes can be found in normal untreated serum, albeit at muchlower amounts, and appear to result from the progressive binding of serumproteins with apatite until reaching saturation, upon which the mineralo-proteincomplexes precipitate. Chemically and morphologically, these complexes arevirtually identical to the so-called nanobacteria (NB) implicated in numerousdiseases and considered unusual for their small size, pleomorphism, and thepresence of HAP. Like NB, serum granulations can seed particles upon transfer toserum-free medium, and their main protein constituents include albumin,complement components 3 and 4A, fetuin-A, and apolipoproteins A1 and B100, aswell as other calcium and apatite binding proteins found in the serum. However,these serum mineralo-protein complexes are formed from the direct chemicalbinding of inorganic and organic phases, bypassing the need for any biologicalprocesses, including the long cultivation in cell culture conditions deemednecessary for the demonstration of NB. Thus, these serum granulations may resultfrom physiologically inherent processes that become amplified with calciumphosphate loading or when subjected to culturing in medium. They may be viewedas simple mineralo-protein complexes formed from the deployment ofcalcification-inhibitory pathways used by the body to cope with excess calciumphosphate so as to prevent unwarranted calcification. Rather than representingnovel pathophysiological mechanisms or exotic lifeforms, these results indicatethat the entities described earlier as NB most likely originate from calcium andapatite binding factors in the serum, presumably calcification inhibitors, thatupon saturation, form seeds for HAP deposition and growth. These calciumgranulations are similar to those found in organisms throughout nature and mayrepresent the products of more general calcium regulation pathways involved inthe control of calcium storage, retrieval, tissue deposition, and disposal. Nanobacterium, which was initially assigned to the α-2subclass of Proteobacteria Biomineralization is a general phenomenon in nature that is not restricted toskeletal calcification alone. In fact, ectopic calcification has been generallyassociated with aging and other pathological processes. Perhaps nowhere else isectopic calcification more enigmatic and controversial than in the case associatedwith the so-called nanobacteria (NB). These are slow growing, pleomorphic,sub-micrometer (50–500 nm) entities coated with proteins and containingcarbonate hydroxyapatite (HAP) In view of the unusual characteristics of the proposed NB, deemed untenable as seenthrough the prism of conventional microbiology, the NB phenomenology has been widelycontested as lacking evidentiary support Together, these various studies point to the many fallacies inherent in theconclusions drawn from the NB literature, and they question the role proposed for NBin disease pathogenesis. For one, NB can no longer be considered as some novel,primitive, and exotic lifeforms or symbionts, as initially claimed These studies indicate that the entire NB literature must be reevaluated carefully,and that, furthermore, many of the earlier conclusions drawn on their biology andpathophysiology must be deemed as largely uninterpretable, if not flawed.Nonetheless, it is clear that entities that morphologically and chemically resemblethe so-called NB can indeed form from prolonged incubation of cell culture mediumsupplemented with serum and other body fluids A more thorough investigation prompted by these questions is also justified by thefact that calcium granules or inclusion bodies similar to NB are commonly found innature In this context, we demonstrate here that mineralo-protein complexes—in theform of laminated granulations, spindles with needle-like projections, andfilms—are formed directly in both FBS and adult human serum (HS) as aresult of calcium or apatite loading and that these complexes resemble thepreviously described NB as well as calcium deposits and granules seen throughoutnature. That is, NB-like precipitating particles will readily form in the serum whenthis same body fluid is challenged with calcium and/or phosphate ions, a processthat does not require the lengthy incubation usually needed to culture NB Calcification inhibitory factors in the serum with multiple binding sites areconceptualized here to bind avidly to calcium, phosphate, or nascent apatitethat upon saturation may precipitate to become seeding templates for furtherapatite growth. We speculate that the interplay between inhibitory and seedinginfluences should be physiologically relevant insofar as calcium regulation andhomeostasis are concerned. Given that human body fluids are supersaturated withrespect to calcium and phosphate ions Along this line of reasoning, the same calcium/apatite inhibitory and seedingfactors must be readily available in the serum as well as in the other bodyfluids. According to this simple model, these same calcium-binding inhibitoryfactors, whether they be fetuin-A, albumin, or other binding sites, shouldsequester increasing amounts of calcium ions by binding to them until thebinding sites for calcium  reach saturation. At thepoint of saturation, the same calcium-inhibitory complexes would then aggregate,become insoluble, and behave as seeds or nidi for further binding of calciumphosphate. This propagation process would continue until microscopic entitiessimilar to the previously described NB and calcium granules commonly found innature are formed. Assuming this reasoning to be correct, simply addingexogenous calcium or phosphate or a combination of both directly to serum, orperhaps to any body fluid, should produce insoluble mineralo-protein complexes,which when introduced into fresh culture medium should in turn seed even moreapatite as well as give rise to the entire NB phenomenology, including theproperties of pleomorphism and particle propagation. Extending this same logic,it should also be possible to demonstrate that normal, untreated serum containsthese same circulating complexes, albeit at much lower amounts. Structurally andfunctionally, these various calcium granulations as well as the putative NBshould all turn out to be virtually indistinguishable from each other.2,Na2HPO4, or a combination of both, could be addedslowly in a drop-wise manner to either filtered FBS or HS, followed by vigorousshaking, without resulting in immediate precipitation. In this manner,CaCl2 could be slowly added to FBS to concentrations as high as50 mM without inducing precipitation even after one hour of incubation at37°C or after two hours of incubation at room temperature. Likewise,under the same incubation conditions, up to 100 mM of CaCl2 could beadded to several batches of HS without resulting in any precipitation. The lackof precipitation in any one of these experiments could be ascertained bycentrifugation at 16,000×g at room temperature for 1hour, which would have been expected to sediment NB-like complexes 2HPO4 could be added up to 30 mM toseveral lots of serum without any apparent precipitation. In marked contrast,when both CaCl2 and Na2HPO4 were added togetherto either FBS or HS, precipitations appeared readily when as low as 3 mM of eachion were introduced, indicating that the propensity for precipitation greatlyincreased when both ions were added simultaneously.Preliminary experiments showed that concentrated solutions of CaClOn the other hand, these same preliminary experiments also revealed a markedvariability of results depending on the lot of serum used. That is, differentbatches of FBS and HS produced significant differences in the extent ofion-triggered precipitation. Significant differences were also noticed dependingon the age of the serum lot tested. Compared to fresh serum for example, agedserum  tended to produceprecipitates more readily when the same amounts of calcium and/or phosphate wereadded. As a result of aging, some batches of serum were seen to produceprecipitates spontaneously through storage alone and without any ion treatment,but in general untreated serum produced little or no visible precipitation uponcentrifugation.2and Na2HPO4 . Thus, while shortincubations as described above did not lead to any visible precipitation whenindividual ions were added even to artificially high concentrations, prolongingthe incubation after the same serum treatment produced steady precipitation as afunction of time. In fact, either calcium or phosphate produced precipitationwhen added to 6 mM or 3 mM, respectively, provided that the subsequentincubation was prolonged to 2 hours at 37°C or overnight at roomtemperature. Likewise, under these incubation conditions, precipitation wasobserved when both calcium and phosphate were added together to as little as 1mM each. With longer periods of incubation, the precipitating concentrations ofthe same ions could be further reduced. For some serum batches, precipitationwas noticed with as little as 0.1 mM calcium phosphate added when the subsequentincubation was lengthened to 48–72 hours at 37°C.The same experiments also revealed marked differences in precipitation dependingon the length of incubation allowed following the addition of CaClIn all instances of serum treatment with precipitating ions, white, granularprecipitates that could be dispersed in solution became visible. In the presenceof excess calcium, pellets appeared granular and fluffy to the eye, while theexcess of phosphate produced gelatinous, grayish pellets that appeared moreclumpy and resistant to dispersion even by vigorous shaking. These visualcharacteristics could be easily modulated by adding different amounts of calciumand phosphate.650 reading remained low,indicating that some of the complexes may have remained in suspension in theion-treated serum following the lower-speed centrifugation used before.The experiments shown in 650 reading following the same 3-day incubation produced little detectable material that appeared ashighly refringent and heterogeneous particles as well as patches varying in sizemarkedly between 0.1 to 10 µm  Nanobacterium sp. strain Seralab 901045 used by Raoult etal. These images were remarkably similar to