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by: Sabina Hodkiewicz


Sabina Hodkiewicz
GPA 3.77


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This 162 page Class Notes was uploaded by Sabina Hodkiewicz on Monday October 26, 2015. The Class Notes belongs to IDM2004 at University of Pittsburgh taught by Staff in Fall. Since its upload, it has received 66 views. For similar materials see /class/229417/idm2004-university-of-pittsburgh in Microbiology at University of Pittsburgh.




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Date Created: 10/26/15
Human papillomaviruses clinical aspects and basic biology Viral Pathogenesis Fall Term 2007 1 HPVassociated diseases epidemiology pathology detection and prevention 2 HPV genomic organization viral gene transcription and replication 3 HPV oncoproteins and cancer E6 and E7 4 Cofactors for HPVassociated carcinogenesis estrogen and genomic instability 5 Excursion HPV16 E7 and genomic instability 6 A comprehensive model of HPVinduced tumorigenesis Basic biology of HPV infection HPV types 39 small DNA tumor viruses approximately 200 genotypes in humans 0 infect epithelial tissue 0 mucosaassociated and cutaneous HPV types 0 mucosaassociated HPV types lowrisk HPV eg HPV611 genital warts highrisk HPV eg HPV16 18 associated 55 nm with squamous anogenital neoplasias cervix vulva penis anus and oropharyngeal cancer certain skinassociated HPV types eg HPV5 8 cause epidermodysplasia verruciformis and skin cancer HPVassociated diseases Common wartsplantar warts Condyloma acuminata F HPV the most common sexually transmitted virus HPVs are transmitted by direct contact with skin and mucosal surfaces vaginal oral or anal sexual contact high incidence in professional sex workers absent in nuns and virgins 62 Mio Americans acquire new HPV infection annually one in four of all young adults in US 15 24 yrs by the age of 50 at least 80 of sexually active women will have acquired sexually transmitted HPVs vertical transmission mother to child possible but rare if it occurs potentially life threatening recurrent respiratory papillomatosis RRP immunocompromised patients HIV HV men who have sex with men high risk for anal cancer cancer susceptibility syndromes Fanconi Anemia HPVassociated cervical cancer the second most common cause of cancerrelated death in women worldwide mam mmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmm m wikipedia Predominantly in medically underserved populations in developing and Third World countries and minorities with suboptimal access to health care HPV types and disease HPV I w Piunlair AEIHS Common urls 65 13410 13 319 i 76 cpidciumid qua ltllj gtlk CitiCiIIUliidl rruc wrmis mmry pupilinmmcsis Focal epithelial 3me in of Hcck Cnnjuntliml pa illnm Mcinnmns Cundglmna lcumim m Igeniml mrh Carri inlmeplihelmi neopladn ed Burd EM Clin Microbiol Rev 2003 161 New HPV type gt10 sequence difference in the L1 ORF HPVs are sexually transmitted and infect the uterine cervix Normal cervical transformation zone Source IARC HPV infection of the cervix CIN I Source IARC HPV infection of the cervix CIN ll Source IARC HPV infection of the cervix CIN Ill Source IARC HPV infection of the cervix invasive squamous cell carcinoma Source IARC Quick l lmeTM and a TIFF Uncompressed decompressor are needed to see his picture The Papanicolaou Pap test introduced 1949 by Dr George P Quick39l39lmeTM and aTIFF Uncompressed decompressor are needed to see this picture Additional tests HybridCapture ll DNARNA hybrids detected by an Ab typespecific E6 E7 ORFs and general primer L1 PCR the latter followed by dot or line hybridization in situ hybridization mostly performed on biopsies immunohistochemistry The good news most HPV infections are self limiting most infections are transient and are cleared spontaneously by the immune system in a college populations the cumulative infection rate was 43 average duration of infection was 8 months 70 of infections were cleared within 1 year only 9 showed persistent infection after 2 years persistence influenced by HPV type HPV16 particularly difficult to eliminate age gt30 yrs infection by multiple HPV types immune function other cofactors eg oral contraceptives Even better news cervical cancer has become a preventable disease Ef ficacy against Dosing schedule CINS II and III 0 Product manufacturer HPV type VLPs Adjuvant months A 95 CI Gardasil Mercka e 11 16 18 Alum o 2 6 100 32 100 360 Cervarix GlaxoSmithKlineb 16 18 A804 0 1 6 100 42 100 Dunne amp Markuw1tzc11n Wee D15 2006 43 624 L1 viruslike particles VLPs tetravelent 6111618 safe and 100 effective in preventing highrisk HPV associated precancerous lesions Basic biology of HPV infection HPV genomic organization ori Oncoprotein F Oncoprotein Major capsid E6 E7 protein L1 HPV16 E1 Replication DNA helicase activity L2 Minor capSId E2 DFOte39n E Transcriptional control replication E5 tethering of viral episomes Viral life CYC39ea Interaction with tranSforma on cytoskeleton keratin Basic biology of HPV infection HPV replication Field s Virology Knipe DM Howley PM eds 5th edition p 2312 Basic biology of HPV infection HPV transcription 54 858859 3003 41014135 5550 2514 65 2726 3553 3333 3620 97 Early promoter I i t 0 1000 2000 0000 4000 5000 5000 pA4215 5528 670 Late promoter I 7003 79055 pA7321 Early transcripts gt97 r 670 880 225 409 550 A 2709 226 525 359 A 2709 56575255 500575255 E6 IlE7E2E5 E6E7E1 E4E5 5505751A5455 E639E7E1 Ed55 E639IIIE1quotE4E5 eeln5105255 AAA E6E7E1EZE5 AAA E1 E2 E5 N AAA 5154 55 W AAA E1AE4L2L1 m AAA EME4 L1 Current Opinion In Genetics 0 Developmenl t Late transcripts McCance G J Curr 0p Gen Dev 2005 1515 Basic biology of HPV infection Virus integration Only two the two viral oncoproteins E6 and E7 are expressed in cervical cancer as a consequence of virus integration E LCR I 6lE7 L1 E1 HPV16 host cell chromosome How do HPVs cause cancer Basic biology of HPV infection HPV genomic organization Oncoprotein F Oncoprotein Major capsid E6 E7 protein L1 HPV16 E1 Replication DNA helicase activity L2 Minor capsnd E2 prOtem Transcriptional control tethering of E5 viral episomes to host chromosomes Viral life CYC39ea Interaction with tranSfOFma on cytoskeleton keratin Basic biology of HPV infection HPV life cycle 5T CORNEUM Vinon assemth and releasa 3T GHANULOSUM Viral genome ampllficaliun 39 lnitialion at vin on awembty ST SHNDSLIM Viral oncogene induced hypemlasia Basic biology of HPV infection Host cell reprogramming Normal Mucosa HPVpositive CIN 3 a I i t K t s c u v I 39 quot1 V g 4 V r L 39 o r 2 I I g 3 393 t F u an 390 1431 ing K G a p 0 I b n quotJ 39 6 n a g 30 knquot quot i z39 i quot9 N956quot 3 0 v i J v V VP fin 39 39 39 t t u s39 39 393 quotIF P 7 I 5 39 0v WV 3 d It V quotL Lquotg tel V ate rf nhim 71155253 394 I U a I y 1 39 w quot39f339 quotquot13quot4 t39 3 3 t H amp E Ki67 H amp E Ki67 proliferation differentiation HPV oncoproteins Highrisk HPV E6 and E7 can transform rodent fibroblasts Continued expression of E6 and E6 is necessary for the transformed phenotype Highrisk HPV E6 and E7 can immortalize normal human cells HPV oncoproteins Highrisk HPV E7 and its cellular targets pm p107 3130 blndmgllllysan mal 1939 Dysunmal 1992 I I 9R31107su cllslsgmdalmnjuvneselal 1937b Gunzalezelalu 20ml I mm 915791JDLE2F mmplsx dismplunll nang at at l 1993 Wu el al 199 pzml ZenassThmn exnl 1995 I p2ln lFunkarall9971JmIaselnl 1997a I I m Masslm elal was TAFHmMazzerelll elsl 1995 1 CR1 CR2 CKII CXXC CXXC 98 9 T 5 t i lGFEPVGlMannharnlelal 2mm LxCxE nGlumsldnsallwenchxe a x mm Miami seal 114503531 Snmmrl and Mr lnllan was I I mm mammom I Factquot Rey at lm 2000 MPPZ anean YFllLlischar Flr zlall el al 1959 an 265 measome Balezusuaya nnn Bagcm x9971 w mth 1w Skip mman mm 2mm Miinger K Howley PM Virus R5 2002 89213228 0 small phosphoproteln of 98 amIno acrds no enzymatic activity no related proteins in human genome sequence similarities to SV40 T Ag and Ad E1A CR1 and CR2 biological activities are mediated by proteinprotein interactions LxCxE motif required for cellular transformation destabilization of pRB p107 and p130 interaction with cyclindependent kinase inhibitors p21 mp1 and p27Kip1 chromatin remodelling activity binding to HDACs p300 CBP pCAF several other functions transactivation of CD025 HPV oncoproteins Highrisk HPV E6 and its cellular targets E6AP PDZ binding domain Paxillin hD39Q myc IRF3 hSrib Highrisk HPV E6 binds to the HECT domain E3 ubiquitin ligase E6AP and redirects its activity towards p53 proteasomal degradation of p53 HPV oncoproteins disrupt cell cycle checkpoints HPV E6 Telomerase ApoptOSIs p53 Ki 1 HPV E7 a ml 1433mp21mm cyclinCDK2 pr p107 p130 cyclin E cyclin A DNA polymerase a EchZSA m 61 s 62 gt M cell division cycle Oncogenic mechanisms of HPV oncoproteins Aberrant cell proliferation Nontransgenic 39 239 Control Controli F141 L Cmmbm uE haves mi DNA snlhesis in Dammit E6ltmnsgent pSSnu and E7 lnmgenic epidermis after Imlmm w h mdiatioa Shaun are higirpm39er magai wlinn Ipr mans oft10 waitn5 of skin mm mmnnsgcnk KMHI Vl E uranigenic 1353quot KNHFVIEET KliHI VEI J m and KIJVIlPVISEwlYe lmmgenk min stainrd irmnunt iismchcmically w B Uni Mine Wire either not reaped a c 1 3 I am it w reared with 5 Gy of ionizigg udiaizim 24 111 print In mud we lb 439 f i and l Annms mdicale mples of BrdUrdpasitive itmwuslaimd nuclei oeils in 1h cpidermix 16 E6 and E7 HPVassociated carcinogenesis requires cofactors the role of estrogens and genomic instability HPVassociated cancer and estrogens epidemiological in vitro and in vivo evidence increased risk for cervical cancer after long term estrogen use enhanced viral transcription following steroid hormone interactions between HPV16 E7 and proteins involved in estrogen signaling animal studies Lowdose estrogen causes cervical cancer in HPV16 transgenic mice Tmnslorma ou Zane CervlonVaqlnll Vaglna Region r quot CaMcuUlarille K14 HPV16 transgenic mice no reproductive tract malignancies K14HPV 16 transgenic mice treated with lowdose estrogen39 100 neoplasia of cervix and vulva 60 invasive carcinomas Genolype with cancer K147 v 55 33 u 42 Kimmy 20 95 21 Nontransgenic mice treated with 005 mg 17 estradiol over 6 months squamous epithelial hyperplasia no cancer Excursion HPV16 E7 and genomic instability Oncogenic mechanisms of HPV oncoproteins Genomic instability quot f K 1 0 939 Chromosomal Instability i i innitgm n m m mquot r 7 i 3 9 I0 II I Numerical abnormalities Hit M NH H H n 13 u 15 13 18 Structural abnormalities Oncogenic mechanisms of HPV oncoproteins Genomic instability HPVassociated lesions are genomically unstable 0 aneuploidy already in preinvasive precursor lesions 0 multipolar mitoses Oncogenic mechanisms of HPV oncoproteins Genomic instability normal multipolar 7 Centrosome small cytoplasmic organelle pair of centrioles embedded in a pericentriolar matrix 0 major microtubule organizing center in interphase and mitotic cells 0 various other functions cytokinesis cell cycle The centrosome duplication cycle is synchronized with the cell division cycle Quick39l39lmeTM and a TIFF Unc ressor he re cell cycle Oncogenic mechanisms of HPV oncoproteins Genomic instability Pericentrin Rhodamine Red Chromosomes DAPI SIL squamous intraepithelial lesion SCC squamous cell carcinoma 93820 323323 21228233 92030 5 52 3mm 32 am 52322 a 23 Q 5 Egg o 01 o of cells with ngt2 centrosomes s a 2 a r e am F E g 2 amp igpx aa mg 6564 z 6 28 E r we Oncogenic mechanisms of HPV oncoproteins Genomic instability V V E6 V E7 v 25 NHKlvansienl 25 UZOStransient NHK 53 or 5 p m 20 20 w i g aclm E r g g 15 15 U208 p53 5 10 10 3 g n acim m g 5 5 neo E5 axquot 15E 0 D neo E6 E7 nee E5 E7 A2124 nee E7 Rapid induction of abnormal centriole synthesis by HPV16 E7 Control HPV16 E7 7 739 CentrinGFP Mitochondrial DsRed Nucl ei DAPI HPV16 E7 induces abnormal centrosome numbers before nuclear atypia and aneusomy Control HPV16 E7 Mononucleated ceHs 2 copies of chromosome H o oo 03 A l 0c of cells with ngt2 centrosomes 0 1tubulinRhodamineRedF 15 Q3 go Chromosome115pectrumGreen FISH V N Nuclei DAPI HPV16 E6expressing cells accumulate centrosomes in parallel with nuclear atypia HPV16 E6 yTubulin Rhodamine Red Nuclei DAPI HPV16 E7 can stimulate centriole overduplication through the formation of more than daughter at single maternal templates centrinGFP DSRED control 24 h HPV 16 E7 24 h HPV 16 E7 48 h la Centriole overduplication by HPV16 E7 requires cyclin ECDK2 PLK4 and HsSAS6 C elegans CD K2 1 SPD2 l ZYG1 SAS 5 SAS 6 J7 12 10 U 9 H sa iens 393 p E a O lt CDK2 Q 6 C 9 ep192 E l 2 PLK4 8 l 6 HsSAS 6 0 0 SiRNA control control HPV 16 E7 2 HH E1IE2 A2 CDK2 Cep192 PLK4 HsSAS B Human papillomavirus HPV and Fanconi Anemia FA Autosomalrecessive or Xlinked cancer susceptibility syndrome with 13 complementation groups Developmental