Copyright 2004, American Society for Microbiology. All Rights Reserved. High Genetic Diver

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CML

Genetic Mutations that Lead to
Chronic Myelogenoeatments
Marty O’Neill II Carmen Banea
What is CML?
• Chronic Myelogenous Leukemia (CML) is defined as, ―a malignant cancer of the bone marrow. It causes rapid growth of the blood-forming cells (known as myeloid precursors) in the bone marrow, peripheral blood, and body tissues.‖ [2] • CML represents about 14% of all occurrences of leukemias. • Patients who have CML are said to be in one of the following three phases (in order of occurrence):
References
• • • • • • • • • [1] D’Antonio, J. Chronic Myelogenous Leukemia. Clinical Journal of Oncology Nursing. 9(5): 535-8. [2] Faderl et al. Oncology (Huntingt). 1999; 13:169. [3] Genetic Science Learning Center at the University of Utah. /. [4] National Marrow Donor Program overview slide presentation. /NMDP/SLIDESET/sld031.htm. [5] Medline Plus Medical Encyclopedia. . [6] Pasternak et al. Chronic Meylogenous Leukemia: Molecular and Cellular Aspects. J Cancer Res Clin Oncol. 1998; 643-60. [7] Rinsky et al. Leukemia in Benzene Workers. Am J Ind Med. 1981; 2(3): 217-45. [8] Smith, MT & Zhang, L. Biomarkers of Leukemia Risk: Benzene as a Model. Environ Health Perspect. 1998 Aug; 106 Suppl 4:937-46. [9] STI-571 in Chronic Myelogenous Leukaemia. British Journal of Haematology. 2002; 15-24.

SAE J434-2004

CCF推荐的国际学术会议和期刊目录修订版发布

CCF推荐的国际学术会议和期刊目录修订版发布CCF(China Computer Federation中国计算机学会)于2010年8月发布了第一版推荐的国际学术会议和期刊目录，一年来，经过业内专家的反馈和修订，于日前推出了修订版，现将修订版予以发布。

本次修订对上一版内容进行了充实，一些会议和期刊的分类排行进行了调整，目录包括：计算机科学理论、计算机体系结构与高性能计算、计算机图形学与多媒体、计算机网络、交叉学科、人工智能与模式识别、软件工程/系统软件/程序设计语言、数据库/数据挖掘/内容检索、网络与信息安全、综合刊物等方向的国际学术会议及期刊目录，供国内高校和科研单位作为学术评价的参考依据。

目录中，刊物和会议分为A、B、C三档。

A类表示国际上极少数的顶级刊物和会议，鼓励我国学者去突破；B类是指国际上著名和非常重要的会议、刊物，代表该领域的较高水平，鼓励国内同行投稿；C类指国际上重要、为国际学术界所认可的会议和刊物。