those obtained from previouslycharacterized NB and chosen as controls in our study . To makeSerum pellets were next studied by higher resolution imaging techniques likescanning electron microscopy (SEM) and transmission electron microscopy (TEM)–5. As sIn the presence of exogenous phosphate, the round particles were seen to rapidlycoalesce into aggregates that looked like the granular mattresses seen earlierwith dark field microscopy . DiffereControl, untreated serum pellets also displayed a whole array of shapes. Mostappeared as poorly defined structures with amorphous and heterogeneous shapes.As noted earlier, serum that had not been treated with precipitating ionsproduced very little pellet; this pellet gave principally images of amorphous,poorly-defined shapes with heterogeneous sizes not seen with the other serumpellets prepared by ion loading see and filard row, The images obtained for these serum pellets were remarkably similar to thosecollected for bovine and human NB shown in . In the When examined by thin section TEM, serum pellets also displayed either the roundparticle  or the sThese morphologies were similar to those seen for NB as exemplified here by bothNB obtained from serum-containing medium as well as the NB strain“nanons” . As befoA laminated and variegated granular morphology, displaying alternatingelectron-dense and pervious layers resembling “bull'seyes,” could be more clearly seen with NB“strains” that had been maintained through multiple serialpassages in the presence of excess serum (5–10% FBS), asillustrated by thin section images of “nanons” shown in thebottom row of Similar images are also associated with calcium granules commonly found in natureIt should also be noted that the serum pellets studied here display markedpleomorphism, with morphologies that include granular as well as membranous andfilamentous patches, while the calcium granules described earlier were notedmainly for their spherical, laminated shapes 10 HAP crystals. For the small serum pellets obtainedfrom control serum, amorphous patterns predominated  analysisof the serum granulations yielded a wide repertoire of amorphous 2, and HAP controls shown in Next, energy-dispersive X-ray spectroscopy (EDX) was done on the same serumpellets obtained from either control serum or from serum treated with calcium,phosphate, or both. As revealed by EDX analysis, untreated serum pelletscontained various elements, including calcium and phosphorus 2 and HAP shown in Serum granulations obtained from untreated serum or from serum treated withadditions of either calcium, phosphate, or both, gave peaks corresponding tocarbonate groups as well as phosphate moieties . Peaks c−1, they gave much stronger signals at the1,400–1,430 cm−1 position, indicating the clearpresence of amorphous carbonate in these preparations. Compared to the carbonatesignals, the phosphate signals were more prominent in all the serum pellets. Inthe case of untreated FBS granulations, the peak corresponding to carbonategroups at 875 cm−1 was absent . The amide III peak on the other hand overlapped with the broadphosphate group seen on its right 2 and HAP,as shown in −1, 712 cm−1 and1,080 cm−1 2 and HAP controls. On the other hand, HS granulations obtained followingaddition of calcium yielded a low peak of carbonate near 1,080cm−1 whereas the phosphate signals were either absentor barely visible 2, and HAP nowincluded as controls in We examined further the crystalline nature of serum granulations by analyzingtheir electron diffraction patterns obtained by TEM , insets.As can be seen from the diffraction patterns presented in the insets of By SDS-PAGE analysis, the protein profiles associated with serum granulationsconsisted of three main bands . Lanes 1As shown before for FBS-NB For the HS pellet, albumin (2/2), fetuin-A (1/1), and apolipoprotein A1 (1/1)were also identified as indicated in In that earlier study The main proteins obtained using this alternative proteomic approach aredisplayed in th), andapolipoprotein A1 (5th) representing among the five main proteinsidentified in each case. The presence and relative abundance of these threeproteins are in agreement with the protein gel profiles presented earlier (nd) andprothrombin (3rd), were also identified that were not obvious fromthe SDS-PAGE analysis. In other words, with its relative abundance, complementcomponent 3 should have appeared as a noticeable band of 187 kDa in theSDS-polyacrylamide gel while prothrombin (71 kDa) should have been detected inthe albumin region after gel excision and protein determination, but as seenfrom As seen in  earlier . HoweverAlbumin, complement component 3, prothrombin, fetuin-A, and apolipoprotein A1also appeared among the main proteins obtained for FBS-NB . The proth amongproteins found in HS granulations and therefore was not listed in For HS granulations, the three proteins albumin, complement component 3 andapolipoprotein A1 were also among the more abundant proteins identified . However2-macroglobulin, which turned out to be among the threemost abundant proteins for human NB, all other proteins appeared to follow thesame frequency pattern shown for HS granulations  g. After 2 hours of incubation, little orno pellet could be discerned, but with longer overnight incubations, smallpellets appeared with both trypsin-treated FBS and HS. Upon inoculation of thesepellets into serum-free DMEM, it could be seen that the pellets obtained from anovernight trypsin-treatment produced particle seeding following incubation, asevidenced by the clouding of the medium seen both visually and byA650 reading (650reading for these 2 samples). The same particle-seeding effect wastime-dependent and increased over a course of several days, ruling out thepossibility that the initial pellet could have accounted for the turbidityreadings (compare the various panels examined on “Day 4” and“Day 8” with the panel labeled as “Day1” referring to the first day of pellet collection and seeding). Incontrast, neither control DMEM .In our hands and compared to trypsin, chymotrypsin was less effective inproducing serum pellets, with the incubation done under identical conditions. Asexpected, the amount of subsequent particle seeding produced by serumgranulations obtained after chymotrypsin treatment was also significantly lower.On the other hand, when either calcium or phosphate is added individually toboth FBS and HS, saturation is not reached until much higher amounts of eitherion species are present, indicating that the inhibitory mechanisms inherent inthe serum can be effectively overcome only when both calcium and phosphate aresimultaneously present. Short of their dual presence, it is more likely that astate of calcification-inhibition would prevail instead.−6 M2−6 M2, stillbelow the ion-product threshold for calcification. However, the ion-productconcept cannot fully explain the different susceptibilities of serum toprecipitate in response to the various precipitating ions added. Thus, ourexperiments demonstrate that while the precipitation-threshold level of6×10−6 M2 for the ion-product canbe exceeded by adding either calcium, phosphate, or a combination of both, togive the same final ion-product, the simultaneous addition of both calcium andphosphate is much more effective in inducing HAP formation than the addition ofeither calcium or phosphate alone. Accordingly, addition of calcium alone to HSgiving an ion-product that exceeds 6×10−6M2 by as much as 30% has shown to produce little or noprecipitation. Along the same line of reasoning, phosphate is more effectivethan calcium, when added to the same final ion-product. Furthermore,precipitation occurs with the addition of only 1 mM of both calcium andphosphate to serum that would give in turn an ion-product of5.06×10−6 M2, still below thethreshold level for precipitation, provided only that a prolonged incubation isused. It can be inferred therefore that the calcification-inhibitory processesinherent in the serum are geared to dealing more efficiently with loadings ofeither calcium or phosphate alone and are overcome much more easily with thesimultaneous presence of excess calcium and phosphate.The need for both calcium and phosphate to be present to disarm this samecalcification-inhibition reinforces the known concept of ion-product  used todetermine the propensity for calcification in any given fluid system Given that the body fluids are supersaturated with respect to calcium andphosphate ions in vitro or in vivoconditions used, including whether the proteins were tested soluble orimmobilized on solid substrates In this context, the large number of proteins bound to the calcium granulationsobtained from both HS and FBS, along with the earlier spectroscopy datasuggesting an abundant presence of bound proteins, indicate that the apatiteformation seen here corresponds probably to a process of“heterogeneous nucleation”—the formation ofapatite crystals on a protein template invitro experiments done on several body fluids challenged withcalcium phosphate loading The presence in the serum granulation and NB scaffold of proteins like albumin,complement components 3 and 4A, prothrombin, fetuin-A, apolipoproteins A1 andB100, as well as other calcium and apatite binding proteins, appears to beentirely circumstantial, depending on their availability in the serum milieu.This conclusion is supported by the presence of select proteins in the granulescaffold that depend solely on the particular species and type of serum used. That is, theprotein profile seen here reflects largely the relative abundance of these sameproteins in the serum used. Thus, albumin, the predominant protein identifiedhere, is not only one of the more abundant serum proteins but is also known toaccount for about half of the calcium-binding activity measured in HS in vitro requires the presence of fetuin-A fortheir formation and stabilization, but the CPP found in vivocontain a low amount of fetuin-A in vivoThe formation of mineral complexes in pathophysiological conditions has beenillustrated recently by the description of calciprotein particles, and morespecifically, by the so-called “secondary CPP,” which havebeen