defects bone marrow failure hypersensitivity to DNA crosslinking agents 50 times more likely to develop solid tumors Rosenberg et al 2002 500700 fold higher incidence of HNSCC gt50 of solid tumors form at sites of predilection for HPV infection Alter et al 2003 Estimated 80 contain highrisk HPV DNA Kutler et al 2003 Cell cycle deregulation can cause replication stress A CDK activity M G1 S GZ M preRC assembly aa cyclin E lt HPV16 E7 C m The FA pathway responds to DNA replication stress FAAP100 Core Complex 1 Core Complex 2 FANCD2 foci form in keratinocytes expressing the HPV16 E7 oncoprotein FANCDZ DAPI Merge 20 2o 20 m N m E 815 815 815 o E 3 T E U u u It 0910 n m 2 10 5 m 3 E i l 7 g a u g 5 g 5 g 5 to 8 8 2 T E a E gt oi o l 39 0 0 if n 6 a cgt z e K K K I 36 36 669 lt9 0 28 Q8 247 4 39 Qx J Virol 2007 8113265 13270 HPV16 E7 stimulates recruitment of FA associated proteins to chromatin fractions SS P3 control 16E7 control 16E7 SS P3 H 4 L control MMC control MMC 4 3 FANCD2 w nun lt L 39 39 FAN D2 om E Pens NOK U2 OS J Virol 2007 8113265 13270 HPV16 E7 expression in FANCD2deficient human fibroblasts increases chromosomal abnormalities Control keratinocytes FANCDZ deficient broblasts 5 0 Wu of melaphases of melaphases with chromosomal abnormalities Wllh chromosomal abnormalities 01 J Virol 2007 81 13265 13270 HPV16 E7 pRB p107 5 ml p27xm cyclin ECDK2 BRCAZ 6 FAproficient Rdeficient mutation epigenetic Chromosoma nstabmtv Vwra mtegranonv Increase MUIE39polar m39tos39s Te omere dvsfuncton Chromosoma nstabmtv HPV16 E6 Summary Lessons from HPV oncoproteins Cooperation of oncogenic events Estrogen E6E7 l Uncontrolled arrest Gro X3 E2F Proliferation 17X 4 gt gt Rel ivegt Immortal gt Cancer APO osis Se 5 nce Cells Genomic Instability I X E7 E E6 I Telomerase E 5 E6 I g E6 5 duensingpittedu N0vel ProteinaCeous Infectious Particles Cause Scrapie A major unanswered question in mo lecular biology concerns the chemical structure of the scrapie agent Until re cently mysteries surrounding the sCrap ie agent were so cemmonpla ce that investigators had come to accept rather than queStiOn its enigmatic properties The scrapie agent cauSes a degenerative disorder of the central nervous system CNS in sheep and geats 1 The extraordinary resiStance of the scrapie agent to Formalin was responsi ble for the inadvertent inbculation of sheep in Scotland Eighteen thousand Stanley B Prusiner senile dementia was shown by Gibbs Gajdusek andcowOrkers to be caused by a transmissible agent 6 7 A recent study suggests that there may be similarities between the agents Caus ing scrapie and CJD 8 Goats inoculat ed with brain tissue from demented pa tients dying of CJD developed a neuro logical disorder 3 to 4 years after inocula tion Fig 1 Five out of ten C D inocula have produced disease in goats 9 Ex perimental CJD in goats is indistinguish able both clinically and neuropathologi cally from natural scrapie Menkeys Summary After infection anda prolonged incubation period the scrapie agent causes a degenerative disease of the central nervous system in sheep and goats Six lines of evidence including senSitivity to proteases demonstrate that this agent contains a protein that is required for infeCtivity Although the scrapie agent is irreversibly inaCtivated by alkali five procedures with more specificity for medifying nucleic acids failed to cauSe inactivation The agent shows heterogeneity with respect to Size apparently a result of its hydrophobicity the smallest form may have a moleCular weight of 50000 or less Because the novel properties of the Scrapie agent distinguish it from viruses plasmids and viroids a new term prion is proposed to denote a small proteinace0us infectious particle whiCh is resistant to inactivation by most procedures that modify nucleic acids KnoWledge of the serapie agent structUre may have significance for Understanding the causes of several degenerative dis eases animals were vaccinated against louping ill virus With a formalintreated suspen sion of ovine brain and spleen that as Was shown subsequently had been con taminated with the scrapie agent 2 Two years later 1500 sheep developed scrapie Subsequently studies on CNS diseases including scrapie of sheep pro vided the foundation for Sigurdss6n s Concept of slow infectiOns 3 In 1959 Hadlow suggested that kuru a CNS de generative disease of New Guinea high landers might be similar to scrapie be cause the patholOgies of these diSorders Share many features 4 The transmis sion of kuru to chimpanzees in 1965 by Gajdusek Gibbs and Alpers forced a major reconsideratiOn of the etiology of all degenerative disorders and made scrapie a subject of intense medical in terest 5 Subsequently CreutzfeldtJa kob diSease CJD a progressive pre have been used as a common experimen tal host for scrapie and CJD curiOusly chimpanzees are susceptible to CJD but not scrapie 10 Numerous attempts to link scrapie epidemiologically to CJD have been unsuccessful 11 At present there is no direct evidence that the scrap ie agent causes disease in humans In contrast to C D which occurs worldwide kuru is found only in a small mountainouskregion of Papua New Guin ea Epidemiological studies of kUru pro vide evidence for incubation periods cf 20 to 30 years 12 13 Although consid39 erable evidence implicates cannibalism in the spread of kuru no direct observa tions 39of cannibalistic acts in the endem ic region have been recorded Attempts to transmit kuru by feeding infected brain tissue to chimpanzees have been unsuccessful althoUgh one monkey de veloped a kurulike illness 36 months 136 00368075820409 013601000 Copyright 1982 AAAS after oral ingestiOn of the kuru agent 14 In contrast goats fed Scrapiein fected tiSSue frequently develop disease 15 Recently we havetaken advantage of the natural cannibalistic activities of hamSters to develop an experimental model of scrapie transmitted by canni balism 16 Oral tranSmi39ssiOn of the scrapie agent appears to be extremely ine icient Cannibalism requires a dose of agent 109 times greater than that need ed to produce scrapie by intracerebral injection These results provide cempel ling evidence for oral transmission of the scrapie agent and may offer new insights into the spread of kuru by cannibalism among the Fore people and their neigh boring tribes Bioassay of the Scrapie Agent Studies on the scrapie kuru and CJD agents have been greatly limited by the slow tedious and costly bioassays used to detect these agents Since tissue cul ture systems are net available for the replication and assay of these agents and they appear to be nonantigenic in their native forms animal bioassays must be used For many years all assays for the scrapie agent were performed in sheep and goats 1 In 1961 transmission of the scrapie agent to mice tranSformed reSearch 18 but the murine endpoint titration assay was still heroic Quantify ing a single sample required eight to ten serial tenfold dilutions and injection of each dilution into Six mice 19 Then 50 to 60 mice Were held for 1 year and examined weekly for signs of scrapie The number of animals developing scrapie at the highest dilutiOn was used to calculate an end point The time re quired for titration of a sample was re duced to 200 days when a mere rapid form of the disease in hamsters was discovered 20 2 1 Several investigatOrs have estimated serapie titers by measuring the time in terval from inoculation to onset of illness incubation period in mice 22 23 Re luctance to re ne such measurements has prevented its Wideuse in mice With hamsters studies on the scrapie agent have been accelerated by develop ment of a bioassay based on measure ments of incubation time 24 25 It is now possible to assay samples with the use of four animals in 60 to 70 days if the titers of the scrapie agent are high As is shown in Fig 2 the interval from inocu The author is an associate professor in the Depart ments of NeurolOgy and Biochemistry and Biophys ics at the School of Medicine University of Califor nia San Francisco 94143 SCIENCE VOL 216 9 APRIL 1982 lation to onset of illness y was inversely proportional to the dose injected intra cerebrally into random bred weanling Syrian hamsters The logarithm of the mean interval 7 in days minus a time factor of 40 is a linear function of the logarithm of the dose over a wide range the time factor was determined by maxi mizing the linear relation between the time interval and dose With a factor of 40 the regression coef cient of the line is 087 A similar analysis was performed for the time interval from inoculation to death z With a time factor of 61 the regression coef cient of the line is 086 Linear relationships were also obtained when the reciprocals of the time inter vals were plotted as a function of the logarithm of the dose Replication of the Scrapie Agent The kinetics of scrapie agent replica tion in hamsters and mice are well docu mented 21 26 After intracerebral in oculation of hamsters with 107 IDSO me dian infectious dose units about 102 ID50 units can be recovered in the brain 24 hours later During the next 50 days the amount of agent in the brain in creases to 109 IDSO units At this time the agent is widely distributed throughout the brain and no regional differences are apparent 27 The neuropathology is minimal and the animals exhibit no neu rological dysfunction During the next 10 to 15 days the animals develop ataxia di iculty righting themselves from a su pine position generalized tremor and head bobbing By 60 to 70 days vacuola tion of neurons and astrogliosis are found throughout the brain even though the titer of the agent remained constant Thus the pathological hallmarks of this spongiform encephalopathy do not correlate with the extent of agent replica tion In addition the spongiform pathol ogy that characterizes kuru and CJD is inconspicuous in natural scrapie 28 Hypothetical Structures for the Scrapie Agent Investigators have been aware of the unusual properties of the scrapie agent for more than three decades Hypothe ses on the chemical structure of the scrapie agent have included sarcospo ridia parasite 29 lterable Virus 30 small DNA virus 31 replicating protein 32 replicating abnormal polysaccha ride within membranes 33 DNA sub virus controlled by a transmissible link age substance 34 provirus consisting 9 APRIL 1982 Chimpanzee 69 6 Monkey 6 CreutzfeldtJakob disease Goats l i Scrapie Monke jy e Chimpanzee x I Fig 1 Experimental relation between Creutz feldtJakob disease of humans and scrapie of goats and sheep of recessive genes generating RNA parti cles 35 naked nucleic acid similar to plant viroids 36 unconventional virus 12 3E aggregated conventional virus with unusual properties 38 replicating polysaccharide 39 nucleoprotein com plex 40 nucleic acid surrounded by a polysaccharide coat 4 spiroplasma like organism 42 multicomponent sys tem with one component quite small 43 and membranebound DNA 43 44 225 200 Log 3 40 Log 2 61X 01 39 150 125 l l L L l 1 l l 0 2 4 6 8 Log dose Fig 2 Assay of the scrapie agent by measure ments of the incubation time interval The intervals from inoculation to onset of illness y and to death z are plotted as a function of the injected dose Equations were written to describe these linear functions which relate the titer of the inoculum to the time intervals both from inoculation to onset of illness Eq 1 and from inoculation to death Eq 2 Log 1 2666 1299 log 6 40 log D 1 Log Tz 2533 1247 log 2 61 logD 2 where T is the titer expressed in ID50 units per milliliter D is the dilution de ned as the fractional concentration of the diluted sample 7 is the mean interval from inoculation to onset of clinical illness in days and Z is the mean interval from inoculation to death in days The most precise estimate of titer is obtained by calculating a weighted average for T and Tz Puri cation and Hydrophobicity of the Scrapie Agent Several investigators have mounted major efforts to purify and characterize the scrapie agent over the past two dec ades 45 48 Early studies suggested that the scrapie agent was distributed throughout virtually all subcellular frac tions 45 46 The interpretation of those observations was complicated by the im precision of the endpoint titrations of the agent Nevertheless the scrapie agent was reported to be intimately asso ciated with cellular membranes and from this association the membrane hypothesis evolved 33 When various extraction procedures failed to release the agent from membrane fractions it was concluded that the agent is a repli cating membrane fragment that cannot be separated from cellular membranes Several different puri cation proce dures have been reported One involved copuri cation of the scrapie agent and microsomes 49 Another involved iso lation of a membrane free fraction after