这些分类目录每年将学术界的反馈和意见，进行修订，并逐步增加研究方向。

中国计算机学会推荐国际学术刊物（网络/信息安全）一、 A类序号刊物简称刊物全称出版社网址1. TIFS IEEE Transactions on Information Forensics andSecurity IEEE /organizations/society/sp/tifs.html2. TDSC IEEE Transactions on Dependable and Secure ComputingIEEE /tdsc/3. TISSEC ACM Transactions on Information and SystemSecurity ACM /二、 B类序号刊物简称刊物全称出版社网址1. Journal of Cryptology Springer /jofc/jofc.html2. Journal of Computer SecurityIOS Press /jcs/3. IEEE Security & Privacy IEEE/security/4. Computers &Security Elsevier http://www.elsevier.nl/inca/publications/store/4/0/5/8/7/7/5. JISecJournal of Internet Security NahumGoldmann. /JiSec/index.asp6. Designs, Codes andCryptography Springer /east/home/math/numbers?SGWID=5 -10048-70-35730330-07. IET Information Security IET /IET-IFS8. EURASIP Journal on InformationSecurity Hindawi /journals/is三、C类序号刊物简称刊物全称出版社网址1. CISDA Computational Intelligence for Security and DefenseApplications IEEE /2. CLSR Computer Law and SecurityReports Elsevier /science/journal/026736493. Information Management & Computer Security MCB UniversityPress /info/journals/imcs/imcs.jsp4. Information Security TechnicalReport Elsevier /locate/istr中国计算机学会推荐国际学术会议（网络/信息安全方向）一、A类序号会议简称会议全称出版社网址1. S&PIEEE Symposium on Security and Privacy IEEE /TC/SP-Index.html2. CCSACM Conference on Computer and Communications Security ACM /sigs/sigsac/ccs/3. CRYPTO International Cryptology Conference Springer-Verlag /conferences/二、B类序号会议简称会议全称出版社网址1. SecurityUSENIX Security Symposium USENIX /events/2. NDSSISOC Network and Distributed System Security Symposium Internet Society /isoc/conferences/ndss/3. EurocryptAnnual International Conference on the Theory and Applications of Cryptographic Techniques Springer /conferences/eurocrypt2009/4. IH Workshop on Information Hiding Springer-Verlag /~rja14/ihws.html5. ESORICSEuropean Symposium on Research in Computer Security Springer-Verlag as.fr/%7Eesorics/6. RAIDInternational Symposium on Recent Advances in Intrusion Detection Springer-Verlag /7. ACSACAnnual Computer Security Applications ConferenceIEEE /8. DSNThe International Conference on Dependable Systems and Networks IEEE/IFIP /9. CSFWIEEE Computer Security Foundations Workshop /CSFWweb/10. TCC Theory of Cryptography Conference Springer-Verlag /~tcc08/11. ASIACRYPT Annual International Conference on the Theory and Application of Cryptology and Information Security Springer-Verlag /conferences/ 12. PKC International Workshop on Practice and Theory in Public Key Cryptography Springer-Verlag /workshops/pkc2008/三、 C类序号会议简称会议全称出版社网址1. SecureCommInternational Conference on Security and Privacy in Communication Networks ACM /2. ASIACCSACM Symposium on Information, Computer and Communications Security ACM .tw/asiaccs/3. ACNSApplied Cryptography and Network Security Springer-Verlag /acns_home/4. NSPWNew Security Paradigms Workshop ACM /current/5. FC Financial Cryptography Springer-Verlag http://fc08.ifca.ai/6. SACACM Symposium on Applied Computing ACM /conferences/sac/ 7. ICICS International Conference on Information and Communications Security Springer /ICICS06/8. ISC Information Security Conference Springer /9. ICISCInternational Conference on Information Security and Cryptology Springer /10. FSE Fast Software Encryption Springer http://fse2008.epfl.ch/11. WiSe ACM Workshop on Wireless Security ACM /~adrian/wise2004/12. SASN ACM Workshop on Security of Ad-Hoc and Sensor Networks ACM /~szhu/SASN2006/13. WORM ACM Workshop on Rapid Malcode ACM /~farnam/worm2006.html14. DRM ACM Workshop on Digital Rights Management ACM /~drm2007/15. SEC IFIP International Information Security Conference Springer http://sec2008.dti.unimi.it/16. IWIAIEEE International Information Assurance Workshop IEEE /17. IAWIEEE SMC Information Assurance Workshop IEEE /workshop18. SACMATACM Symposium on Access Control Models and Technologies ACM /19. CHESWorkshop on Cryptographic Hardware and Embedded Systems Springer /20. CT-RSA RSA Conference, Cryptographers' Track Springer /21. DIMVA SIG SIDAR Conference on Detection of Intrusions and Malware and Vulnerability Assessment IEEE /dimva200622. SRUTI Steps to Reducing Unwanted Traffic on the Internet USENIX /events/23. HotSecUSENIX Workshop on Hot Topics in Security USENIX /events/ 24. HotBots USENIX Workshop on Hot Topics in Understanding Botnets USENIX /event/hotbots07/tech/25. ACM MM&SEC ACM Multimedia and Security Workshop ACM。