isolated from the ascites of a patient suffering from calcifyingperitonitis linked to kidney disorders Evidence that mineral complexes similar to the ones described in this study mayform in pathological conditions also comes from the observation that treatingrats with etidronate, a biophosphonate known to decrease the mineralization ofbones, or vitamin D, a liposoluble vitamin known to be required for theabsorption of intestinal calcium and phosphate ions, led to the formation ofprotein-mineral particles in the blood of rats That fetuin-A is indeed involved in the formation and clearance of the mineralcomplexes has been elegantly established by observations on fetuin-A knockoutmice, shown to spontaneously develop ectopic calcification of soft tissues An analysis of the propensity for CPP to form in the body also needs to take intoaccount the amount of albumin and other calcium and apatite binding proteins aswell as the calcium phosphate ion product present at the site of calcificationOnce formed, the calcium and apatite granulations seen here are probably siphonedfor clearance, probably through the RES, as it has also been suggested to be thecase for CPP The potential involvement of other proteins and factors associated with thecalcium granulations seen here suggests that these entities are much morecomplex than what fetuin-A alone can possibly explain. Thus, while the elegantstudies performed by Price, Jahnen-Dechent, Heiss, Schäfer, Ketteler,and their colleagues have been centered on the role of fetuin-A as the basis forthe formation of mineralo-protein complexes, it appears that the role of otherproteins and factors must also be taken into account to explain the biology ofthe serum-derived calcium granulations observed here.It appears that the serum calcium granulations seen here are also closely linkedto normal bone formation as well as ectopic calcification. Electron microscopystudies have established that the mineral precursor of bone and enamel actuallyconsists of round amorphous nanoparticles of calcium phosphate with small sizesranging from 5 to 50 nm which take form inside the collagen fibrils and thatfuses in a co-linear fashion until filaments are formed From a yet another perspective, the calcium and apatite complexes seen hereappear to resemble calcium granules found widely in nature Calcium granules have also been found in a number of other mammalian tissues,including cartilage, atherosclerotic lesions, kidney and cancer tissues as wellas in association with Randall's plaque While there is marked morphological resemblance between the serum granulationsobserved here and the earlier described calcium granules normal, rather thananomalous or even pathological, calcium metabolism! These same authors hadearlier suggested that the same electron-dense granules observed near thebasement membranes of collecting ducts and the interstitium showed gradualmigration into a subepithelial location in the renal papillae that then form theso-called Randall's plaque As for the involvement of these same granules (or NB) in human pathologies, onearea that deserves mention is kidney stone and its link with the formation ofRandall's papillary plaque fetal bovine antigens presumably detected by the samestudies may have inadvertently been used to assert the presence of NB in anincreasing number of human diseases Regarding the involvement of NB in the development of kidney stones as well asother disease conditions, it should be noted that our earlier study provided adetailed analysis questioning NB both as living organisms and as pathogenicagents of disease In further support of this alternative position on NB that questions theirproposed involvement in the development of kidney stones via Randall'splaque, it should be pointed out that Ryall's review The NB field  has probably garnered more mediaattention and controversy than any other field in recent memory The calcium granulations described in this study also share some characteristicswith the so-called matrix vesicles (MV), which represent another kind of calciumphosphate template implicated in the formation of mineralized tissues in thebody. Skeletal cells such as osteoblasts, chondroblasts, and odontoblastscontrol the deposition of minerals in the body by releasing MV which initiatecalcification in selected and restricted areas destined to be mineralized An analogy can also be drawn between the formation of the calcium granulationspresented here and the formation of polymer gels from dissolved organic matteras described earlier Finally, it should be noted that similar granulations have been observed in humanblood specimens and likewise described over the past century as“inclusion bodies,” “filterable bodies,”“microzymas,” “Granuligera,”“protids,” “somatids,” “bloodbacteria,” among others gfor 15 min at room temperature. The supernatant corresponding to HS was savedinto another tube. FBS  andHS were successively filtered through 0.2 µm and 0.1 µmmembranes prior to use. Serum pellets were prepared by adding sterile solutionsof either 0.25 M CaCl2, pH 7.4, or 0.25 MNa2HPO4 (adjusted to pH 7.4 with 0.25 MNaH2PO4), or a combination of both, into filtered FBSor HS (2.5 ml) at final concentrations ranging from 12 mM to 48 mM forCaCl2, 6 mM to 24 mM for Na2HPO4, or 1 mMto 2 mM of each when both CaCl2 and Na2HPO4were added. The serum pellets shown in 2 (labeled as FBS-2/HS-2), 24 mMNa2HPO4 (labeled as FBS-3/HS-3), or 2 mM of bothCaCl2 and Na2HPO4 (labeled as FBS-4/HS-4)to the serum. Ion solutions were added drop-wise with vigorous agitation toavoid precipitation. Treated or control sera were incubated at 37°C for2 hours  or HEPES buffer  using the same centrifugation steps.Human blood was collected from healthy human volunteers using a conventionalvenipuncture method. The use of human samples was approved by the InstitutionalReview Board of Chang Gung Memorial Hospital  and supported by written informed consents. Whole blood was collectedinto Vacutainer tubes without anticoagulant  and centrifuged at 1,500×used for and 4 orused for  with geng at 4°C for 2hours using a L8-80M ultracentrifuge . Thepellets were then washed twice with either filtered DMEM, HEPES buffer, ordouble distilled water using the same centrifugation steps.Alternatively, the untreated serum pellets were prepared by incubating filteredFBS or HS (38 ml) with gentle agitation overnight at room temperature, followedby centrifugation at 140,000×650 weredone as before 2 to FBS, followed byeither a short incubation of 1 hour (FBS-2′) or overnight(FBS-2″) at room temperature, and were processed as describedabove.For Nanobacterium sp. strains designated as DSM 5820 andDSM 5821, initially isolated from commercially available FBS Nanobacterium sp. strain Seralab901045” 2 flask  showing abundant culture and an aliquot of 2 ml wascentrifugated at 16,000×g at room temperature for 15min. The pellet was washed twice with HEPES buffer or double distilled waterusing the same centrifugation steps. NB were then resuspended in doubledistilled water and used as such for the different microscopy, spectroscopy, andproteomic analyses.Putative NB were obtained from FBS or HS as described earlier g at room temperature for 1hour. The pellets were then washed twice with HEPES buffer and were resuspendedin 100 µl of the same buffer. Small aliquots (10 µl) of theresuspended pellets were inoculated into DMEM and incubated in cell cultureconditions. FBS and HS that received no treatment with trypsin or chymotrypsinwere processed exactly the same way as controls.Serum pellets used for the experiment described on Washed serum pellets and NB samples were resuspended in double distilled waterand aliquots were deposited on glass slides. The samples were visualized withoutfixation or staining with a BX-51 optical microscope equipped with a dark field condenser  and a 100× oil immersion objective withiris . The specimens were visualized at a magnification of1,000× and the images were acquired with a Spot Flex color,charge-coupled device (CCD) camera . Commercially available HAP  was used here as well as throughout this study as acontrol.For scanning electron microscopy (SEM), washed serum pellets and NB specimenswere resuspended in double distilled water and deposited on formvarcarbon-coated grids. After drying overnight under a laminar flow hood, thespecimens were coated with gold for 90 seconds and observed with a SEM S-5000field-emission scanning electron microscope . For transmission electron microscopy (TEM), washed serum pellets and NBsamples were deposited on formvar carbon-coated grids without staining; theexcess liquid was removed with an absorbent paper, and the grids were then driedovernight. For thin sections, the same specimens were washed twice in doubledistilled water and dehydrated with two washes of 100% ethanol. Thesamples were then embedded in Epon 812 resin  and incubated at 72°C for 2 days to allow resinpolymerization. Thin sections were cut using a Leica Ultracut UCT microtome and were transferred on formvarcarbon-coated grids. Specimens prepared for TEM were observed without stainingwith a JEOL JEM-1230 transmission electron microscope operated at 120 keV.Washed serum pellets and NB specimens resuspended in double distilled water weredeposited on glass slides and dried overnight. Powder X-ray diffractionspectroscopy (XRD) was performed using a Bruker AXS D5005 X-ray diffractometer equipped with a sealed 2.2 kW copper X-raysource, a diffracted beam monochromator, and a scintillation counter detector.Experimental diffraction spectra were compared with the database of the JointCommittee on Powder Diffraction and Standards (JPCDS) in order to identify thechemical formula of each crystalline compound. Electron diffraction patternswere acquired with a JEOL JEM-1230 transmission electron microscope (JEOL)operated at 120 keV.3  and calcium phosphate tribasic, also labeled asCa3(PO4)2 in the figures, were used forcomparison with the experimental specimens.Aliquots of washed serum pellets and NB specimens resuspended in water weredeposited on formvar carbon-coated grids and dried overnight. Energy-dispersiveX-ray spectroscopy (EDX) was performed using a SEM