prolonged ultracentrifugation 50 This fraction contained 1 to 10 percent of the scrapie agent and was precipitated with ammonium sulfate Sodium dodecyl sulfate SDS gel electrophoresis was used to obtain a further puri cation 51 Although the results of these studies seemed encouraging initially subse quent work has been disappointing 52 Using equilibrium sucrose and sodium chloride density gradients Siakotos Gajdusek Gibbs and coworkers have attempted to purify the scrapie agent from murine brain 53 They suggested that there was a peak of infectivity at a sucrose density of 119 gcm3 However multiple peaks of infectivity were found throughout the gradients an indication of considerable heterogeneity with re spect to density and showing that the technique when applied to crude suspen sions of membranous material from brain is probably not useful in isolating the scrapie agent Other studies from the laboratory of Gajdusek have shown con siderable heterogeneity of the agent in metrizamide and cesium chloride density gradients 54 Since the initial puri cation of many biological macromolecules involves a se ries of differential centrifugations 55 we began our studies on the scrapie agent by de ning its sedimentation prop erties in xedangle rotors in order to develop a preparative protocol 56 These studies showed that the agent from both murine spleen and brain sedi mented over a range of particle sizes from 605 to 10005 19 137 Table 1 Molecular properties of the scrapie agent evidence that it contains a protein Abbreviations PMSF phenylmethylsulfonyl uoride DEP diethyl pyrocarbonate T X100 Triton X100 OGS octylglucoside SB 314 sulfobetaine 314 ET12H 1dodecyl propane diol3phosphorylcholine SDS sodium dodecyl sulfate LDS lithium dodecyl sulfate Gdn guanidinium NP40 Nonidet P40 Sarkosyl sodium dodecyl sarcosinate T CA trichloroacetic acid SCN thiocyanate Treatment Stable Labile Protease digestion Chemical modi cation Detergents TX100 NP40 OGS SB 314 Proteinase K trypsin DEP butanedione PMSF SDS LDS ET12H cholate Sarkosyl Ions PO4 3 Denaturants Organic solvents Na K Cl SO4 2 EDTA 4 Methanol ethanol Gdn SCNquot TCA Urea Phenol On the basis of the information de rived from these sedimentation pro les a partial puri cation scheme for the mu rine scrapie agent from spleen was de rived 57 The preparation was devoid of cellular membranes and enriched for the scrapie agent 20 to 30fold with respect to protein and DNA Studies on the agent by rate zonal sucrose gradient centrifugation gave sedimentation coef cients for the agent ranging from 40S to gt 500S Sucrose density gradient cen trifugation revealed a particle density ranging from 108 to more than 130 g cm3 an indication that some forms of the agent might be associated with lipids Further sedimentation studies showed that the agent aggregated with cellular elements on heating the agent in a par tially puri ed fraction 58 The agent was stable in nonionic and nondenatur ing anionic detergents but was inactivat ed by SDS Free ow electrophoresis showed that most of the agent has a net negative charge but signi cant charge heterogeneity was found Heterogeneity of the scrapie agent with respect to size density and charge suggested that hydrophobic domains on its surface might be responsible for these phenomena Such domains are usually formed by the juxtaposition of nonpolar side chains of amino acids within a pro tein 57 59 These initial studies on the murine agent from spleen revealed the complex ities of scrapie agent puri cation We then developed an improved assay based on measurements of the incubation time With this new bioassay we created a puri cation scheme for the agent from hamster brain where the titers are high est 21 The initial steps of the puri ca tion were similar to those for the murine agent 57 Deoxycholate extracts P4 were digested sequentially with micro coccal nuclease and proteinase K The digestions were performed at 4 C to pre vent aggregation of the agent which is 138 observed at elevated temperatures 59 The digested preparations were then subjected to cholateSarkosyl extraction followed by ammonium sulfate precipita tion P5 MoSt of the remaining digested proteins and nucleic acids were separat ed from the scrapie agent by Sarkosyl agarose gel electrophoresis at 4 C 60 Such preparations of the eluted scrapie agent E6 were 100 to 1000fold puri ed with respect to cellular protein 60 With these enriched preparations we demon strated that a protein within the agent is required for infectivity 60 Fraction E6 contains per milliliter 106395 to 108395 IDSO units of agent 20 to 50 pg of protein lt 1 pug of DNA and lt 10 pug of RNA Since the ratio of particle number to infectivity unit for the scrapie agent is unknown the extent of puri cation re quired to obtain homogeneous prepara tions is unknown Each hamster brain contains 109 IDSO units of the scrapie agent and 100 mg of protein Assuming an average molecular weight of 50000 for all proteins and a ratio of particle number to infectivity unit of 1 a 107fold puri cation will be required to prepare a pure preparation of the agent However if the particle to infectivity ratio is 1000 then a39 104fold puri cation will be re quired Experience with assaying con ventional viruses in animals suggests that the particle to infectivity ratio for the scrapie agent may be considerably greater than 1 These calculations indi cate the need for a source of the scrapie agent with considerably higher titers Attempts to propagate the scrapie agent in cell culture have been disappointing The hydrophobicity of the scrapie agent has complicated puri cation as de scribed above Further evidence for the hydrophobicity of the scrapie agent comes from its binding to phenylSepha rose 60 The agent could not be eluted in 85M ethylene glycol however inclu sion of 4 percent Nonidet P40 and 2 percent Sarkosyl in the ethylene glycol eluate resulted in the almost quantitative recovery of the agent from phenyl Sepharose In addition the hydropho bicity was re ected by diminished titers when detergent was removed from frac tion E6 60 Presumably this decrease in infectivity was due to aggregation Not only is the hydrophobic nature of the scrapie agent important with respect to puri cation it may also explain some of its enigmatic properties That hydro phobic interactions increase with elevat ed temperature may be re ected in the extreme heat stability of the agent 47 Numerous unsuccessful attempts to de tect antibodies against the native scrapie agent in fraction E6 might be explained by its hydrophobicity 61 Hydrophobic proteins in their native state are some times poor antigens 62 An alternative and attractive explanation for the ap parent lack of immunogenicity of the scrapie agent evolves from the possibili ty that the agent may be closely related to a normal cellular protein to which the host does not produce antibodies Scrapie Agent Contains Protein Six separate and distinct lines of evi dence show that the scrapie agent con tains a protein that is required for infec tivity i inactivation as a result of diges tion with proteinase K ii inactivation by chemical modi cation with diethyl pyrocarbonate iii inactivation by SDS iv inactivation by chaotropic salts such as guanidinium thiocyanate v inactiva tion by phenol and vi inactivation by urea 60 The cumulative evidence for a protein within the scrapie agent appears to be compelling Table 1 Digestion with crystalline proteinase K inactivated the scrapie agent from hamster brain 60 the decrease in titer was a function of enzyme concentration temperature and time of digestion Prior treatment of proteinase K with the prote ase inhibitor phenylmethylsulfonyl uo ride PMSF completely abolished the proteasecatalyzed degradation of the agent Digestion with trypsin also de stroyed the scrapie agent The protease sensitivity of the scrapie agent was re vealed only after considerable puri ca tion fraction E6 Other investigators have occasionally observed decreases in scrapie titers after addition of proteases 48 63 Carbethoxylation by diethyl pyrocar bonate also inactiVated the puri ed scrap ie agent 60 64 but activity was re stored by treatment with hydroxylamine This reversibility of the inactive chemi cally modi ed agent provides a further SCIENCE VOL 216 Table 2 Resistance of the sCrapie agent to procedures that attack nucleic acids Abbreviations AMT 439aminomethyl45398trimethylpsoralen HEP 1oi4 hydroxyethylpsoralen HMT 4 hydroxymethyl 45 8 trimethylpsoralen MMT 4 methoXymethyl45 8 trimethylpsoraleri TMP 45 39 8trimethylpsoralen Procedure Resistant Labile Possible explanations pH H OH Hydrolysis of RNA genome denaturation of dsDNA genome or protein denaturation Nucleases Ribonucleases Enzymes unable to penetrate protein shell deoxyribonucleases I UV irradiation 254 nm Shielded by protein shell or no critical nucleotide dimers Divalent cation hydrolysis Zn2 Psoralen photoreaction Chemical mOdi cation AMT HEP HMT MMT TMP Hydroxylamine formed Ions unable to penetrate protein shell Monoadducts of singlestranded genome do not inactivate or psoralen s unable to penetrate protein shell Nucleophiles react only with surfaCe protein and are unable to penetrate the shell or react minimally with doublestranded genome argument for a protein target More re cent data on chemical modi cation indi cate that the scrapie agent is also inacti vated by 10 mM bUtanedione and 2 mM PMSF Butanedione modi es arginine 65 lysine and histidine residues modi cations of one or more of theSe amino acid residues may explain its effect on the sCrapie agent 39 Three reagents used to denature pro teins and isolate biologiCally active nu cleic acids 66 also inactivate the agent First SDS diminished the scrapie agent titer when the ratio of SDS grams to protein grams exceeded 18 24 In contrast the agent was stable in various nondenaturing ionic and nonionic deter gents Table 1 Simultaneous addition of a nonionic detergent and SDS to a prepa ration Containing the scrapie agent pre vented the inactivation observed with SDS alone SecOnd studies with Chao tropic ions have shown that in low Con centrations they inactivate the agent 24 67 Irreversible inactivation of the agent was foUnd upon exposure to IM guani dinium thiocyanate at 4 C for 3 hours Higher concentrations 39of less potent chaotropic salts Were required to achieve irreversible inactivation Third phenol useful in the isOlation 0f nucleic acids inactivates the sCrapie agent 68 In conf trast the agent is stable in methanol and ethanol but is readily precipitated Ex traction with phenol a potent denaturant of protein under various salt and pH conditions destroyed infectivity 24 In the above Studies partiallypu ri ed prep arations were rSt digeSted with protein ase K to prevent the formation of an interface in which the agent might be trapped We have attempted to restore scrapie agent infectivity from phenol ex tracted preparations by incorporation into liposomes and by transfection into cultured cells Using reversephase lipo some formation 69 no infectivity was recovered from the aqueous phase dia 9 APRIL 1982 lyzed phenol phase or Combination of these tWo phases 70 Preparations of phenol extracted DNA and RNA from scrapieinfected murine Spleen failed to produce infectious scrapie agent upon transfectionof L cells 71 Similar trans fection experiments with murine fetal brain Cells and embryonic broblasts also failed to produce infeCtiOus agent 72 From all of these studies with chemical reagents that denature prbteins bUt permit isolation of biologically active nucleic acids we conclude that denatur ation of a protein 1 within the scrapie agent leads to inactivation of the infec tious particle Moreover CJD agents adapted to guinea pigs and mice are also inactivated when eXtraCted with phenol 73 Hunter and coworkers showed that exposure of the scrapie agent to 60M urea decreased the titer by a factor of 100 74 This high COncentration of urea COuld have denatUred p39rotein 0r nucleic acid We haVe found that exposure of the fscrapie agent in partially pUri ed frac tions to 3M urea at 46C decreases the titer by a factor of 50 70 Removal Of the urea after 2 hoUrs Was not accompa nied by a return of infectivity This ob servation contraSts with other ndings where removal of the KSCN Was accom panied byan apparent return of infectng ity 67 Whether urea or cyanate ions are responsible for the loss of scrapie infectivity in these experiments 75 is not known From our data the most likely target within the Scrapie agent for denatUration by urea is a protein The funCtiOns of a protein or proteins within the scrapie agent are unknown The hydrophobicity of the protein should allow it to penetrate membranes but whether or not there are speci c recep tors on cell sUrfaCes to which