ASME_V_ART1_2004

04SUBSECTION A NONDESTRUCTIVE METHODSOF EXAMINATIONARTICLE1GENERAL REQUIREMENTST-110SCOPE(a)This Section of the Code contains requirementsand methods for nondestructive examination(NDE),which are Code requirements to the extent they are spe-ciﬁcally referenced and required by other Code Sectionsor referencing document.These NDE methods areintended to detect surface and internal imperfections inmaterials,welds,fabricated parts,and components.Theyinclude radiographic examination,ultrasonic examina-tion,liquid penetrant examination,magnetic particleexamination,eddy current examination,visual examina-tion,leak testing,and acoustic emission examination.SeeNonmandatory Appendix A of this Article for a listingof common imperfections and damage mechanisms,andthe NDE methods that are generally capable ofdetecting them.(b)For general terms such as Inspection,Flaw,Dis-continuity,Evaluation,etc.,refer to Mandatory Appen-dix I.T-120GENERAL(a)Subsection A describes the methods of nondestruc-tive examination to be used if referenced by other CodeSections or referencing documents.(b)Subsection B lists Standards covering nondestruc-tive examination methods which have been accepted asstandards.These standards are nonmandatory unless spe-ciﬁcally referenced in whole or in part in Subsection Aor as indicated in other Code Sections or referencingdocument.(c)Any reference to a paragraph of any Article inSubsection A of this Section includes all of the applicablerules in the paragraph.1In every case,reference to aparagraph includes all the subparagraphs and subdivisionsunder that paragraph.(d)Reference to a standard contained in SubsectionB is mandatory only to the extent speciﬁed.2(e)For those documents that directly reference thisArticle for the qualiﬁcation of NDE personnel,the quali-ﬁcation shall be in accordance with their employer’s writ-ten practice which must be in accordance with one of thefollowing documents:(1)SNT-TC-1A,3Personnel Qualiﬁcation and Cer-tiﬁcation in Nondestructive Testing;or(2)ANSI/ASNT CP-189,3ASNT Standard forQualiﬁcation and Certiﬁcation of Nondestructive TestingPersonnel1For example,reference to T-270includes all the rules contained inT-271through T-277.3.2For example,T-233requires that Image Quality Indicators be manu-factured and identiﬁed in accordance with the requirements or alterna-tives allowed in SE-747or SE-1025,and Appendices,as appropriatefor the style of IQI to be used.These are the only parts of either SE-747or SE-1025that are mandatory in Article2.3SNT-TC-1A(2001Edition),“Personnel Qualiﬁcation and Certiﬁca-tion in Nondestructive Testing;”and ANSI/ASNT CP-189(2001Edi-tion),“ASNT Standard for Qualiﬁcation and Certiﬁcation ofNondestructive Testing Personnel;”published by the American Societyfor Nondestructive Testing,1711Arlingate Lane,P.O.Box28518,Columbus,OH43228-0518.--`,,`,,,```,,,,``````,,`,,,``,-`-`,,`,,`,`,,`---T-1202004SECTION V T-160(f)National or international central certiﬁcation pro-grams,such as the ASNT Central Certiﬁcation Program (ACCP),may be alternatively used to fulﬁll the examina-tion requirements of the documents listed in T-120(e)as speciﬁed in the employer’s written practice.(g)When the referencing Code Section does not spec-ify qualiﬁcations or does not reference directly Article1 of this Section,qualiﬁcation may simply involve demon-stration in routine manufacturing operations to show that the personnel performing the nondestructive examina-tions are competent to do so in accordance with the Manu-facturer’s established procedures.(h)The user of this Article is responsible for the quali-ﬁcation and certiﬁcation of NDE Personnel in accordance with the requirements of this Article.The Code User’s4 Quality Program shall stipulate how this is to be accom-plished.Qualiﬁcations in accordance with a prior edition of SNT-TC-1A,or CP-189are valid until recertiﬁcation. Recertiﬁcation or new certiﬁcation shall be in accordance with the edition of SNT-TC-1A or CP-189speciﬁed in Footnote3.(i)Limited certiﬁcation of nondestructive examination personnel who do not perform all of the operations of a nondestructive method that consists of more than one operation,or who perform nondestructive examinations of limited scope,may be based on fewer hours of training and experience than recommended in SNT-TC-1A or CP-189.Any limitations or restrictions placed upon a person’s certiﬁcation shall be described in the written practice and on the certiﬁcation.(j)Either U.S.Customary Units or SI Units may be used for compliance with all requirements of this edition, but one system shall be used consistently throughout for all phases of construction.