S-3000N scanning electronmicroscope (Hitachi Science Systems) equipped with an EMAX Energy EX-400 EDXdevice  as described before −1 and represented an average of 32 consecutivescans.Washed and dried serum pellets and NB specimens were mixed 1∶100 (w/w)with KBr powder and compressed with a hand press to form a thin pellicle. FTIRspectroscopy was performed using a Nicolet 5700 FTIR spectrometer  equipped with a deuterated triglycine sulfate(DTGS) detector. Each spectrum was obtained at a resolution of 4cmWashed serum pellets and NB samples were resuspended in double distilled waterand deposited on glass slides. The specimens were then dried overnight at roomtemperature under a laminar flow hood. Micro-Raman spectroscopy was performedusing the inVia Raman confocal microscope  equippedwith a CCD detector. A laser beam of 633 nm operated at 17 mW was focused on thespecimens and provided the excitation source.2 µl aliquots of each 100 µl pellet suspension describedearlier were successively mixed with 14 µl of 50 mM EDTA and 4µl of concentrated “loading buffer” . Theprotein samples were heated at 95°C for 5 min prior to gelelectrophoresis using a mini-gel system . The gelswere then stained with Coomassie blue as described Protein bands from gels were excised, reduced and alkylated, in-gel digested withtrypsin, and further processed for mass spectrometry (MS) 18 column . Elution wasperformed with a succession of different solutions containing an increasingconcentration of formic acid (starting with 0.1% formic acid dilutedin 99.9% acetonitrile). The liquid chromatography column was placedin line with a 2-D linear ion trap mass spectrometer  operated using the Xcalibur 2.0 software (Thermo Fisher).In addition, serum pellet and NB samples were submitted to in-solution trypsindigestion as described earlier Mass fingerprints were searched against the Swiss-Prot database using the MASCOTsearch engine  as described before


To facilitate whole-genome association studies (WGAS), several high-density SNP genotyping arrays have been developed. Genetic coverage and statistical power are the primary benchmark metrics in evaluating the performance of SNP arrays. Ideally, such evaluations would be done on a SNP set and a cohort of individuals that are both independently sampled from the original SNPs and individuals used in developing the arrays. Without utilization of an independent test set, previous estimates of genetic coverage and statistical power may be subject to an overfitting bias. Additionally, the SNP arrays' statistical power in WGAS has not been systematically assessed on real traits. One robust setting for doing so is to evaluate statistical power on thousands of traits measured from a single set of individuals. In this study, 359 newly sampled Americans of European descent were genotyped using both Affymetrix 500K (Affx500K) and Illumina 650Y (Ilmn650K) SNP arrays. From these data, we were able to obtain estimates of genetic coverage, which are robust to overfitting, by constructing an independent test set from among these genotypes and individuals. Furthermore, we collected liver tissue RNA from the participants and profiled these samples on a comprehensive gene expression microarray. The RNA levels were used as a large-scale set of quantitative traits to calibrate the relative statistical power of the commercial arrays. Our genetic coverage estimates are lower than previous reports, providing evidence that previous estimates may be inflated due to overfitting. The Ilmn650K platform showed reasonable power (50% or greater) to detect SNPs associated with quantitative traits when the signal-to-noise ratio (SNR) is greater than or equal to 0.5 and the causal SNP's minor allele frequency (MAF) is greater than or equal to 20% (N = 359). In testing each of the more than 40,000 gene expression traits for association to each of the SNPs on the Ilmn650K and Affx500K arrays, we found that the Ilmn650K yielded 15% times more discoveries than the Affx500K at the same false discovery rate (FDR) level. Advances in SNP genotyping array technologies have made whole-genome association studies (WGAS) a readily available approach. Genetic coverage and the statistical power are two key properties to evaluate on the arrays. In this study, 359 newly sampled individuals were genotyped using Affymetrix 500K and Illumina 650Y SNP arrays. From these data, we obtained new estimates of genetic coverage by constructing a test set from among these genotypes and individuals that is independent from the SNPs and individuals used to construct the arrays. These estimates are notably smaller than previous ones, which we argue is due to an overfitting bias in previous studies. We also collected liver tissue RNA from the participants and profiled these samples on a comprehensive gene expression microarray. The RNA levels were used as a large-scale set of quantitative traits to calibrate the relative statistical power of the commercial arrays. Through this dataset and simulations, we find that the SNP arrays provide adequate power to detect quantitative trait loci when the causal SNP's minor allele frequency is greater than 20%, but low power is less than 10%. Importantly, we provide evidence that sample size has a greater impact on the power of WGAS than SNP density or genetic coverage. It has been estimated that the human genome contains more than 5 million common single nucleotide polymorphisms (SNPs) with minor allele frequencies (MAF) ≥10% There are two strategies to building whole-genome SNP arrays. One is to randomly select SNPs that are relatively evenly spaced across the genome, not taking into account the inter-SNP linkage disequilibrium (LD) patterns, such as the Affymetrix 100K and 500K SNP arrays  The identification of tag SNPs is essentially a feature selection problem. It has been well established in the machine learning field that using an independently sampled test dataset is necessary to guarantee an unbiased assessment of the selected features' operating characteristics. It has also been shown that if the evaluation takes place on the training dataset itself, then the quality of the features' performance is often anti-conservatively over-estimated, commonly referred to as the overfitting problem The key operating characteristics of several whole-genome SNP arrays have been evaluated recently on HapMap data We utilized two commercially available high-density SNP arrays on an American Caucasian cohort to obtain estimates of genetic coverage for these different SNP panels that are robust to overfitting. The estimates we obtain in this cohort are lower than previous reports. In addition, liver RNAs were extracted and profiled on a comprehensive gene expression microarray. By simultaneously utilizing these thousands of gene expression traits scored on a fixed set of genetic backgrounds, we obtain estimates of the relative power of the different SNP genotyping arrays to detect quantitative trait SNPs of varying effect sizes Human liver tissue samples were collected as described in a companion article −4. In total, 286K and 545K genotyped autosomal SNPs on Affx500K and Ilmn650K, respectively, were used in the analysis. Genotypes from Ilmn300K and Ilmn550K were also derived as subsets of the Ilmn650 data, resulting in 296K and 514K genotyped SNPs, respectively.DNA specimens were extracted and sent to Perlegen Sciences Inc. and Illumina Inc. for genotyping services using Affx500K and Ilmn650K, respectively. There was an overlap of >80,000 SNPs tiled on both SNP arrays. We found the two platforms gave highly consistent (98.7%) genotypes on their shared SNPs, suggesting both genotyping arrays are fairly accurate. We filtered out SNPs with MAF <5%, call rate <90%, or Hardy-Weinberg Test p-value <10Additionally, we purified RNA from the tissue samples and measured the 39,280 gene transcription levels using the Agilent platform. We adjusted the expression values for gender, age, and medical center by using a standard linear model. See The HapMap data are comprised of 270 individuals from four ethnic groups: (i) 30 trios from the Yoruba group in Ibadan, Nigeria (YRI); (ii) 30 trios from the CEPH collection, which are Utah residents with Northern and Western Europe ancestry (CEU); (iii) 45 unrelated Han Chinese individuals from Beijing, China (CHB); and (iv) 45 unrelated individuals from Tokyo, Japan (JPT). The CHB and JPT samples are often considered as a single East Asian sample Recall that the Affx500K platform harbors 90K common SNPs that were not utilized in the HapMap project (referred to here as Affx NonHapMap SNPs). The genotypes from the Affx500K platform measured on the 359 Liver Study subjects therefore provide two key sources of independent data: (i) genotypes of SNPs identified independently from the HapMap project (the Affx NonHapMap SNPs) and (ii) individuals sampled independently from the HapMap project. This allowed us to study SNP selection overfitting and sample overfitting, respectively, in calculating genetic coverage.Given that SNPs on the Affx500K were randomly chosen, the 90K Affx NonHapMap SNPs can be considered as a random sample of the entire 5.3M NonHapMap SNPs. We therefore utilized these Affx NonHapMap SNPs as a set of SNPs identified independently from the HapMap data. Also note that the complementary Affx HapMap SNPs (SNPs on Affx500K that are included in HapMap data) represent a random subsample of the 2.2M HapMap SNPs. We utilized these two classes of SNPs genotyped in our independently sampled human cohort scored for thousands of gene expression traits to estimate the true genetic coverage of the different SNP panels as well as assess the power these panels afford to detect associations between SNPs and traits (details below).2 cutoff (r2cutoff) with at least one SNP typed by the microarray 2cutoff, 0.8 and 0.9. Results were qualitatively equivalent in the 0.7 to 0.9 range for r2cutoff.Genetic coverage was calculated as the fraction of SNPs exceeding a pre-defined rOverfitting of genetic coverage estimates was assessed separately for SNP overfitting and sample overfitting.To systematically evaluate whether there has been SNP overfitting in