the scrapie protein might bind is unknOWn Studies on transmission of scrapie by cannibal ism in hamsters suggest that the scrapie agent is transported across epithelial cells and then presumably enters the bloodstream I6 Manuelidis and Co workers have found the CJD agent in white blood cells 76 Studies by Kim berlin and Walker suggest that the agent may be transported within axons much like rabies virus 77 One possibility is that the proteinis a polymerase that is necessary for replication of a putative nucleic acid within the agent This wou1d explain the protein requirement for in fectivity and would be similar to negative 39strand Viruses We also must COnsider the possibility that the scrapie protein acts as an inducer or as a template for its Own synthesis Search for Nucleic Acid in Scra pie Agent In our search for a nucleic aCid genome within the scrapie agent we subjected the agent to Changes in pH Table 2 Although other investigators had indicated that the agent Was stable oVer a pH range from 2 to 105 78 our observations do not agree with some of these earlier studies We have found that the titer of the scrapie agent is irrevers ibly reduced by alkali 67 The titer was reduced by a faCtor of 1000 on exposure to pH 10 for 1 hour at 4 Cor by a f actOr of 100 on exposure to pH 9 for 1 hourlat 37 C 70 Neutralization with acid did not restOre infectivity In COntrast no 1033 of infectivity at pH 3 was observed over a 16hour period at 37 C 0ne inter pretation of these studies is that alkali hydrolyzed a few phOsphodiester bonds within a scrapie nucleic acid rendering the agent inactive The covalent back bone in RNA is labile to alkali while that in DNA is generally stable however base modi cations suCh as methylation of purines render DNA labile in alkali 79 Denaturation of double stranded DNA dsDNA in alkali is also well doc 139 umented 80 Alternatively inactivation by alkali under 39rather mild Conditions could be due to protein denaturation Unfortunately the lack of speci city in these pH stability studies does not allow us to make a de nitive statement con cerning the presence or absence of a nucleic acid within the scrapie agent Over the paSt 15 years two techniques with high degrees of speci city have suggested that the scrapie agent might not contain a nucleic acid The scrapie agent in crude preparations has been found to be resistant to nuclease diges tion 46 48 59 and to ultraviolet UV irradiation at 254 nm 81 82 The Objec tion to these studies was that a protec tive coat prevented nucleases from pene trating the agent as well as shielding it from radiation At several different stages of puri ca tibn we have searched for susceptibility of the agent to nuclease digestion No deCrease in scrapie infectivity has been observed with microc0ccal nuclease nu clease P deoxyribonucleases I and II ribonucleases A and T1 and phosphodi esterases I and II at 10 100 and 500 ug ml for 3 to 30 hours at 37 C Ribonucle ases III and H at 1 and 10 unitml also showed no effect Although nuclease sensitivity has been described for the scrapie agent 44 we have been unable to con rm this observation 52 V The complete lack of scrapie agent sensitivity to nucleases in view of inacti vation by proteases is of interest Nu merous viruses are resistant to nu cleases presumably these enzymes do not penetrate the viral protein coats 83 In contrast addition of ribonuclease A at 01 rigml to a crude nucleic acid extract containing potato spindle tuber virOid PSTV decreased the PSTV titer39by a factor of gt106 in 1 hour at 25 C 84 Hydrolysis of a single phosphodiester bond within a viroid probably inactivates it 85 86 There are many examples of proteins that retain their biological activ ities after limited proteolysis 87 We do not know in the case of the scrapie agent how many peptide bonds must be cleaved to cause inactivation Studies with the optically clear frac tion E6 have con rmed the resistance of the scrapie agent to UVinactivation 81 82 Fractions 82 P5 and E6 Were irradi ated at 254 nm with increasing doses Although no inactivatiOn of the agent in fraction Sziwas observed a minimal but prObably signi cant decrease was found in fractions P5 and E6 as a function of dose 88 The kinetics of inactivation by irradiation at 254 nm suggest a singlehit process The survival of 37 percent of the scrapie agent in fractions P5 and E6 140 Table 3 Inactivation of small infectious agents by UV irradiation at 254 nm D37 Example Jmz Bacteriophage T2 4 Bacteriophage S13 20 Bacteriophage X174 20 Rous sarComa virus 150 Polyoma virus 240 Friend leukemia virus 500 Murine leukemia virus 1400 Potato spindle tuber viroid 5000 Scrapie agent 42000 Data from 82 85 88 D37 is the dose of irradiation that permits 37 percent survival was observed after a UV dose D37 of 42000 Jmz The resistance of the scrap ie agent to irradiation at 254 nm is com pared to that obServed for viruses and viroids in Table 3 Clearly the inaC tivation of the scrapie agent at these extreme energy levels indicates a photo chemistry of a far different nature from that observed for virus inactivation through the formation of thymine or ura cil dimers Proteins are relatively resist ant to irradiation at 254 nm 89 and are probably the target within the scrapie agent in these irradiation studies Observations on the resistance of the scrapie agent to procedures attacking 100 F 1 020 C 80 g lt 016 E 012 Q A I 2 l f V E 008 8 g lt2 3 i 004 quotmm 000 Fraction number Fig 3 Molecular sieve chromatography of the scrapie agent in sulfobetaine 314 Frac tion P5 was exposed to 10 percent weight to volume sulfobetaine 3 14 for 16 heurs at 4 C and then chromatographed in 60 mM tris acetate pH 72 containing 1 mM EDTA and 0024 percent sulfobetaine 3 14 over a molec ular sieve Toya Soda TSK 4000 column 075 cm inside diameter by 50 cm length The column was developed at 4 C Varian 5000 HPLC at 1 mlmin Fractions 1 ml were collected and aSSayed in hamsters by measurements of incubation time intervals Blue dextran bovine serum albumin and tryptophan under identical conditions eluted in fractions 7 15 and 22 respectively nUcleic acids have been extended by means of three other techniques Table 2 The agent has been incubated at pH 7 in the presence of 2 mM ZnNO3392 at 65 C for periods as long as 24 hours without loss of infectivity 70 Under these conditions polymers of RNA are completely reduced to mononucleotides and polymers of DNA undergo consider able hydrolysis 90 Photochemical in activation of the scrapie agent with psor alens was attempted with samples at several levels of puri cation both frOm murine spleen and hamster brain Five different psoralens of varying degrees of hydrophobicity were used 91 It was expected that the most hydrophobic psoralens readily partiti0ned into the scrapie agent No inactivation of the scrapie agent was observed with any of these psoralens over a wide range of dosages 92 Psoralens may form diad ducts upon photoactivation within base paired regions of nucleic acids and mono adducts within singlestranded regions 93 Psoralens have several advantages in searching for a nucleic acid genome i low reactivity with proteins ii pene tration of viral prOtein and lipid coats and iii formation of stable 39covalent linkages on photoactivation Psoralens have been found to inactivate numerous viruses but not for example piCornavi ruses 94 Psoralens like acridine or ange and neutral red dyes 95 do not penetrate the protein coat of poliovirus PhotoaddUCts with viral RNA Were formed when psoralens or the above tricyclic dyes were added to Cultured cells replicating the poliovirus In contrast to psoralens hydroxyl amine readily inactivates poliovirus at neutral pH 96 Hydroxylamine does not generally react withproteins at neu tralpH but it does decarbethoxylate modi ed proteins and it does modify cytosine bases 97 At concentrations up to 05M at neutral pH hydroxylamine failed to alter scrapie agent infectivity 64 Under these conditions most ani mal and plant viruses as well as bacterio phage are inactivated by hydroxylamine 98 except for the paramyxoviruses which are resistant In contrast inactiva tion of the scrapie agent by carbethoxy lation upon treatment with diethyl pyro carbonate was found to be reversible with NH20H 64 The extreme resistance of the scrapie agent to inactivation suggests that its structure is different from that of viruses While there are examples of viruses that are resistant towinactivation by two or even three of the six procedures in Table 2 we are unaware of any viruses which like the scrapie agent are resistant by all SCIENCE VOL 216 of these procedures However the pos sibility must be considered that the puta tive genome of the scrapie agent is bur ied within a tightly packed protein shell which excludes nucleases 39UV irradia tion Zn psoralens and NHZOH Al so we cannot exclude an unusual nu cleic acid with a different base structure or polymer packing that might exhibit the resistant characteristics described for the scrapie agent 39 Of interest are studies showing a large 1 oxygen effect upon exposure of the scrap ie agent to ionizing radiation 99 Vi ruses and nucleic aCids characteristically show a small oxygen effect Biological membranes and probably lipoproteins show large oxygen effects The increased sensitivity of the scrapie agent to ioniz ing radiation in the presence of oxygen presumably re ects the hydrophobic protein With bound lipids that is required for infectivity 60 These data do not eliminate the possibility that the agent also contains a nucleic acid Molecular Size of the Scrapie Agent 39 The extreme resistance of the scrapie agent to inactivation by ionizing radia tion raised the possibility that the agent is quite small 100 Target calculations haVe given minimum molecular weights ranging from 64000 to 150000 82 100 However two important factors could not be taken into account in these calcu lations The rSt is the possibility that multiple copies of the agent might exist within a single infectious particle as would occur with aggregation We have good evidence that the agent readily as sociates with cellular elements and prob ably aggregates with itself in puri ed preparations 47 57 58 The second is the ef ciency of the cellular repair pro cesses For example polyoma virus dsDNA 3 X 106 daltons has been found to be almost as resistant to ionizing radi ation as either viroids or the scrapie agent 10 i The extreme ef ciency of the cellular repair processes for the poly oma virus dsDNA genome accounts for its apparent resistance to damage by ionizing radiation 82 Studies on the scrapie agent in murine spleen have shown a continuum of sizes ranging from 405 or less to more than 5008 by ratezonal sucrose gradients 4 7 57 Parvoviruses are among the small est viruses identi ed and they have sedi mentation coef cients of 1005 to 1105 83 The scrapie agent in preparations extracted with sodium deoxycholate as sociated with cellular elements when heated to form large infectious particles 9 APRIL 1982 Table 4 Properties of the scrapie agent Stable at 90 C for 30 minutes Low molecular weight infectious particles minimum estimate 50000 daltons or less Hydrophobic protein or proteins is re quired for infectivity Resistant to ribonucleases and deoxyribonu cleases 39 Resistant to UV irradiation at 254 nm Resistant to psoralen photoadduct formation Resistant to Zn catalyzed hydrolysis Resistant to NH20H chemical modi cation of gt10000 47 58 Such particles are the size of mitochondria Sedimentation studies of CJD agents adapted to both guinea pigs and miCe suggest that the sizes of these agents are similar to that observed for the scrapie agent 73 Gel electrophoresis has also Shown that the scrapie agent exists as a succes sion of particles OfVarying size 52 59 Sarkosyl agarose gel electrophoresis of partially puri ed fractions showed that some forms migrated more slowly than DNA restriction endonucle39ase fragments of 15 X 106daltons Some smaller forms of the agentmigrated ahead of 3 X 105 dalton DNA fragments Digestion of crude preparations with nucleases and proteases facilitated the entry of the agent into these gels One report showed that most of the scrapie agent migrated with SS RNA molecules in the presence of SDS 51 We were unable to con rm these ndings since SDS inactivated the agent 24 52 Until recently gel ltration studies with anionic detergents and chaotropic ions have given reSults similar to thOse described for ratezonal sucrose gradi ents and gel electrophoresis Typically most of the agent eluted in the void volume follOwed by a continuum of par ticles apparently of decreasing size 59 67 In contrast incubation of the scrapie agent overnight with 10 percent weight to volume sulfobetaine 314 a zwitter ionic detergent appears to