(1)Either the U.S.Customary Units or SI Units that are listed in Mandatory Appendix II are identiﬁed in the text,or are identiﬁed in the nomenclature for equations shall be used consistently for all phases of construction (e.g.,materials,design,fabrication,and reports).Since values in the two systems are not exact equivalents,each system shall be used independently of the other without mixing U.S.Customary Units and SI Units.(2)When SI Units are selected,U.S.Customary values in referenced speciﬁcations that do not contain SI Units shall be converted to SI values to at least three signiﬁcantﬁgures for use in calculations and other aspects of construction.4In this Code Section,“Code User”is any organization conducting nondestructive examinations to the requirements of this Section.T-130EQUIPMENTIt is the responsibility of the Code User to ensure that the examination equipment being used conforms to the requirements of this Code Section.T-150PROCEDURE(a)The nondestructive examination methods included in this Section are applicable to most geometric conﬁgu-rations and materials encountered in fabrication under normal conditions.However,special conﬁgurations and materials may require modiﬁed methods and techniques, in which case the Manufacturer shall develop special procedures which are equivalent or superior to the meth-ods and techniques described in this Code Section,and which are capable of producing interpretable examination results under the special conditions.Such special proce-dures may be modiﬁcations or combinations of methods described or referenced in this Code Section,and shall be proved by demonstration to be capable of detecting discontinuities under the special conditions,and such demonstrated capabilities shall be equivalent to the capa-bilities of the methods described in this Code Section when used under more general conditions.Depending on the quality assurance or quality control system require-ments of the referencing Code Section,these special pro-cedures shall be submitted to the Inspector for acceptance where required,and shall be adopted as part of the Manu-facturer’s quality control program.(b)When an examination to the requirements of this Section of the Code is required by other Sections of the Code,it shall be the responsibility of the Manufacturer, fabricator,or installer to establish nondestructive exami-nation procedures and personnel certiﬁcation procedures conforming to the referencing Code requirements. (c)When required by the referencing Code Section,all nondestructive examinations performed under this Code Section shall be done to a written procedure.This proce-dure shall be demonstrated to the satisfaction of the Inspector.The procedure or method shall comply with the applicable requirements of this Section for the particular examination method.Where so required,written proce-dures shall be made available to the Inspector on request. At least one copy of each procedure shall be readily available to the Manufacturer’s Nondestructive Examina-tion Personnel for their reference and use.T-160CALIBRATION(a)The Manufacturer,fabricator,or installer shall assure that all equipment calibrations required by Subsec-tion A and/or Subsection B are performed.--`,,`,,,```,,,,``````,,`,,,``,-`-`,,`,,`,`,,`---T-160ARTICLE1—GENERAL REQUIREMENTS T-190(b)When special procedures are developed[see T-150(a)],the Code User shall specify what calibration is necessary,when calibration is required.T-170EXAMINATIONS ANDINSPECTIONS(a)The Inspector concerned with the fabrication of the vessel or pressure part shall have the duty of verifying to his satisfaction that all examinations required by the referencing Code Section have been made to the require-ments of this Section and the referencing document(s). He shall have the right to witness any of these examina-tions to the extent stated in the referencing document(s). Throughout this Section of the Code,the word Inspector means the Authorized Inspector who has been qualiﬁed as required in the various referencing Code Sections. (b)The special distinction established in the various Code Sections between inspection and examination and the personnel performing them is also adopted in this Code Section.In other words,the term inspection applies to the functions performed by the Authorized Inspector, but the term examination applies to those quality control functions performed by personnel employed by the Manu-facturer.One area of occasional deviation from these distinctions exists.In the ASTM Standard Methods and Recommended Practices incorporated in this Section of the Code by reference or by reproduction in Subsection B,the words inspection or Inspector,which frequently occur in the text or titles of the referenced ASTM docu-ments,may actually describe what the Code calls