estimated genetic coverage, we compared genetic coverage rates of Affx NonHapMap SNPs to Affx HapMap SNPs on the Illm650K platform. A higher genetic coverage of Affx HapMap SNPs is evidence for SNP overfitting. The SNPs represented on Ilmn300K and Ilmn550K are subsets of Ilmn650K, and we computed their genetic coverage in a similar manner.To determine whether there has been sample overfitting in previous estimates, we compared genetic coverage of the CEU individuals versus the Liver Study individuals. A higher level of genetic coverage in the former is evidence of overfitting. We made this comparison among Affx HapMap SNPs, among Affx HapMap SNPs, and among all Affx SNPs combined. Evidence for sample overfitting was present for all three sets of SNPs. Finally, for both types of overfitting, we stratified the AffxSNPs into MAF bins, and computed coverage for each bin to investigate whether the overfitting bias is also a function of MAF.2), where σ2 was constantly set to 1 and μ varied among genotypes. We set  = , , or  to investigate a range of signal strengths. Second, we conducted single-marker tests, which examined association between each SNP and each simulated trait. We surveyed three choices of α level  that are reasonable for WGAS. Kruskal-Wallis and Spearman rank correlation tests were employed because these non-parametric methods were robust to the underlying genetic model and trait distribution, thereby allowing our simulation to be useful for non-normal traits and non-additive models. We defined a “true discovery” to be any association detected within 200 kb of the causal SNP. We calculated statistical power  and the average number of true discoveries (NTD) over the set of simulated datasets. Two million simulation runs were conducted for each parameter setting.The statistical power of the SNP array platforms for WGAS were first estimated from simulation studies. First, we randomly selected a SNP from Affx500K and used its genotype to simulate trait value. Over the range of simulations, SNP genotypes from both Affx HapMap SNPs and Affx NonHapMap SNPs were utilized. We assumed the trait followed a Normal distribution N for each of the ∼40,000 gene expression traits measured. Furthermore, we repeated the tests on three permuted gene expression datasets and calculated the false discovery rate (FDR). In each permutation run, we first randomized the patient IDs in the expression file, breaking any association between expression traits and genotypes while leaving the respective correlation structures among gene expression traits and SNP genotypes intact. Second, we repeated the association tests for every expression trait and genotype pair, resulting a set of null statistics for each permutation. A standard FDR estimator was then applied to the resulting association statistics, as previously carried out on observed and permutation null statistics Based on the Liver Study subjects, we obtained new estimates of genetic coverage for the Illumina and Affymetrix SNP genotyping arrays , which aBy comparing estimates of genetic coverage derived from HapMap CEU to those from Liver Study subjects , we alsow = 2.2/7.5) and that on NonHapMap SNPs (w = 5.3/7.5). Among the Liver Study Caucasian subjects, the Ilmn300K and Ilmn650K's weighted genetic coverage was 64% and 76% at r2cutoff = 0.8, which is lower than previous reports . Furthermore, we found that the tagSNP arrays cover low MAF SNPs (e.g. MAF<15%) worse than the high MAF ones , and importantly, the overfitting bias appears to be more severe for the low MAF range  was proportional to the number of SNPs employed in the WGAS, with NFDcreening .Finally, we conducted association tests on actual traits, namely RNA expression levels or “expression traits.” It has been shown that comparing the ND from many related traits, all conditional on the same set of individuals , can lead to meaningful empirical power comparisons, where simple models often used for simulations do not have to be assumed The number of discoveries (ND) obtained on both observed data and permuted data followed the same pattern: Ilmn650K>Ilmn550K>Ilmn300K>Affx500K. Because the true and false discoveries were no longer distinguishable, we could not directly infer the SNP arrays' statistical power using ND. Instead, we compared the relative power using ND conditioning on FDR  and S3. After filtering SNPs based on MAF, call rate, and HWE p-values , a similcis-associations. In brief, for a given expression trait, only SNPs within ±1 Mb of the corresponding gene are tested. By taking these steps, the number of tests is substantially reduced and the statistical power increased, illustrated in more cis-association discoveries in cis-association genes also reflect the relative power. Consistent with cis-association. Surprisingly, Affx500K+Ilmn650K (relative power = 115%) only slightly outperforms Ilmn650K, indicating the limited return of adding additional SNPs on top of Ilmn650K.As a novel feature of our study, we were also able to investigate the power of combining Affx500K and Ilmn650K arrays for a single analysis . Since tcis-association discoveries , implying a respective 31% and 33% boost in relative power. In contrast, conditioning on the same sample size, Ilmn650K yields about 10% more cis-associations than Affy500K. This is potentially an important observation that sample size has a more profound impact on statistical power than the difference in genetic coverage among current SNP arrays. Since arrays vary greatly in price, and argument has been raised whether to choose high genetic coverage arrays or cheaper ones and run more subjects.We collected 68 additional liver samples from Caucasian donors. We performed RNA expression profiling as before and obtained SNP genotypes using the Affy500K array only. We then we pooled the sample  and reran the association tests on the Affx500K SNPs. Surprisingly, this increase in sample size (19%) results in 31% and 33% more Whole-genome association studies using high-density SNP arrays are viewed as a powerful approach to elucidating the genetic bases of common human diseases. We provided a novel investigation of two key properties for determining the performance of SNP array genotyping platforms in WGAS: genetic coverage and statistical power. The availability of (i) 90K genotyped SNPs which were identified independently from the HapMap, (ii) a new, independently sampled cohort of subjects, and (iii) thousands of related “expression traits” measured simultaneously on these subjects, yielded the opportunity to provide new insights into genetic coverage and statistical power, and make comparisons to previous results.Two strategies are usually taken in selecting SNPs and constructing genome wide arrays: random SNPs and tag SNPs. These strategies might result in different levels of performance in terms of genetic coverage and statistical power 2r≥0.8) among the “pseudo” HapMap I SNPs Since there are a limited number of common SNPs in the human genome, tag SNPs selected from the complete set  would be robust to SNP overfitting in assessing genetic coverage. At the current stage, tag SNPs are usually selected from an incomplete initial SNP set , and the remaining SNPs  would be “hidden” from the training procedure. Previous simulation studies showed that 26% of the common ENCODE SNPs in CEU had no good proxies . Certainly, larger sample size is necessary to detect weaker effects . Illumina tag SNP arrays were designed to capture common HapMap SNPs. Therefore, most of these tag SNPs have MAF ≥5%. In contrast, Affx500K harbors over 100K rare SNPs (MAF ≤5%). Because we used common SNPs to simulate quantitative traits, the Affx500K rare SNPs provided little statistical power and were therefore excluded from the analysis. Rare SNPs might be useful when the disease-causing polymorphism was also rare. However, Our SNP filtering resulted in similar number of SNPs on Affx500K and Ilmn300K, which enabled a fair comparison between random SNPs and tag SNPs. We found random SNPs actually achieved larger ND , but man2r≥0.8) among the simulated HapMap II datasets, and those SNPs could only be adequately captured by searching beyond the HapMap data. In another words, the HapMap-derived tag SNP struggles to reach the 90% genetic coverage level. Through simulations, we were able to directly test the causal SNP, allowing us to calibrate the upper bound of a SNP array's performance in WGAS. At SNR = 0.5 Click here for additional data file.Figure S2The null distribution of p-values for the Kruskal-Wallis test and Spearman Rank Correlation test. The p-value of Spearman test (p.spearman) follows the uniform distribution under the null, whereas the p-values of the Kruskal-Wallis test shows a lower density in the  range, indicating this test is conservative.(0.33 MB TIF)Click here for additional data file.Figure S3Using the expression traits, we surveyed a wide range of p-value cutoffs and the corresponding FDR values for simulated WGAS.(1.23 MB TIF)Click here for additional data file.Figure S4(A) and (B): the number of discovery (ND) observed in the expressiongenotype association screening. Please note that one expression trait sometimes showed significant association with multiple mutually proximal SNPs, because these SNPs were in strong LD. (C) and (D): the number of expression traits that showed at least one significant association with SNP(s).(1.00 MB TIF)Click here for additional data file.Figure S5The statistical power of the Affymetrix array, Illumina arrays, and “direct genotyping.” (A) SNR = 1/4 and Kruskal-Wallis test; (B) SNR = 1/4 and Spearman rank correlation test; (C) SNR = 1 and Kruskal-Wallis test; (D) SNR = 1 and Spearman rank correlation test.(0.63 MB TIF)Click here for additional data file.Table S1Genetic Coverage of tagSNP Arrays.(0.11 MB DOC)Click here for additional data file.Table S2Power and Number of Discoveries (ND) of Whole Genome SNP Genotyping Products.(0.21 MB DOC)Click here for additional data file.Table S3Number of False Discoveries in Mapping Quantitative Trait.(0.04 MB DOC)Click here for additional data file.Text S1Supplementary Materials.(0.03 MB DOC)Click here for additional data file.