have dissoci ated the agent Fig 3 70 Under these conditions the scrapie agent eluted as a peak behindquot bovine serum albumin BSA but slightly ahead of ovalbumin If the agent has a globular shape in sulfobetaine 314 then it may have a molecular size of 50000 daltons or less How much detergent is bound to the agent and how the detergent in uences the apparent molecular weight of the agent remains to be determined 102 Similar observations have been recorded with another detergent 1d0decyl pro panediol3phosphorylcholine which is a synthetic derivative of lysolecithin Con rmation of these ndings by rate zonal sucrose gradient centrifugation is awaited since anomalous behavior of proteins during gel ltration is well known 103 Thus the monOmeric form of the scrapie agent may indeed be con siderably smaller than that of a viroid which until now has been the smallest infectious agent known 39 If the scrapie agent does have a molec ular weight of 50000 or less then a nucleic acid within such a globular struc ture will be too small to code for a protein A spherical scrapie agent of molecular weight 50000 would have a diameter of 4 to 6 nm 104 Let us assume that the agent has a protective protein which is 1 nm 10 A thick The Volume of the core will be 14139 nm3 From measurements of DNA packing in crystals and bacteriophage 105 there is space for a 12 nucleotide polymer con sisting of six base pairs Dehydration of the polymer would permit 32 nucleotides to be encapsidated Indeed if such oligo nucleotides exist within the agent they must have a function other than that of a template directing the synthesis of scrap iecOat proteins v Novel Properties of the Scrapie Agent The foregoing summary of experimen tal data indicates that the molecular properties of the scrapie agent differ from those of viruses viroids and plas mids Table 4 Its resistance to proce dures that attack nucleic acids its resist ance to inactivation by heat and its apparent small size all Suggest that the scrapie agent is a novel infectious entity Because the dominant characteristics of the scrapie agent resemble those of a protein an acrOnym is introduced to emphasize this feature In place of such terms as unconventional virus or un usual slow viruslike agent the term prion pronounced preeon is sug gested Prions are small proteinaceous infectious particles which are resistant to inactivation by most procedures that modify nucleic acids The term prion underscores the requirement of a protein for infection current knowledge does not allow exclusion of a small nucleic acid within the interior of the particle Our data and that of other investiga tors suggest two possible models for the scrapie agent i a small nucleic acid surrounded by a tightly packed protein coat or ii a protein devoid of nucleic acid that is an infectious protein While the rst model might seem the most plausible there is no evidence for a nucleic acid within the agent The sec 141 ond model is consistent with the experi mental data but is clearly heretical Skepticism of the second model is cer tainly justi ed Only puri cation of the scrapie agentto homogeneity and deter mination of its chemical structure will allow a rigorous conclusion as to which of these two models is correct There seems to be little advantage in championing one model over another however several previously postulated structures for the scrapie agent can now be discarded The requirement of a pro tein for infectivity eliminates the poSsi bilities that the scrapie agent is com posed entirely of polysaccharide or nu cleic acid Thus the replicating polysac charide and naked nucleic acid viroid hypotheses are no longer viable The hypothetical nucleic acid surrounded by a polysaccharide coat can also be elimi nated Studies demonstrating the small size of the scrapie agent clearly distin guish it from conventional viruses spiro plasma like organisms and parasites such as sarcosporidia 39 Rigid categorization of the scrapie agent at this time would be premature Determination of its molecular structure will be required prior to deciding wheth er prions represent a distinct subgroup of extraordinarily small viruses Or a com pleter different type of pathogen which lacks a nucleic acid genome How Do Prions Replicate One of the fascinating questions about prions concerns their mode of replica tion If prions do not contain a nucleic acid genome then studies on the replica tion of prions may reveal unprecedented mechanisms of reproduction The rst possibility is that prions con tain a protected nucleic acid and that like a viral genome it codes for the protein shell Table 5 The hypothetical prion genome could derive its protection from the proteinlipid coat or from an unusual chemical structure Such an un usual genomic structure might Confer upon prions the characteristics of pro teinaceous particles that are resistant to most procedures that attack nucleic ac ids Alternatively prions may contain an oligonucleotide that acts as a regulatory element instead of a39 coding template This oligonucleotide might act as an in ducer to promote the synthesis of prions Small nuclear RNA s are thought to be regulatory elements controlling the splic ing of genes 106 If the postulated nu cleic acid within the scrapie agent does not code for the protein or proteins in 142 Table 5 Possible mechanisms of prion repli cation Prions contain undetected nucleic acids Code for prion protein or proteins Activate transcription of host genes coding for prion protein 39 Prions are devoid of nucleic acids Activate transcription of host genes coding for prion protein Code for their own replication by Reverse translation V quot Protein directed protein synthesis its coat then this would be a major feature distinguishing prions from virus es I The second possibility is that prions are in fact devoid of nucleic acid if this is the case then alternative modes of replication for these infectious proteins must exist Table 5 The macromolecu lar information required for the synthesis of priOns must be contained either in the host cell or in the prion itself If cellular genes coding for the scrapie prion do exist then they are highly regu lated not readily activated and present in various mammalian cells ranging from mice to monkeys It is pertinent that hundreds of mice and hamsters inoculat ed with homogenates from the brains of control animals have never developed a neurological diSorder 9 107 These ani mals have been observed for Up to 1 year a period of time su icient to detect one infectious unit in the inoculum An occasional activatiOn of sUch cellular genes might explain the sporadic occur rence of CJD with an incidence of 1106 11 A few clusters of CJD with higher rates of incidence have been identi ed and 10 percent of CJ D cases are familial The molecular mechanism by which prions might activate cellular genes which code fOr their biosynthesis is un known The emerging story of oncov genes within retroviruses and their cellu lar counterparts provides an interesting analogy 108 y In addition we must account for the evolutionary pressure that preserves such hypothetical cellular genes that code for prions Perhaps these hypo thetical genes code for some necessary related protein or proteins when they are under normal regulation It may be that tolerance to a normal crossreacting gene product might allow the scrapie prion to replicateunnoticed by the im mune system Another explanation for tolerance toward the scrapie agent in volves selective suppression of small populations of potentially reactive lym phocytes 109 39 i 39 Alternatively prions could code for their own biosynthesis This hypothesis contradicts the central dogma of mo lecular biology 110 Unorthodox mech anisms such as reverse translation or protein directed protein synthesis would allow prions to replicate 111 We have no precedents for either of these 39synthet ic processes in biology The possibility that prions are devoid of nucleic acid should be compared to early studies on crystalline tobacco mosaic virus where no RNA Was found and Stanley suggest ed that the protein of tobacco mosaic virus was autocatalytic 112 Relevant to the mechanism by which the scrapie agent replicates are two ob servations First the various strains of the scrapie and CJD agents have been identi ed by repeated passage at limiting dilution and by their host range in experi mental animals 45 113 114 Second adaptation of the agent has been ob served upon repeated passage in the same host species as evidenced by a reduction in the length of the incubation period 21 113 115 Hadlow observed that the scrapie agent when passaged in mink retains its ability to infect goats but loses its ability to infect mice 116 The agent causing mink encephalopathy has a similar host range 20 While adap tation is mostreadily39explained by modi cation of a nucleic acid genome within the agent multiple host genes coding for several agents could also explain these observations The presence of multiple genes coding for different proteins with the same biological activities is empha sized by the occurrence of differing inter ferons 113 The genetics of the host clearly in u ences the length of the inCubation period for scrapie Dickinson t al have identi ed in mice two genetic loci that in u ence the length of the incubation period 23 118 In a survey of immunode cient mice we found that NZB and NZB x W F1 mice inoculated intracerebrally have an incubation period of similar magni tude to that found in BALBc and C57 B1 mice 61 In chtrast NZW mice have a signi cantly shorter incubation period Further studies with F2 back crosses are required to determine if a single gene is responsible for these dif ferences From these studies and those on the murine CJD agent we conclude that longer incubation time alleles are autosomal dominant Murine CJD stud ies have shown that the D subregion of the H2 complex39plays a Central role in controlling the length of the incubation period 119 The q allele in this subre gion resulted in shorter incubation times while the d allele resulted in longer 39 ones 39 SCIENCE VOL 216 Conclusion The consequences of understanding the structure function and replication of prions are signi cant If prions do not contain a nucleic acid genome which codes for its protein or proteins al ternative mechanisms of replication and information transfer must then be enter tained A knowledge of the molecular struc ture of prions may help identify the etiol ogies of some chronic degenerative dis eases of humans Development of sensi tive probes for detecting prions in such diseases is needed Diseases where 39 prions might play an etiological role include Alzheimer s senile dementia multiple sclerosis Parkinson s disease amyotrophic lateral sclerosis diabetes mellitus rheumatoid arthritis and lupus erythematosus as well as a variety of neoplastic disorders 12 The importance of prion research in the potential elucidation of a wide vari ety of medical illnesses underscores the need for puri cation of the scrapie agent to homogeneity and the subsequent iden ti cation of its macromolecular compo nents Only then can we determine with certainty whether or not prions are de void of nucleic acids Indeed recent progress in scrapie research has trans formed an intriguing yet forbidding prob lem into an exciting and productive area of investigation References and Notes S Stockman J Comp Pathol Ther 26 317 1913 W S Gordon Vet Rec 58 516 1946 B Sigurdsson Br Vet J 110 341 1954 W J Hadlow Lancet 1959 11 289 1959 D C Gajdusek C J Gibbs Jr M Alpers Nature London 209 794 1966 C J Gibbs Jr et al Science 161 388 1968 C J Gibbs Jr and D C Gajdusek ibid 182 67 1973 W J Hadlow S B Prusiner R C Kennedy R E Race Ann Neurol 8 628 1980 W J Hadlow and S B Prusiner unpublished observations C J Gibbs Jr and D C Gajdusek Nature London 236 73 1972 C L Masters J 0 Harris D C Gajdusek C J Gibbs Jr C Bernoulli D M Asher Ann Neurol 5 177 1979 12 D C Gajdusek Science 197 943 1977 13 M P Alpers in Slow Transmissible Diseases of the Nervous System S B Prusiner and W J Hadlow 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60 267 1950 C M Eklund W J Hadlow R C Kennedy Proc Soc Exp Biol Med 112 974 1963 H J Cho Nature London 262 411 1976 R H Kimberlin and G D Hunter J Gen Virol 1 115 1967 J S Gri ith Nature London 215 1043 1967 I H Pattison and K M Jones Vet Rec 80 1 1967 P Lewin Lancet 1972 1 748 1972 Can Med Assoc J 124 1436 1981 R A Gibbons and G D Hunter Nature London 215 1041 1967 G D Hunter R H Kimberlin R A Gibbons J Theor Biol 20 355 1968 D H Adams and E J Field Lancet 1968 11 714 1968 D H Adams Pathol Biol 18 559 1970 H B Parry Heredity 17 75 1962 in Virus Diseases and the Nervous System C W M Whitty J T Hughes F O MacCallum Eds Blackwell Oxford 1969 p 99 