The presence of full-length complements of viral genomic RNA is a hallmark of RNA virus replication within an infected cell. As such, methods for detecting and measuring specific strands of viral RNA in infected cells and tissues are important in the study of RNA viruses. Strand-specific quantitative real-time PCR (ssqPCR) assays are increasingly being used for this purpose, but the accuracy of these assays depends on the assumption that the amount of cDNA measured during the quantitative PCR (qPCR) step accurately reflects amounts of a specific viral RNA strand present in the RT reaction. To specifically test this assumption, we developed multiple ssqPCR assays for the positive-strand RNA virus o'nyong-nyong (ONNV) that were based upon the most prevalent ssqPCR assay design types in the literature. We then compared various parameters of the ONNV-specific assays. We found that an assay employing standard unmodified virus-specific primers failed to discern the difference between cDNAs generated from virus specific primers and those generated through false priming. Further, we were unable to accurately measure levels of ONNV (−) strand RNA with this assay when higher levels of cDNA generated from the (+) strand were present. Taken together, these results suggest that assays of this type do not accurately quantify levels of the anti-genomic strand present during RNA virus infectious cycles. However, an assay permitting the use of a tag-specific primer was able to distinguish cDNAs transcribed from ONNV (−) strand RNA from other cDNAs present, thus allowing accurate quantification of the anti-genomic strand. We also report the sensitivities of two different detection strategies and chemistries, SYBR® Green and DNA hydrolysis probes, used with our tagged ONNV-specific ssqPCR assays. Finally, we describe development, design and validation of ssqPCR assays for chikungunya virus (CHIKV), the recent cause of large outbreaks of disease in the Indian Ocean region. Although the genomes of RNA viruses occur in a variety of conformations, all must be efficiently copied within the infected cell. These copies are essential to the production of messenger RNA (mRNA) that can be translated by host ribosomes, and as a source of genomic RNA for packaging into mature progeny virions. The alphaviruses are a group of enveloped, positive (+) strand RNA viruses in the family Togaviridae Members of the alphavirus genus pose a serious or potential threat to public health in many areas of the world. Nearly all alphaviruses are maintained in nature by transmission cycles that involve alternating replication in a susceptible vertebrate and invertebrate host. Because infection of the vertebrate host is acute and often associated with disease, continual transmission depends on life-long persistent infection of the invertebrate vector host, for many alphaviruses a mosquito. It is presently unclear how persistent alphavirus infections are maintained in the vector host, after (−) strand synthesis terminates in the infected cells. One of the difficulties in addressing this question has been the limitations of methodologies for detecting and measuring (−) strand RNA in infected cells. Competition between viral (+) strands and labeled probe makes nuclease protection assays problematic for the detection of (−) strand RNA, particularly late in the infection when (+) strands are much more abundant Although a variety of strand-specific quantitative real-time PCR (ssqPCR) assays utilizing different designs, detection strategies and chemistries have been reported Aedes albopictus C6/36 cell monolayers were grown to 80% confluency in 12-well plates, washed twice with PBS, and infected with ONNV at a multiplicity of infection (MOI) of 5. Virus was diluted with Dulbecco's Modified Eagle Medium (DMEM)  to a total volume of 0.5 mL/well and placed on the cells at 4°C for one hour. After one hour, fresh medium was added to the wells bringing the total volume to 1 mL, and cells were placed at 28°C for 1 hour. Total RNA was isolated from ONNV-infected cells with TRI Reagent RT®  at 1 hour post infection.in vitro from a plasmid containing a portion of the nsP1 gene from either ONNV or CHIKV. The plasmids pblue-nsP1 (ONNV) and pblue-nsP1 (CHIKV) were produced by cloning the 5′ terminal 853 and 669 nucleotides of the respective viral nsP1 gene into pBluescript II SK (−) (Stratagene). Minus strand RNA was synthesized with T7 RNA polymerase from HindIII-digested plasmid templates in a standard in vitro transcription reaction. Positive strand RNA was synthesized with SP6 RNA polymerase from KpnI-digested plasmid templates in a standard in vitro transcription reaction. The RNA generated during the in vitro transcription reactions was isolated with TRI Reagent RT®, as per manufacturer's instructions. The absence of template DNA was confirmed through PCR. The concentration of RNA transcripts was determined with a NanoDrop spectrophotometer (Thermo Scientific). The cloned ONNV nsP1 gene fragment has a molecular weight of 273,545 g/mol, while the cloned CHIKV nsP1 gene fragment has a molecular weight of 214,574 g/mol. One µg of RNA transcribed from pblue-nsP1 (ONNV) equals approximately 2.2×1012 molecules, while one µg of RNA transcribed from pblue-nsP1 (CHIKV) equals approximately 2.8×1012 molecules.To generate strand-specific standard curves for ssqPCR, (+) and (−) strand RNA was transcribed 10–102 gene copies/µl) and stored at −20°C until further use.cDNAs of both polarities were transcribed with primers containing a 5′ tag sequence To increase fluorescent signal strength during ssqPCR reactions, an AT-rich 12-nucleotide flap sequence (5′AATAAATCATAA 3′) was added to the 5′ end of tagged primers ssqPCR was performed with the appropriate forward or reverse and tag-specific primer pair . When a 2R) of 0.994, and amplification efficiency (Eff%) of 96.8%. The standard curve for the assay using a combination of tag-specific and ONNV-specific primers had a slope of −3.4, an 2R of 0.999, and amplification efficiency of 96.8%. Both assays gave values for the quantity of ONNV (−) strand RNA that were within the acceptable ranges of their respective standard curves: 8.0×105 copies of (−) strand RNA/µg of total RNA for the ssqPCR assay using unmodified primers, and 1.1×105 copies of (−) strand RNA/µg of total RNA for the ssqPCR assay using a tagged primer system , suggesting that one or both of the ssqPCR assays was prone to error.The ability of ssqPCR assays that employ a tagged primer system to detect and quantify viral RNA of a specific polarity has been well demonstrated r system . However6 copies of ONNV RNA/µg of total RNA (the polarity of the RNA is unclear) was determined by standard curve when the two unmodified ONNV-specific primers were used together  strand cDNA, the slope was −1.4, the 2R value was 0.809, and the amplification efficiency was 417.9%. Only at dilutions in which ONNV (−) strand cDNA was present in excess of ONNV (+) strand cDNA were CT values comparable between the two standard curves  strand, suggesting amplification of cDNA from both the intended target strand and the competing (+) strands. Standard curves generated with the tag-specific primer set were similar in the absence or presence of ONNV (+) strand cDNA: the slopes were −3.6 and −3.7, the 2R values were 0.995 and 0.990, and amplification efficiencies were 89.6% and 86.3%, respectively  strand RNA ssqPCR assays in the presence or absence of a fixed amount of cDNA corresponding to ONNV (+) strand RNA. Standard curves produced with the unmodified primer set were very different depending on whether or not ONNV (+) strand cDNA was present during qPCR . In the d curves . Higher ectively . OverallSYBR® Green emits a strong fluorescent signal upon binding to dsDNA. Because the intensity of this fluorescent signal increases with the amount of dsDNA present, the dye can be used to detect and measure the accumulation of qPCR amplicons. However, amplification of nonspecific templates after many PCR cycles can result in additional fluorescence unrelated to any specific target. This nonspecific fluorescence has been shown to limit the sensitivity of dsDNA specific dyes for the detection and quantification of low-copy number targets 3 copies of ONNV cDNA/reaction  or (−) strands were synthesized in an in vitro transcription reaction. Following reverse transcription with a tagged forward or reverse primer , ONNV +. New tagreaction . Howeverreaction . These r3 copies of CHIKV cDNA/reaction  strand cDNA, and a slope of −3.5,an 2R value of 0.973, and amplification efficiency of 93.07% in the presence of CHIKV (+) strand cDNA  strand RNA is inhibited by the presence of relatively higher levels of cDNA generated from the competing (+) strand RNA, when standard unmodified ONNV-specific primers are used for reverse transcription and qPCR . FalselyThe sensitivity of our ONNV (−) strand ssqPCR tag-specific assays was determined with serial 10-fold dilutions of cDNA generated from in vitro transcribed ONNV RNAs. With our SYBR® Green assay we were able to detect 8000 copies per reaction but could not detect 800 copies per reaction, indicating a lower limit of detection somewhere within this range. However, the limit of low-copy number detection decreased to somewhere between 800 (which could be detected) and 80 (which could not be detected) copies per reaction when a TaqMan® DNA hydrolysis probe was used to monitor amplification. The increased sensitivity likely resulted from eliminating the non-specific amplification of dsDNA products that is common when monitoring the generic fluorescence emitted by dsDNA-binding dyes. The sensitivities of our ONNV (+) strand ssqPCR tag-specific assays were determined to be identical to those of the (−) strand assays, with both detection strategies and chemistries. In the case of alphaviruses, the increased sensitivity of assays incorporating DNA hydrolysis probes should be useful, particularly when quantifying much less abundant (−) strands.Finally, using the information obtained by directly comparing various parameters of multiple ONNV-specific ssqPCR assays, we developed and validated assays to detect and quantify CHIKV (−) and (+) strand RNAs. Serial dilutions of cDNA generated from in vitro transcribed CHIKV RNAs were used to confirm strand-specificity and determine sensitivity. Amplification of dsDNA was undetectable when the (+) strand primer set was used with cDNA derived from the (−) strand at all concentrations tested. The reverse was also found to be true using the (−) strand primer set and cDNA generated from (+) strand RNA. The accuracy of the CHIKV (−) strand assay was confirmed in the presence of cDNAs transcribed from competing (+) strand RNAs . The senIn summary, we have developed and validated two new ssqPCR assays for the medically important alphaviruses, CHIKV and ONNV. These assays will be useful in studies to determine how persistent alphavirus infections are maintained in the vector host, and in the detection and quantification of replicating virus from clinical specimens and potential reservoir hosts. In the course of developing these assays, we have shown that accurate quantification of cDNAs generated from specific strands of viral RNA, in the presence of higher levels of falsely-primed cDNA products generated from competing RNA strands, requires incorporation of a unique tag sequence during reverse transcription, which in combination with a tag-specific primer can be used to specifically amplify cDNAs corresponding to the intended target strand during qPCR. While it was also possible to quantify specific strands of viral RNA with assays employing unmodified virus-specific primers, the accuracy of these measurements depends on conditions in which lower relative levels of cDNA generated from the competing strand are present during qPCR. Therefore, previously reported results obtained with assays of this type should be interpreted with caution, particularly when the assay in question has been used to measure amounts of anti-genomic strands, which are typically less abundant than genomic RNAs in cells and tissues infected with RNA viruses.


The HIV-1 epidemic in sub-Saharan Africa is driven largely by heterosexual transmission of non-subtype B viruses, of which subtypes C and A are predominant. Previous studies of subtype B and subtype C transmission pairs have suggested that a single variant from the chronically infected partner can establish infection in their newly infected partner. However, in subtype A infected individuals from a sex worker cohort and subtype B individuals from STD clinics, infection was frequently established by multiple variants. This study examined over 1750 single-genome amplified viral sequences derived from epidemiologically linked subtype C and subtype A transmission pairs very early after infection. In 90% (18/20) of the pairs, HIV-1 infection is initiated by a single viral variant that is derived from the quasispecies of the transmitting partner. In addition, the virus initiating infection in individuals who were infected by someone other than their spouse was characterized to determine if genital infections mitigated the severe genetic bottleneck observed in a majority of epidemiologically linked heterosexual HIV-1 transmission events. In nearly 50% (3/7) of individuals infected by someone other than their spouse, multiple genetic variants from a single individual established infection. A statistically significant association was observed between infection by multiple genetic variants and an inflammatory genital infection in the newly infected individual. Thus, in the vast majority of HIV-1 transmission events in cohabiting heterosexual couples, a single genetic variant establishes infection. Nevertheless, this severe genetic bottleneck can be mitigated by the presence of inflammatory genital infections in the at risk partner, suggesting that this restriction on genetic diversity is imposed in large part by the mucosal barrier. Previous studies of HIV transmission have yielded conflicting results regarding the genetic heterogeneity of the virus establishing infection in the newly infected individual. In this study of populations from Zambia and Rwanda that are infected by two distinct viral genetic subtypes, we compared viral sequences that encode the entry-mediating envelope glycoproteins from newly infected individuals (recipients) and their spouses (donors) very early after infection, as well as newly infected individuals infected by someone other than their spouse. In spite of the genetically diverse virus population in the donor, approximately 90% of newly infected individuals were infected by a single viral variant, while the rest were infected by multiple viral variants. The homogeneity of the virus population in the newly infected recipient, as well as the presence, in some cases, of identical virus variants in the donor, allowed us to precisely identify the transmitted variant. We were able to examine the clinical history of each newly infected individual and observed that all individuals infected by multiple variants also showed evidence of inflammatory genital infections. Our results suggest that the genital mucosa provides a natural barrier to infection by multiple genetic variants of HIV-1, but that this barrier can be lowered by inflammatory genital infections. Subtype B viruses predominate in North America and Western Europe, and as a result, research efforts have focused primarily on this subtype. However, almost 25 million individuals in sub-Saharan Africa have been infected with non-subtype B viruses via heterosexual transmission, leading to a significant gap in our understanding of the most predominant HIV-1 variants worldwide. In order to achieve protection against infection with a globally effective vaccine, the origin and nature of variants that establish a new infection must be better defined.Nearly 35 million people across the globe are infected with the human immunodeficiency virus type 1 (HIV-1) and an additional 2.5 million new infections occur annually The mechanisms by which HIV-1 is transmitted sexually across a mucosal barrier remain poorly understood. Characterization of viral sequences in newly infected individuals has produced mixed results. For instance, studies of individuals newly infected with subtype B have demonstrated a relatively homogenous viral population, suggesting infection by a single variant A previous study in this laboratory examined 8 epidemiologically linked heterosexual transmission pairs participating in a HIV-discordant couple cohort in Lusaka, Zambia env gene sequences within the quasispecies of the donor and recipient. To investigate transmission in a setting analogous to those that are associated with transmission of multiple variants, we included a limited number of epidemiologically unlinked transmission events, in which the seronegative partner was infected by someone other than their spouse. The results indicate that, in the majority of cases, HIV-1 infection is initiated by a single viral variant from a complex quasispecies in the transmitting partner. The marked reduction in genetic diversity that is observed during HIV-1 transmission appears to be imposed in large part by the mucosal barrier, since this extreme genetic bottleneck can be mitigated when inflammatory infections are present in the recipient partner.Here we have expanded our studies of HIV-1 transmission to include 12 additional transmission pairs from the Lusaka cohort and 8 pairs from a similar cohort in Kigali, Rwanda env sequences from the latter formed a distinct cluster that is equidistant from known subtypes (data not shown). All of the 8 transmission pairs from the Kigali cohort were identified as encoding subtype A Env sequences (data not shown). Importantly, 14/20 of the newly infected partners were identified as p24-positive or viral RNA-positive prior to or on the date of seropositive testing and sample collection from both partners, and for 11 of these subjects, the samples analyzed were collected within an estimated 3–5 weeks (17–40 days) following infection  and plasma virus RNA (20 amplicons). SGA was employed to minimize the potential for in vitro recombination during the PCR reaction and re-sampling of genomes env gene. Neighbor-joining phylogenetic trees were constructed using the sequences derived from the 10 subtype C transmission pairs and ZM248 (subtype unknown)   and the unknown) . Sequencunknown) . In 18 oenv genes, indicating that they did not arise de novo in the recipient. The branching pattern and the presence of two distinct populations of sequences that cannot be explained by viral evolution (see below), suggests that 2 different but closely related viral variants were involved in the establishment of infection in RW57F. The neighbor-joining tree for couple ZM229  were not monophyletic. Examination of the neighbor-joining tree for RW57  reveals le ZM229  showed r−5 per day or 3.4×10−5 per replication cycle To evaluate the extent of sequence diversity in the newly infected individuals, V1–V4 sequences from the recipient were examined using the Highlighter tool on the Los Alamos National Laboratory website. This tool allows a comparison of each recipient Env sequence to a reference recipient sequence and graphically depicts any nucleotide differences between the two. Examples of the output from this tool for one subtype C and one subtype A recipient are given in env diversification from a single most recent common ancestor (MRCA) sequence as described previously  similar to that observed in the linked recipients. In contrast, three of the unlinked recipients  have a highly diverse virus population, where multiple divergent branches appear to be present. The Highlighter Tool output for ZM247F is shown in In contrast to the results presented above, analyses of newly infected sex workers in Kenya and STD clinic participants in North Carolina suggested that, in up to 50% of cases, multiple variants established new infections To further quantitate the genetic diversity in the seven unlinked individuals, nucleotide pairwise distances were calculated for the sequences within each subject . As mighThus, in the context of both linked and unlinked transmissions, conditions do exist that mitigate the genetic bottleneck, allowing for transmission of multiple variants. However, this occurred more frequently in the unlinked recipients. An analysis of the clinical history for each of the 7 unlinked recipients showed a significant association (p = 0.0286) between the presence of vaginal/urethral discharge, which is evidence of an inflammatory genital infection, and the establishment of infection by more than one genetic variant in the three females in which this was present . Lower aThis analysis of clinical histories was expanded to include a total of 42 newly infected individuals for whom we had established whether infection in the recipient was initiated by a single or multiple genetic variants from the donor, in order to identify any significant associations between clinical criteria and the establishment of infection by more than one genetic variant. This group included the 27 newly infected individuals presented