C D Darling ton in ibid p 133 A T O Diener Nature London 235 218 1972 Ann Clin Res 5 268 1973 I H Pattison J Comp Pathol 75 159 1965 J T Stamp Br Med Bull 23 133 1967 G D Hunter J Infect Dis 125 427 1972 D H Adams Biochem Soc Trans 1 1061 1973 D C Gajdusek in Human Diseases Caused by Viruses H Rothschild F Allison Jr C Howe Eds Oxford Univ Press New York 1978 p 231 D C Gajdusek and C J Gibbs Jr in Viruses and Environment E Kurstak and K Maramorosch Eds Academ ic Press New York 1978 p 79 R G Rohwer and D C Gajdusek in Search for the Cause of Multiple Sclerosis and Other Chronic Diseases of the Central Nervous Sys tem A Boese Ed Verlag Chemie Wein heim 1980 p 333 E J Field Br Med J 2 564 1966 Dtsch Z Nervenheilkd 192 265 1967 R Latanet B Muel D A Haig M C Clarllte T Alper Nature London 227 1341 197 D H Adams and E A Caspary Br Med J 3 1973 1967 H K Narang Acta Neuropathol 29 37 1974 A N Siakotos D Raveed G Longa J Gen Virol 43 417 1979 F O Bastian Arch Pathol Lab Med 103 665 1979 A Gray R J Francis C L Scholtz Lancet 198011 152 1980 F O Bastian M N Hart P A Cancilla ibid 1981 I 660 1981 G D Hunter R H Kimberlin S Collis G C Millson Ann Clin Res 5 262 1973 R A Somerville G C Millson G D Hunter Bio chem Soc Trans 4 1112 1976 R F Marsh T G Malone J S Semancik Nature London 275 146 1978 R H Kimberlin Scrapie in the Mouse Mea dow eld Durham England 1976 pp 1 77 G D Hunter in Slow Transmissible Diseases of the Nervous System S B Prusiner and W J Hadlow Eds Academic Press New York 1979 vol 2 p 365 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 66 67 68 69 70 71 72 73 74 39 75 76 77 78 79 80 81 82 S B Prusiner et al in ibid p 425 G C Millson G D Hunter R H Kimberlin in Slow Virus Diseases of Animals and Man R H Kimberlin Ed Elsevier New York 1976 p 243 I J S Semancik R F Marsh J L Geelen R P Hanson J Virol 18 693 1976 T G Malone R F Marsh R P Hanson J S Semancik ibid 25 933 1978 Nature London 278 575 1979 SB Prusiner D F Groth C Bildstein F R Masiarz M P McKinley S P Cochran Proc Natl Acad Sci USA 77 2984 1980 A N Siakotos D C Gajdusek C J Gibbs Jr R D Traub C Bucana Virology 70 230 1976 P Brown E M Green D C Gajdusek Proc Soc Exp Biol Med 158513 Hadlow Eds Academic Press New York 1979 vol 2 p 387 83 J A Rose Compr Virol 3 1 1974 F L Schaifer and C E Schwerdt Adv Virus Res 6 159 1959 84 T O Diener and W B Raymer Virology 37 351 1969 85 T O Diener Viroids and Viroid Diseases Wiley New York 1979 86 H L Sanger K Ramm H Domdey H J Gross K Henco D Riesner FEBS Lett 99 117 1979 87 E Mihalyi Application of Proteolytic Enzymes to Protein Structure Studies CRC Press Cleveland 1972 88 S B Prusiner J Cleaver D F Groth unpub lished observations 89 A D McLaren and D Shugar Photochemis try of Proteins and Nucleic Acids Pergamon New York 1964 90 J J Butzow and G L Eichhorn Biopolymers 3 95 1964 Nature London 254 358 1975 91 S T Isaacs C J Shen J E Hearst H Rapoport Biochemistry 16 1058 1977 92 M P McKinley F R Masiarz JHearst S B Prusiner in preparation 93 J E Hearst and L Thiry Nucleic Acid Res 4 1339 1977 C V Hanson J L Riggs E H Lennette J Gen Virol 40 345 1978 94 C Hanson personal communication 95 D Crowther and J L Melnick Virology 14 11 1961 96 K Borgert K Koschel Wecker J Virol 8 1 1971 97 P Bornstein and G Balian J Biol Chem 245 4854 1970 98 R M Franklin and E Wecker Nature Lon don 184 343 1959 E Freese E Bautz Freese E Bautz J Mol Biol 3 133 1961 H Schuster and HG Wittman Virology 19 421 1963 J H Phillips and D M Brown Prog Nucleic Acid Res Mol Biol 7 349 1967 I Tessman Virology 35 330 1968 99 T Alper D A Haig M C Clarke J Gen Virol 41 503 1978 Biochem Biophys Res Commun 22 278 1966 H Tauber E 100 101 R Latanet R Cramer L Montagnier Virolo gy 33 104 1967 J S Semancik T J Morris L G Weathers ibid 53 448 1973 102 J A Reynolds in Membrane Receptors S Jacobs and P Cuatrecasas Eds Receptors and Recognition Series B vol 11 Chapman amp Hall London 1981 p 33 103 P Andrews Methods Biochem Anal 18 1 1971 Y Nozaki N M Schechter J A gyrglds C Tanford Biochemistry 15 3884 104 A C T Nozth and A Rich Nature London 191 1242 1961 C Tanford Physical Chemis try of Macromolecules Wiley New York 1961 pp 317 456 105 R Langridge H R Wilson C W Hooper M H F Wilkins L D Hamilton J Mol Biol 2 19 1960 G Giannoni F J Padden Jr H D Keith Proc Natl Acad Sci USA 62 964 1969 W C Earnshaw and S R Casjens Cell 21 319 1980 106 V W Yang M R Lerner J A Steitz S J Flint Proc Natl Acad Sci USA 78 1371 1981 107 J M K Mackay Nature London 219 182 1968 I H Pattison K M Jones J N Jebbett Res Vet Sci 12 30 1971 108 J M Bishop Cell 23 5 1981 109 W O Weigle in Autoimmunity Genetic Im munologic Virologic and Clinical Aspects Ac ademic Press New York 1977 p 141 B H Waksman Clin Exp Immunol 28 363 1977 L F Qualtiere and P Meyers J Immunol 122 825 1979 39 110 F Crick Nature London 227 561 1970 111 R Craig J Theor Biol 88 757 1981 H Kleinkauf and H Von Dahren Curr Top Microbiol Immunol 91 129 1981 112 W M Stanley Science 81 644 1935 113 C J Gibbs Jr D C Gajdusek H Amyx in Slow TransmissibleDiseases of the Nervous System S B Prusiner and W J Hadlow Eds Academic New York 1979 vol 2 p 87 E E Manuelidis and L Manuelidis in ibid p 147 J Tateishi M Ohta M Koga Y Sato Y Kuroiwa Ann Neurol 5 581 1979 D T Kingsbury D A Smeltzer H L Amyx C Dominance in Fishes The Relation Between Environment and Abundance Marine shery studies are replete with comparisons of environmental factors and population abundance or recruit ment and recently correlation matrices have been used to compare responses coefficients of different species to the same factor I 2 Although the studies have included dominant and subordinate species that interact 3 4 the responses of these species have not been related speci cally to their positions in the dom inance hierarchy The purposes of this articles are to compare the relation of temperature to the catch of Atlantic her ring Clupea harengus and Atlantic mackerel Scomber scombrus species that have alternated as dominant and subordinate in the pelagic biomass off Bernard Einar Skud New England and the Canadian Mari time Provinces and to relate their re sponse to dominance The relation also is examined for other species including the California sardine Sardinops sagax caerulea and anchovy Engraulis mor dax To paraphrase Daan 5 the dominant species is de ned as the more abundant of two species that have a functional relation interact and whose densities are maintained at distinctly different lev els He speci ed that replacement or a change in dominance required at least a 50 percent reduction in abundance of one stock and a comparable increase in the other and that the change be persistent for a number of years 144 00368075820409014401000 Copyright 1982 AAAS 39 J Gibbs Jr D C Gajdusek in preparation 114 A G Dickinson and H Fraser in Slow Virus Infections of the Central Nervous System V ter Meulen and M Katz Eds SpringerVer lag New York 1977 p 3 R H Kimberlin and C A Walker J Gen Virol 39 487 1978 115 S B Prusiner S P Cochran D F Groth D Hadley H Martinez W Hadlow in Aging of the Brain and Dementia L Amaducci A N Davison P Antuono Eds Raven New York 1980 p 205 116 W J Hadlow unpublished observations R P Hanson R J Eckroade R F Marsh G M Zu Rhein C L Kanitz D P Gustafson Science 172 859 1971 117 G Allen and K H Fantes Nature London 287 408 1980 S Nagata N Mantei C Weissmann ibid 287 401 1980 M Rubin stein et al Arch Biochem Biophys 210 307 1981 118 A G Dickinson and H Fraser in Slow Trans missible Diseases of the Nervous System S B Prusiner and W J Hadlow Eds Academic Press New York 1979 vol 1 p 367 119 D T Kingsbury and J D Watson in prepara tion 120 This article is dedicated to Dr Francis A Sooy on the occasion of his completing a decade as Chancellor of the University of California San Francisco I thank F Elvin F R Masiarz L Gallagher S P Cochran D F Groth M P McKinley K A Bowman D E Downey N 1 Mock D P Stites and J R Baringer for continuing help in these studies Drs R C Williams R C Morris Jr P Bendheim D Bolton T O Diener W J Hadlow A Gor don H Fields B M Alberts T B Kornberg I F Diamond W J Rutter M Rubinstein and R M Stroud for discussions during the preparation of this manuscript and Drs F Seitz R Schmid L H Smith Jr and J R Krevans for support and encouragement Sup ported by NIH research grants NS14069 and AG 02132 NSF grant PCM7724076 a gift from the R J Reynolds Industries and past funds from the Howard Hughes Medical Insti tute Interaction Between Herring and Mackerel Landings catchand other estimates of abundance and biomass of herring and mackerel in the Gulf of St Lawrence since 1960 indicated that the species in teract and have alternated as the domi nant species in the pelagic biomass 4 6 The evidence was based on 15 years of data and on the results obtained from simulation models The estimates of abundance were from cohort analyses and recruitment surveys The conclu sions of the investigators 4 6 were tempered by the constraints of their models and by the need for a longer series of empirical data Supporting evi dence of this interaction is apparent in data from Georges Bank 7 and from the North Sea 8 In order to satisfy the need for a long term empirical series I compared the landings of mackerel and herring from the Gulf of Maine to the Gulf of St Lawrence from the late 1800 s to 1960 Fig 1 9 I 0 I assumed that long term The author 11112008 Slow Viruses Lecture AmiL Hartman run nannianl cur medu 1125488755 31112001 Virai Pathogenesis 111301 What is a slow virus Slow diseases caused by prions in humans 11112008 Slow diseases caused by prions in animals mm L Pmunu 1 up Nrumumxmunu mm m lMH Mum y mm m m m m Inapon am pm WWW I mH mww W um m My mmmu mu nmmmn 1N N I mm x Huummmw a 1mm 4 r DuJ m 111 1111 Transmissible spongiform encephalopathies TSES Three classes 7 lnlemuuslransmwsswb E1 Transv aHtstanmhahsmmnsumvtmn a maf nm Nam Emma s 7 Famma 114 1mm autusnma ammantmmatmn 7 Spuramctssaa N n evmamm ngmnmtatmns 11112008 Common features of Prion Diseases 1 Progressive and fatal no treatments 2 Spongirorm degeneration swiss cheese 3 nervous system 4 Amyloid plaque formation aggregates 5 Cerebellar ataxia loss of coordination amewangnarml n nncel wnmeeseitenotr inmehninanmwwnh mz ca yak Yhehninannmemwnntreumei zkah isezkluhmzsz simiorznnemm Signs of TSE Defective motiongait cerebellar ataxia Dimentia Death takes YEARS 11112008 Scrapie 1st TSE identified Signs in sheep wavering gait staring eyes paralysis Weight loss and death within 4 6 weeks after n first signs of in ectIo Recognized 200 years ago in Europe Endemic in UK Can pass from infected to uninfected herd 1939 sheep brain homogenates contained infectivity could not be filtere Still can not purify scrapie agent to homogeneity Kuru 1st TSE of humans 1957 Carleton Gajdusek Studied strange disease in New Guinea 39kuru39 shivering disease trembling Kuru stages of disease Prodrome headache aching Ambulatow stage unsteadiness tremors Sedentew stage inability to walk uncontrollable laughter quotlaughing deathquot Tertiaw phase muscle weakness inability to speak unable to control bowelsbladder Similarities Scrapie vs Kuru Spongiform holes in gray matter Difficulty standing Motor imbalance 11112008 What is the infectious agent Resistant m formalin 57m m m Mimi m WWW mm xation i i v 4 i blmmvwhic Wquot my mum i a ReSIStant m proteolySIs qubr formimitii Romam whomm quota mmm w m th i i uuiwm a womltn nhanvarbluzl fumian Hard 7 k39 quot Kmmm u mquot mmny imlmlwx Proposed structures for Scrapie agent 2 Uncunventiunalvirus filterable virus Conventionalvirusw manDN Virus unusualpru enies Replicatingpmgin Nudeupmteinmmplex Replicating Nucleicaci w palysaccharide palysaccharide coat DN subviru 39 Spimplasmar ike arganis ruvirus Naked nucleic acid Membrane39buund DNA ivim d5 Can infectious agents exist without nucleic acid Identification of the pmp gene teat protein complexes visualized in infected sneep brains Concentrated protein remained infectious Stanley 5 Prusinerdeveloped fractionation procedure to isolate protein Cloned seduenced prnp gene Highly conserved in manyanimals 11112008 Genetic relationships of pmp genes pmp gene PrP or PrPt 39 Neurons pmprr mice develop normally 18 mutations in human prnp gene associated w familial TSE diseases Genetic mutations associated w genetic TSE 11112008 JS Griffith started the quotproteinonly hypothesis Proposed that scrapie was mediated by a host t us pro ein not avir Prions Prusiner named scrapie agent altered form of PrP causes scrapie proteinonly hypothesis Essential pathogenic component is hostrencoded PrP protein w aitered conformation Called PrP or PrP39ES PrPSK can convert normal PrP to the pathogenic orm pmpl mice are resistant to infection lt93quot n 5 t inka Alt 1 25quot 1quot1 z 4quot y 1quot 7 v I c 7 g PrPC proteins from different