here, and fifteen, additional linked recipients that have been described In studies that have characterized the virus population in newly infected individuals, the complexity of the virus is higher in some cohorts than in others. Most of these studies were unable to examine the viral population of the sexual partner of that individual. The samples used in the studies presented here were collected from both the chronically infected donor and the newly infected recipient partner enrolled in discordant couple cohorts in Lusaka, Zambia and Kigali, Rwanda. This has provided a unique opportunity to compare epidemiologically linked virus populations in both the donor and recipient very near the time of virus transmission and to use this information to define the nature and origin of the virus establishing infection. The studies presented here reinforce our previous finding that a severe genetic bottleneck occurs during heterosexual transmission of subtype C HIV-1 de novo viral evolution in the new host.Variants could be identified within the donor quasispecies that were identical (ZM221 and RW53) or highly related (less than 2% amino acid differences) to the variant establishing infection in the recipient in several transmission pairs . HoweverA previous study in this laboratory reported that the genetic bottleneck involved a selection for variants with length-constrained hyper-variable domains compared to the median of corresponding donor sequences In only a minority (10%) of the linked transmission events examined, were recipient sequences present on multiple branches of the donor phylogenetic tree  is 1 in 300 to 1 in 600 unprotected coital acts The observation, that multiple variants established infection at a higher frequency following epidemiologically unlinked transmission, suggested that factors capable of mitigating the severity of the genetic bottleneck could be more common in these cases. Examination of the clinical history of the newly infected partner in unlinked pairs revealed a striking association between the presence of multiple variants and vaginal/urethral discharge or lower abdominal pain . As the Genital tract inflammation could compromise the mucosal barrier during sexual exposure such that multiple variants can be transmitted and ultimately establish a systemic infection. While HIV-1 transmission from a chronically infected individual to their partner has been documented to be a relatively rare event  for the donor partners are provided in The transmission pairs characterized in this study were enrolled in the Zambia Emory HIV research Project (ZEHRP) in Lusaka or Projet San Francisco (PSF) Kigali, which provide voluntary HIV-1 testing and counseling, long-term monitoring and health care to cohabitating heterosexual couples in the capital cities of Zambia and Rwanda The estimated number of days between infection and sample collection for each newly infected individual was calculated according the following algorithm. For individuals who were identified as antibody positive and p24 antigen negative at the date of sample collection, the estimated date of infection is presented as less than the number of days between the last seronegative test date and the first seropositive test date (day of sample collection). For individuals identified as antibody positive and p24 antigen positive at the date of seroconversion, the estimated date of infection was determined as 31 days prior to the date of seropositive testing  using the Qiagen DNA Blood Mini Kit. For amplification of plasma HIV-1 env genes, RNA was purified from plasma samples using the Qiagen Viral Isolation Kit and cDNA prepared using the SuperScript III reverse transcriptase according to the manufacturer's instructions. All reverse transcription reactions were primed using an HIV-specific primer . We were able to amplify viral sequences for all individuals from whom we attempted PCR amplification.For amplification of proviral HIV-1 env amplicon. At this dilution, approximately 90% of the reactions will have originated from a single virus genome in the reaction 5′ – GGGTTTATTACAGGGACAGCAGAG – 3′), antisense primer OFM19 (5′ – GCACTCAAGGCAAGCTTTATTGAGGCTTA – 3′); second round: sense primer EA1 (5′ – CCTAGGCATTTCCTATGGCAGGAAGAAGC – 3′), antisense primer EN1 (5′ – TTGCCAATCAGGGAAGTAGCCTTGTGT – 3′). PCR conditions were performed in 50 µl reaction volume with cycling parameters as previously published Full-length gp160 amplicons were obtained from genomic DNA or plasma cDNA using nested PCR amplification by single genome amplification 5′–TATGGGACCAAAGCCTAAAGCCATGTG–3′) and Rev16 (5′–ATGGGAGGGGCATACATTGCT–3′) were used to generate overlapping sequences of the V1–V4 region of the HIV envelope.A portion of the final PCR product was analyzed by 1% agarose gel electrophoresis. Positive PCR reactions were purified using the QIAquick PCR Product Purification Kit or Exosap according to the manufacturer's instructions. Amplicons were directly sequenced by Lone Star Labs, Inc. , Macrogen, Inc.  or the UAB CFAR DNA Sequence Analysis Core . Sequencing primers: For14 (http://www.hiv.lanl.gov/content/hiv-db/HIGHLIGHT/highlighter.html) was used to further analyze the diversity in viral variants transmitted to the recipient partner. Aligned sequences for each recipient were imported into the Highlighter tool and the master was selected as the sequence representing the most highly abundant variant in that virus population. Most recent common ancestor (MRCA) calculations were determined as previously described env sequences from each newly infected and chronically infected individual were deposited in GenBank under the accession numbers FJ185853-FJ187678. Full length gp160 sequences have been deposited for ZM184F (EU166413-EU166438), ZM215F (EU166544-EU166575), ZM229M (EU166576-EU166604), ZM247F (EU166779-EU166856), and ZM249M (EU166857-EU166916) To confirm PCR amplification of a single template, nucleotide sequence chromatograms were examined for multiple peaks, and any such amplicons were discarded. Sequences were trimmed to the first cysteine in V1 and the final cysteine in V4 for further analysis. Sequences were hand-aligned using MacClade 4.06 then gap-stripped to generate neighbor-joining trees using Clustal W. Unrelated reference sequences from the Los Alamos National Laboratory HIV Sequence Database were used as an outgroup for phylogenetic trees. Reference sequences represented Subtype A sequences from Rwanda and Subtype C sequences from Zambia . The reliability of branching orders was assessed by bootstrap analysis with 1,000 replicates. Nucleotide and amino acid pairwise distance calculations for each recipient virus population was determined using PAUP 4.0b10. The Highlighter tool  were recorded systematically at routine quarterly study visits and at interim sick visits, with full physical and/or genital exams conducted as clinically indicated; physical and genital exams were routinely conducted on the visit date when lab test results indicated HIV-1 seroconversion. A self-reported symptom was considered present whether or not the patient sought medical treatment and included treatment administered at external clinics. Composite variables were created from physical exam, medical and laboratory data for genital ulceration and genital inflammation. An STI sign or symptom was included in this analysis if it was reported on the visit date when HIV-1 seroconversion was detected or within one quarterly study visit prior to this visit date. Base SAS® and SAS/STAT® statistical software was used for data management and analysis; Fisher's exact test was used to assess statistical significance of difference in STI proportions between groups.Figure S1Diversity in individuals infected with multiple variants. Aligned linked recipient sequences were analyzed by the Highlighter tool  for (A) RW57F and (B) ZM229F (C) ZM224F and (D) ZM215F. Tic marks indicate nucleotide differences from the indicated master sequences derived from the recipient. Nucleotide differences are color-coded and are marked according to their genetic location along the length of V1–V4. Colors are as follows: A: green, T: red, G: yellow, C: blue and gaps: gray. Red box indicates signature of 5 synonymous nucleotide differences between variants that established infection in RW57F.(1.72 MB PDF)Click here for additional data file.Figure S2Diversity in newly infected individuals. Aligned linked recipient sequences were analyzed by the Highlighter tool  for (A) ZM216M and (B) ZM292M. Tic marks indicate nucleotide differences from the indicated master sequences derived from the recipient. Nucleotide differences are color-coded and are marked according to their genetic location along the length of V1–V4. Colors are as follows: A: green, T: red, G: yellow, C: blue and gaps: gray. Red box indicates CTL-escape footprint.(1.36 MB PDF)Click here for additional data file.Table S1Enrolled Partner(0.05 MB PDF)Click here for additional data file.Protocol S1Criteria for defining infection by a single or multiple variant(0.04 MB PDF)Click here for additional data file.