animals 11112008 Neuropathology sunshnwmg ivnng nrm lemma iixrni l lzgllnili Lrlgml V 55 daeieihnwmg Svnnglmrm Ude m rrr vliweslrgml Mun shawlquot l rpnngrnrmllmlm 7 VerliqueiLrgml 11112008 Carleton Gajdusek Nobel Prize 1976 for discovering slow Viruses39 in humans lTSEl Stanley Prusiner Nobel Prize 1997 for discovery of Prions we l39nIrlrunvm mm mm m Salim Differences in PrPC and PrPSc PrPC PrPs 2723 s mummy Em How is PrPc converted to PrPSC Two main models of molecular conversion of PrPCQPrPS 1 Templatedirected refolding aka quotrefnl ingmn el 2 NucleationDependent Polymerization 7 aka quotseedingmn elquot 11112008 TemplateaDirected Refolding Model PrP uniuided and refolded using PrPS astemplate High activation energy barrier gt molecular chapemne ipratein x Raterlimiting step binding of PrPE and chaperone Evidence for Template Model Chaperone studies increase formation of PrPS N ucleatlonsDependent Polymerization Model PrP and HP in reversible monomeric equilibrium Several PrPS monomers needed to form stable nucleus r see Rapid autocatalytic growth of PrP5 opolymer amyioid Infectivernaterial contains PrPS seed a 3 Al 9 r L Ri e 11112008 Evidence for Nucleation Model Excess wit can convert recombinant wt to protease resistant form helical and Bssheet folds Current Research on Prions Expensive difficult time consuming Many unanswered questions Rapid culture assay for infectivity Regulation of pmp gene expression BSE or Mad Cow Disease progressive neurological disorder of cattle infectious ltransmiss 124000 cases in UK Peaked in 1993 11112008 sheep e MBM pmcess changed in 1930 1992 7 UK banned feeding protein from any ruminants to cattle Ml MlnaquotLlzlda1537 1quot Yhh lV A m1 W Hi i Effects ofUK Mad Cow quot L 39 loss otnearly 200000 diseased cattle 45 million older cattle slaughtered British livestock industry crippled industries that make bovinerderived products severelya ected r harmacelmal r lealln Cases outside UK due to export otcattle and cattle products BSE cases in cattle in UK 11112008 VCJD cases in UK and Europe ass Cases in quotarm America by Year and Country of mm 1993mm 3 US cases of BSE Dec 23 2003 7cow from Washington sate m 39 71mpmammm Canada Jun 24 2005 7cow from Tea 7 pm endemm case Mar 15 20067 cow from A abama 7 Unknuwnungm Canada 15 cases of BSE in cam e 2 11112008 vCJD in humans in US 3 cases total 2 exposed in UK 39 1 in SaudiArabia Viral Immunology T cell or not T cell using HIV as the model IDM 2004 Lecture 4 September 2008 Charles R Rinaldo PhD quotU 9 o 3 Are CTL important 1 L7 in HIV infection r1 239 09 H Exposed 1 L Lquot7 uninfectedsquot 3 03 11 0 H 839 L a 07 Q 2 g 0396 Logrank P 01 a 0 1 2 3 4 No at risk CTL negative 31 27 25 24 1B 11 CTL positive 8 7 6 6 5 5 Time to positive HIV1 ELISA years Time to HIV1 seroconversion by cytotoxic TIymphocyte status at baseline Between 1996 and 1997 HIV1Envspecific CTL was assessed in HIV seronegative female sex workers Although baseline CTL were associated with a reduced HIV incidence in univariate analysis a multivariable model that included the duration of previous e work and levels of HIV exposure found no independent association between HIV1 protection and baseline HIVspecific CTL P 09 Kaul et al 2004 Koup et al J Virol 684650 1994 DF39DSK Hmn Wampum Pam Cl Neutralizing Ab 39titre1 5 3 J I C3 C1 l1 1 39539 13139 II D w umrsald ENE iLMH EWSEId F105 F105 134 x Survival time of CD8 lymphocytedepleted and control antibodytreated monkeys after primary Sleac infection Significantly different survival time from control antibodytreated monkeys nnng E 3 D 32 Days I CD Sdepleted all n 6 9 Controls n s 6 D CD Bdepleted gt28 days n 3 ltgt CDB depleted lt21 days 1 3 Schmitz etral Science 1999 Plasma SIVmac Gag p27 antigen levels and the appearance of virusspecific CTLs during primary SIVmac infection of CD8 lymphocytedepleted rhesus monkeys 39Enntml a CDE dnplaiad H21 days CDEdapla ted 33923 days IE 7 3 C 5W Eng 27 inglml n r is 21 an as n7 3951 131 21 325735 E 7F m t 139 3 7 r i Pi 39 m u m E M F 39 u E E 2 L 1quot wquot If xiii quotr 1 g E E 5 I i 5 39 a t quot l quot 31 v I 1 1 1 a I I a g 33 n II Hm a i I H H I a gt 7 In 6 7 E i U 39n f 14 11 2393 35 a r w 21 25 as in r M 21 23 35 39 Days Schmitz et al Science 1999 lL2 receptor VA Q RECOGNITION gt Figure 345 lmmunobiology 7ed Garland Science 2008 Table l Assays culmmly used Io detect HIVspecilic CTLs Assay Bulk lysis Liming dilution analysis LDA Temmlerlc complexes IFNll assay lloiv cytomatryl IFNi assay ELlSpotJ Polyrunctional T s cell a say ICS ow cytoni etry Based on Ability of CTLs to lvse taiget c it Ability oi ms to lvse target cells Abiliiv or LTLS 0 39ecognlse HCrpepllde complexes Abili v of CTLs to produce this cyloklne Abilty o CTL to procuce tnis oytokine Ability of CTLs to produce cytokines and cytotoxic molecules Advantages Measuies tie tinal e eclor m nation Measules the twat eWeClor function sensilivily Speclhclty FleXibility at How cytomelry Sensitivity Specilioity Flexlbwly of new cymmelry Tne wncle pioteome can be testec Sensitivity Speoilioity Tiie wiiute pioleoiiie can a tester Eesilv automated and standardized Sensitivity speci city exibi ty of ow cytoni etry Disadvantages Seml39quanlllallve Low sensitivity time consuming Time consuming Detects only set s min a man Drohfelallve mtential Not a tuntnonat assay Pesmo ed few epitopes 0an a Danlcular furcllorl of CTL is tested Onlv a pariiculai turction of CTL is tested Expensive and sophisticated procedures Benito el al 2004 DCs are needed to induce an efficient CD8 CTL response need DCs It takes two to tango It takes two to tango DCs T cells Tango II by Giclee class II 39 Figure 814 lmmunabiology6e Garland ScieIKe 2005 MHC class I antigen processing and antigen presentation pathways 3 Gel membrane E 3 gt 175 Gdgl 1 73 Gymml nature 7 MHC GEES A we a V Eugenest IEHGUIUIT39I rims du raeal 7 f I l FEWquotLEE quotquot If 39 1 TA 1 1 TAPE 599 THYf 13913 7 re V 39J r Fmtaasoma Intraceuular prawn u b CEquot I39I39lEITDI39EI39IB E L Enlg v2 PEP n Cyml Endoplwmlc reuwlun 7 Mature 39 x MHC 39 39 classl E quot l HHS235 I 3 vi 7 3 x Phagosun39na i39 Andersen et al 2006 SFC106 PBMC 20000 15000 10000 8000 6000 4000 2000 20000 15000 10000 5000 1000 D p lt005 no DC Single 3032 6062 B Nef no DC DC CD40L 0 0 0 Single A10 B10 mc10 mE10 EFZ5 324 EEEEUH49 109 2524 49 Single Peptide pools Moral Tango is for 2 not 1 Huang et al unpublished p17SL9 Gag GQZO MODC RTAIVQ EBV GL9 A 3 p17SLQ GagG020 RT lV9 EEV GLB FluGL9 FluGL9 E s W l u 3 l g 1 x Functions 5 4 C 7 O O O O C O O O O O O O O O O 0 FN O O O I O O O C O C O O O O O C ILZ O O O O C O O O O O C O O O O MKPgtll O I C U C O O O O O C D O O O O TNFiu O O O O O O O O O O O O O C O O p17SL9 Gag6020 RTIV9 EBV GLQ p17rSL9 GangOZO RTerB EBVVGLS FlurGLB FUgtGL9 Equot 5 U 3 Functions S 4 CD1 3 D O O O O I C O O O O O O O C IF I C O O O O I O O O D O O I I O 1L2 I O I O I I I O O O O C 0 C O MIPil O O O I O O O O O O C O O O O O TNFu O O O C 0 O O O O O O I O O O O p17SL9 Gag GQZO RT IVQ EBVGLQ p17rSL9 65976020 RTerS EBV GL9 FlurGLS FIu GL9 aa39 1 a E F Functions 5 4 CD107 O O O O O O O O O O O O O O O 0 IFN 0 O O O O O O O O O O O O I 0 O LZ C O U O O O O O C O O O O O O MIPlh O C O O C O O O O O O O O O O O TNF u I O C O D O O O O O O O C O O 0 BBC prime the broadest and greatest number of muiltifunctional CD8 T cells from 4 HLA A0201 newborn cord bloods with autologous DC loaded with viral peptides representing different viral dominant epitopes The T cells were primed for 2 weeks with the different DC loaded with HLA A0201restricted 9mer and 20mer viral peptides and then assessed in a standard ICS assay Huang et al unpublished LANA 411427 KKEDEEDGG DGNK rlSI NmIFNAvSFC par m an PBMC DGGDGNKTL G L D T E L DA sum Ema mum lnmer 3 um um DGGDGIIKH Lepone et al unpublished CD8 T cell differentiation Nalve Memory EWecior comm cuzao cmsaoo 5mm cmus CD25 cmsnoo cuzr cmsnoo 002539 ms 5m war war cnzrccar cuzro ccRr CD27 ccRr cozrccnr ch71cRr iwycrpemw iFNr39fGr periunrr iFNr39fGrperiormquot iFanGrperiunnquot iFNrr GNperiunn IFN f Gf perinnn Andersen at a was 1 was cull Nywxrlllu mm alcbr v m mnumnumn AWWM mm 5mm M u u 4w cm muuh umw w mmu In an Mum my mmwm w cm v mwmm v mm Ca7 mama llmnmlm mm cw r m n ma lo spuiMy EBV Hcv cw w HIV an M nmv mm mm Appay a a 2mm HIV1 Dysfunctional State G CD27hi9hCD57390W Early Stage Intermediate Stage Late Stage CD27hi9hCD57 CD2739 WCD5739 W CD27 CD57hi9h Na39I39ve CD8 Memory Stages antigenexperienced Hoji et al 2007 Figure 825 lmmunobiology 6e Garland Science 2005 Getting kinky Dancing with 3 partners Some CD8 T cells require both DC and CD4 T cell help Figure 828 lmmunobiology 7ed Garland Science 2008 5 3 Nil 2 5 31mlnd Ii 5i 3 cum 7 5h 3 Hum want 3 v r 1 nunsan jr 3 E I quot 39 1am 391 I s f quotWWW 3 51 T L i i v E J z I i w A a r39s V 39 l g u 1m x I v f E mam r4 3 E a 3 In n V 39 l1 an g s l I i E E 1D 3 E LE 4 i E 1 i aquot gig 1 mJ GTL pmumr I IJ nty CIT 1329 CTL Em chm CTL Ne pl 311 13 115 1 Requirement for IFN gammamatured DCs CD4 T cells Priming of HIV1 specific CD8 T cells Spots10 CD8 T DCs matured with CD40L CD8CD4 effects in priming conditions a 208 I CD8 CD4 02 meow CD4 2 DCs matured with CD40L IFNg CD8CD4 effects in priming conditions El CD8 I CD8 CD4 02 E CDB2 CD4 2 19 V W x c HLAA2 HIV1 peptides Colleton et al unpublished I Before T cells can be primed HIV1 infection of LC and DDC 9 a smash f 93 Q Q Q Q Q Q Q Q C9 7 LC Langerhans neHs cow CD47 lymphocytes Subrepnheha level ympnncytes a DDC Dandvmc 25H 0 O Dendmm ceHs ngrahnn m pmmal ymphnndes Dennrmccens W E W m K m ym n K 0 mm 0 com ymbhocyles Q nlelmun or he has KGALT Werm gas 7425qu m ymmmwgy Priming of HHV8 specific CD8 T cells LC are better than DDC IDDC 350 ILC 300 250 5200 E150 E W i l J J 50 01 1 ll Jr ii lJi 0 QB overlapping peptides 130 39 T cell IFNI 39 39 overlapping 15mer gB peptides CEF CMV EBV Flu A9mers Primi ng of HIV EBV Flu specific CD8 T cells LC are better than DDC zooo m 1500 mom 8 1000 39 LC 39 500 I i DDC L no oo D 3 0 o 200 O 100 LL 0 0 53 70 53 L9 L9 RNA 6 NA 30 NA 66quot G F G Pep de CD40L I 2 1 rJlrr1mature Mature Dc DC b IFNi I L2 IFNi DC maturation Antiviral reactivity DC maturation factors TABLE 2 V39irus Egeci e FNquotr39 Production in 13th T Cells Stimulated hjquot PepIideLmded DC HIV 1 1 HIV 1 jl n ART HWl Ijjl nor on ART EMS realmam K 39 P i39t39quot39 P 1v quotquot39 P pnlyl39 12 Nquot NE 10quot 1 391 FNo NS NS mm F39va NS NS 3 35 L13 NS Ifu N5 2MB TNF c NS it ll 6 N5 BEL 2 pzuly c 1FNCt FN r L I TNF c n I5 fill396 a5 EEMLIZI i 05 Qfllr l 13k EEMEIL JEN1r NS lIILI39IIlI III 11quot139l39 139quot393 Number all results stealth equal In OI law rm Ill3y atimulaled Iquot almt 239 Wilcox2115 aigner mnk lesl 397 NE quotat signi cml Huang et al 2008 Maturation of DC for stimulation of viralspeci c lFNI production by autologous CD8 T cells A mvn quot J quot31va rnnlmn pasmslrwysrcIvu39cwl u m Huang el al 2qu HlV1specific lFNy production 2500 No DC 2000 1500 1000 500 GagA GagB GagC GagD NefA NefB PolA PolB PolC PolH PolI PolJ Rev Tat VifA VifB Vpr Vpu III quotHit0 gtgt cc mm Med IIII it gtgtgtgt ccx LIJLIJUJLIJ 2500 SFC106 PBMC DCCD40L 2000 1500 1000 500 Env Gag Nef Pol RevTat Vipreru HIV1 person on ART CD4 T cell 703mm3 HIV1 RNA lt 40 copiesml DC CD40L induced higher levels of peptide specific lFNi production PBMCDCCD40L HIV1 peptide pools revealed more T cell responses than PBMC HIV1 peptide pools I said the moral is tango with 2 not 1 Huang et al unpublished Now the tango is over you can go it alone 3 mechanisms of CTLmediated cytotoxicity Andersen etaL ZEIEIE Patient A1 1 66 MK35 study HIV1 HLAA0201 1 Paradigm of HIV1 disease progression and host response nE am I V s ML m r 5392 0 917 3 V V 39 w m E 39 m1 wm a I r W 25 M g E a E 2 g 3 I I w r1 1391391391391 395 39 cmmusmr 1 3i u o m 1 WI i3 1 c n rm E5 1 MW 5 3 F a 8 5 39 i W i I 533 E l w I39l39 quotl39l39l39 gi39 39 p amp Years Weeks 4 2 a a a at man Yum m um BTW MC JJNAW Some potential mechanisms of CTL failure in controlling HIV replication Deiicit oi helper activity 4 CTL dysiunctionanergyapoptosis r 7i Altered phenolyplc maturation lt Dmnshed cytokine producllon neiicii oi HLA expression J CTL epitope escape mutation Alteration in antigen processing Benito et al 2004 1Leu kapheresis r l lt 18 subj dose 1 cells a ects will be enrolled 6 at low 3 million nd 12 at high I dose 510 million cells 2 PBMCs are placed flasks and separated by plastic adherence to derive monocytes V5 5 Vaccination Subjects are randomized bl to receive vaccine either i subcutaneously or intravenously 4 DCs are matu d overnight in lL13 V l Gag 386394 VLAEAMSQV HIV1 Env 134142 KTLPLCVTL HIV1 Pol 498506 ILKEPVHGV InflAMP 58 66 GILGFVFTL V Peptides chosen were HLAA2 supertype highly conserved immunodominant epitopes 3 Monocytes are grown for 6 days under GMP conditions in lL4 and GMCSF to transform into DCs mm wm mm mm Wm quotquotquotW39M Connoiiy et ai 2008 Mean ELISPOT responses per 105 PBMC of all 18 DCHlV 1 vaccine participants Tough guy cuts in Regulatory T Cells Thymus Lymph node If 0 o o 5 Q k T g A 00 2 i M 39 Treg APC T helper cell lL2 0f lt qnotoxic T cell R III R RI C025 eoogn on 0009 0quot GITR o In of sell am In thymul Regulmory In pulpingn T cells unuu 0x40 Inhlbltlon of lnhlbltlon of T cell T cell actlvatlon a ector lunctlons HLADR inhibition of T cell activation gt Emelor Toolls Naive AFC T I Suppress selfreactive T cells regulate immune response May originate from thymus or induced in periphery CD4CD25quoti9thoxP3 as marker Multiple mechanisms of suppression Tregs and HIV MHC II HIVspeci c Toeil expansion HIV CD4CDZ5 t MHC IV peptideTOR Natural Tregulatory cell CD4CDZS G gt HIV endotherstimuii l CTLs response Infected cell 73909quot induoe Toell activation AmiVE ed C8 ltlt l Antibody production Higher susceptibility immune dysfunction v to HIV infection Cell death T oell Sempere et al 2007 Detrimental or Protective Impairment of HIVspecific response or Limiting immune activation Tregs are increased in HIV1 elite suppressors A p emu parJ1 Tregs inversely correlate E m F u I with activated CD4 T cells g 4w in HIV1 infection 53939 a nlcr H 1593 7 E I I F ED ma 3 39r g a I 3 I 1 it 5 I m E l l E 239 t 1 a 393 H 1 1 D i 1 I vn u 7 A 39 4 I A g 2L iii 1 qu uf FOXPM cmzm cow T cumin I r iimam llhH39 am J39Inle ad NdthVU 9t 3L 2008 Fa39i ilz u l39alml 39Flu u mSif l Chase et al 2008 Thus do Tregs dampen T cell activation thereby decreasing HIV1 replication Tregs and HIV Disease stale Treg phenotype No ART CD4 CD2S39 CD4 Foxp339 CD4 CD2 S Foxp339 CD4 CDZSMC062 Lhl CD4 CDZS39C012739 CD4 CD25 C0127 45RO39 On ART CD4 CD25 CD4 Foxp339 CD4 CDZS Foxp3 CD4 C025 CDIZ739 Change I Increase Increase Decrease Increase Increase Increase Decrease Increase Increase Increase Increase Organ Peripheral blood Lymph node Peripheral blood Peripheral blood Peripheral blood Mucosa Tonsils Lymph node Peripheral blood Peripheral blood Peripheral blood Peripheral blood Peripheral blood Lymph node spleen Mucosa Peripheral blood Tonsils Peripheral blood 13 I16 251 1539 44 14 10 1012 ml 16 Seddiki and Kelleher 2008 Conflicting results probably due to technical issues Important in immunebased therapeutic strategies sum AND 3 PATIENT 1 20030424 HutuIn FL2 LUGrFLd LOG ADC PCS 04 Gating for CD4 Tregs Hungaiedl PATIENquot 1 200301121 H07 LMD FL3 lOGSS L1H ADC HA1 PAT ENY1 209399213 1107LMD FL3 LOGI FL U35 MIC 1323 PCS C04 l y 1039 10 10 m ECU E03 EDD 003 leAAllU BAND 6 FATIEH I 200843424 107LMD n1 LDGJFLz LDGA Ant PE CD 25 HIH i u m yuy IVWIIHII ml yl y 10 0 4 1 FE 5125 FITC FDXP3 Macatangay et al unpublished IPCPOO1 DC therapy clinical trial Treg immunophenotype increases at 6 wk I nTregs pre 7 I nTregs post i 14 15 16 17 96 nTregs N H O Uquot l wquot 2 10 11 12 13 Subjects Treg values average of 5 readings Macatangay et al unpublished IPCP 002 Preparation of DC loaded with autologous HIV1 as therapeutic vaccine 5 Vaccination Dose route of administration and vaccination schedule vary by trial Largely empiric at this point 1 PBMC for DC preparations Substantial quantities neede Obtained by leuktheresis for 4 DC matured with L2 IFNI IFNX TNFX poly lC Has profound effect on the ultimate DC product and its action on T cells in vivo preparation of multiple vaccines 2 lm mature DC are differentiated from monocytes GMCSF and L4 Automated process 3 Autologous HIV1 antigen is loaded into immature DC Type of antigen will influence both DC maturation and T cell response Balance isthe answer to an HIV vaccine Not too much and not too little le swamquot Law sensmwly ya mum Wen cm 1 quotH cm 1 quotm amp wwsonshw lylmhamznl gcns Actmummy a mmmm cm I anus allarpnmuryH v n mm rwanw I 3 Emma w mm W swam m r cevs 1 Luis m HIV mm W mmquot m IHICWECDE39YCEHSIW a a E 1 w an unewm mam s K Appay et aL 2008 FN Herpesviruses Herpesviruses From Greekmeans to creep Records from ancient Greek times Hippocrates Shakespeare Romeo and Juliet Reference to Queen Mab midwife of the fairies O er ladies lips who straight on kisses dreams which oft the angry Mab with blisters plagues because their breaths with sweetmeats tainted are Human Herpesviruses HHV HSV 1 Herpes simplex virus HSV2 VZV VaricellaZoster Wrus CMV Cytomegalovirus EBV EpsteinBarr ViI39US HHV6 Human Herpesvirus 6 Variants A and B HHV7 HHV8 KSHV Kaposi s SarcomaAsscciated Herpesvirus Identification of Herpesviruses oMorphology Core DS DNA Genome lcosadeltrahedral Capsid 162 Capsomers Amorphous often asymmetrical tegument Envelope Cederived Membrane with glycoprotein spikes Hallmark ofALL HHV Establishment of LATENCY Herpes Virion Herpesviridae Vi rions th ogenic Tree Genelic Helatedness Of Different Herpesviruses HSV1 HSV2 I alphaherpesvirinae VZV 39 CNN HH J BA HHVGB EBV betaherpesvirinae l HVS Rheum HH JS Rhadin WWI gammherpesvirinae Herpes Latency Lytic Replication Latency HSV Reactvation HERPES SIMPLEX VIRUS REACTIVATION SENSORY NERVE GANGLFON HSV Life Cycle Gene Transcription Human Herpesviruses HHV Alphaherpesviruses Neurotrophic Rapid life cycle HSV1 Herpes simplex virus HSV2 VZV VaricelIaZoster rus Alphaherpesviruses Herpes Simplex Virus HSV 1 amp 2 Enters through the mucosal membrane of abraded skin and establishes infection in local epithelial cells viral replication formation of virus lled blisters transport to the nuclei of the sensory neurons establishes latency Reactivation through stress stimuli such as UV light fever hormonal changes surgical trauma to the neuron How Common 5 HSVaZ One in ve people in the USA are infected with HSVZ quot wank w NH m Pregnant Wamen HSVEZ 6 Japan 0 Ta 39Im u39a n Austria I i a Iceland Sweden l ta My mm a HSV 2 Facts HSV 2 infection is most common in people aged 20 to 30 years The number of people infected with HSV 2 in the USA increased by 30 per cent between 1980 and 1994 There is a similar trend in other parts of the world Yet four in five people who are infected with HSV 2 do not even know it either because they and their doctors do not recognize the symptoms or because they are infected with the virus but never suffer from any symptoms httpwww herpesweb netgenerafactsgenera htm 9 20 of people infected with HSV Z have no sympto ms at all 5mg m penplle infected with HEM2 have I ay mpmms but 2 in mm knnw they are mfacted with the virus mquot Antavira Therapy HSV p Hunk W quot 1va 1 XTP39I GLmnFFiv Acyclovir Pharmacology The effect of acyclovir is based on a selective inhibition of the replication of herpes viruses The drug is phosphorylated by viral thymidine kinase As a triphosphate acyclovir inhibits the DNA polymerase and thus the formation of viral DNA The host cells are not considerably affected VaricelIazoster viruses are not as sensitive as herpes simplex viruses Resistant herpes viruses that lack the thymidine kinase have been observed almost exclusively in immunocompromised subjects httpwww infom edorg1 OOdrugslacycfram html Alphaherpesvirus Varicella Zoster Virus VZV Considered endemic gt95 infected Transmitted with direct contact or airborne droplets 0 late winter amp spring Disease 39Primaiy infection chicken pox 39reoccurring infection shingles 50009000year hospitalized 100year deaths Communicable 12 days prior to onset of rash and during the presence of the rash 50 of all individuals over 80 years old or immunocomprimised will develop shingles o Posttherapeutic Neuralgia PMN extremely painful due to nerve damage a Vaccine live attenuate OKA strain Live Attenuated Vaccine is estimated to be 95 effective in children fails to protect in up to 30 of adults with close emosure to someone with ol39iioken pox anyone over 12 years of age will need two shots can protect for more than four to ve years 39 million cases of chicken pox occur annually in the United States killing an estimated 90 persons 0002396 death rate adults comprise only 2 of chicken pox cases but are responsible for 475 of deems highrisk children with leukemia or persons receiving steroids 39 patients with cancer arthritis kidney disease organ transplants or asthma Herd Immunity could reduce transmission of varioella to immunooompromised or other susceptible persons and may reduce the risk of congenital or neonatal varicella resulting from infection of pregnant women Vaccination program might shift the incidence of chicken pox to older ages adults where the complication and death rate rise sharply 7 l Kristine M Severyn RPh PILD Human Herpesviruses HHV Betaherpesviruses Lymphotrophic T cells CMV Cytomegalovirus HHV6 Human Herpesvirus 6 Variants A and B HHV7 Betaherpesvirus Cytomegalovirus CMV l39ransrnission through saliva birth canal breast milk blood products and sexual exchange of body uids 4080 world population are CMV positive lnfects epithelial cells polymorphonuclear leukocytes and T Cells Congenital CMV infection in 1 of all newborns 10 of infected newborns will exhibit clinical symptoms 0 Cytomegalic inclusion disese CID leading cause of mental retardation deafness and other neurological defects Reactivation immunosuppressed can result in interstital pneumonia gastroenteritis hepatitis and leukopoenia Betaherpesvirua Human HerpesvirusG HHV 6 Discovered in 1986 gt90 of the world population by age 2 are HHV 6 positive lnfects Toells epithelial cells natural killer cells and monocytes Causative agent of the childhood disease roseola infantum Primary infection in adults can result in mononucleosis hepatitis and lymphadenopathy HHV 6 Betaherpesvirus Human Herpesvirus7 HHV7 Discovered in 1990 85 of the world population by age 2 are HHV7 positive Infects T cells epithelial cells natural killer cells and monocytes Not associated with any de ned disease at this time Human Herpesviruses HHV Gammaherpesviruses Lymphotrophic B cells Tumorigenic EBV EpsteinBarr Virus HHV8 KSHV Kaposi s SarcomaAssociated Herpesvirus Ga39mmaherpesvirus EpsteinBarr Virus EBV gt90 of Be world population are EBV positive i mostly asymptomatic infections children in developing countries become infected immediately afterthe mother s antibodies passed in the milk begin to wane children in developed countries are usually not exposed to the virus until adolescence Signs and symptoms 39 fever pharyngitis lymphadenopathy splenomegaly hepatocellular impairment and occassional skin rash 0 primary infection can result in mononucleosis 39 strong association with Nasopharyngeal Carcinoma Endemic Burkitt39s Lymphoma Hodkin s Disease PTLD and Aids Lymphoma EBV Infections Transmitted primarily through saliva Infection and replication in the epithelial cells of the nasopharynx spreads to Bcells Gammaherpesvirus Kaposi sassociated Herpesvirus KSHV HHV 8 Discovered 1994 Chang amp Moore Transmission through exchange of body uids sexual contact organ transplantation o saliva inef cient 350 of world population are HHV8 positive adults Associated with Kaposi s Sarcoma gt95 Multioentric Castleman s Disease and Primary Effusion Lymphoma HHV8 Tumor Association Associated with development of Kaposi s sarcoma Bodycavity based lymphoma Multicentric Castleman s disease Kaposi s Sarcoma 3gt Endemic Africa gt Classical Mediterranean gt AidsRelatedImmunosuppressive gt Iatrogenic Induced inadvettently by the medical treatment or procedures or activity of a physician Perivenular in ltrate of KS a mixture of spindle cells in ammatory cells and vascular spaces The Johns Hopkins Aumpsy Resource JHAR Image Archive Kaposi s Sarcoma Invasive multifocal neoplasm of unknown cellular or1g1n Contains a mixture of cell types broblasts endothelial cells dendritic cells and leukocytes Spindle cells are a distinctive histological marker High power view showing 391 j 3quot 539 spindle cell formation with 39 39 39 39 variable size irregular vascular channels and quotslit likequot spaces Complements of M Micheals KS Lesion stained with antiLANA antibody HHV 8 Serology Jenkins et a1 SAMPLE SEROPREVALENCE Kaposi s Sarcoma 98 HIV Infected Men No KS 60 HIV Uninfected Homosexual Men 41 Healthy Adults 310 BodyCavity Based Lymphoma Found in pleural pericardial and peritoneal cavities Lymphomatous effusion generally no tumor mass B cell genotype have immunoglobulin gene rearrangements E rearrangement of cmyc gene Generally EBV coinfected Mixture of BCBL1 and BJAB cells 1 2 stained with AntiLANA antibody Multicentric Castleman s Disease Atypical lymphoproliferative disorder 39 Close association with Kaposi s sarcoma HHV8 found in 40 60 of tumors 39 Presence of HHV 8 results in poorer outcome H HV8 Adapted from Russo JJ et al PNAS 1996 93 25 14826 HHV 8 Cellular Homologues vlL6 vFLlP leP1 2 and 3 vaclinD vbcL2 VGPCR leF


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