exami-nation or examiner.This situation exists because ASTM has no occasion to be concerned with the distinctions which the Code makes between inspection and examina-tion,since ASTM activities and documents do not involve the Authorized Inspector described in the Code Sections. However,no attempt has been made to edit the ASTM documents to conform with Code usage;this should cause no difﬁculty if the users of this Section recognize that the terms inspection,testing,and examination in the ASTM documents referenced in Subsection B do not describe duties of the Authorized Code Inspector but rather describe the things to be done by the Manufacturer’s examination personnel.T-180EVALUATIONThe acceptance standards for these methods shall be as stated in the referencing document(s).T-190RECORDS/DOCUMENTATION Records/Documentation shall be in accordance with the referencing document(s)and the applicable require-ments of Subsection A and/or B of this Code Section. The Code User shall be responsible for all required Records/Documentation.ARTICLE1 MANDATORY APPENDIXAPPENDIX I—GLOSSARY OFTERMS FOR NONDESTRUCTIVEEXAMINATIONI-110SCOPEThis Mandatory Appendix is used for the purpose of establishing standard terms and deﬁnition of terms com-mon to all methods used in Nondestructive Examination. I-120GENERAL REQUIREMENTS(a)The Standard Terminology for Nondestructive Examinations(ASTM E1316)has been adopted by the Committee as SE-1316.(b)SE-1316Section A provides the deﬁnition of terms listed in I-130(a).(c)Paragraph I-130(b)provides a list of terms and deﬁnitions,which are in addition to SE-1316and are Code speciﬁc.I-130REQUIREMENTS(a)The following SE-1316terms are used in conjunc-tion with this Article:defect,discontinuity,evaluation, false indication,ﬂaw,ﬂaw characterization,imperfection, interpretation,nonrelevant indication,relevant indi-cation.(b)The following Code terms are used in conjunction with this Article:area of interest—the speciﬁc portion of the object that is to be evaluated as deﬁned by the referencing Code Sectionindication—the response or evidence from a nonde-structive examination that requires interpretation to deter-mine relevanceinspection—the observation of any operation per-formed on materials and/or components to determine its acceptability in accordance with given criterialimited certiﬁcation—an accreditation of an individu-al’s qualiﬁcation to perform some but not all of the opera-tions within a given nondestructive examination method or technique that consists of one or more than one opera-tion,or to perform nondestructive examinations within a limited scope of responsibilitymethod—the following is a list of nondestructive examination methods and respective abbreviations used within the scope of Section V:RT—RadiographyUT—UltrasonicsMT—Magnetic ParticlePT—Liquid PenetrantsVT—VisualLT—Leak TestingET—Electromagnetic(Eddy Current)AE—Acoustic Emissionnondestructive examination(NDE)—the development and application of technical methods to examine materials and/or components in ways that do not impair future usefulness and serviceability in order to detect,locate, measure,interpret,and evaluateﬂawsoperation—a speciﬁc phase of a method or technique procedure—an orderly sequence of actions describing how a speciﬁc technique shall be applied sensitivity—a measure of the level of response from a discontinuity by a nondestructive examination technique—a technique is a speciﬁc way of utilizing a particular nondestructive examination(NDE)methodARTICLE1 NONMANDATORY APPENDIXAPPENDIX A—IMPERFECTION VSTYPE OF NDE METHODA-110SCOPETable A-110lists common imperfections and the NDE methods that are generally capable of detecting them. Caution—Table A-110should be regarded for gen-eral guidance only and not as a basis for requiring or prohibiting a particular type of NDE method for a speciﬁc application.For example,material and product form are factors that could result in differences from the degree of effectiveness implied in the table.For service-induced imperfections,accessibility and other conditions at the examination location are also signiﬁcant factors that must be considered in selecting a particular NDE method.In addition,Table A-110must not be considered to be all inclusive;there are several NDE methods/techniques and imperfections not listed in the table.The user must con-sider all applicable conditions when selecting NDE meth-ods for a speciﬁc application.2004SECTION VTABLE A-110IMPERFECTION VS TYPE OF NDE METHODSurface Sub-surf.[Note(1)][Note(2)]Volumetric[Note(3)]VT PT MT ET RT UTA UTS AE UTT Service-Induced ImperfectionsAbrasive Wear(Localized)᭹᭾᭾᭹᭾᭾᭾Bafﬂe Wear(Heat Exchangers)᭹᭾Corrosion-Assisted Fatigue Cracks᭺᭾᭹᭺᭹᭹Corrosion-Crevice᭹᭺-General/Uniform᭺᭾᭾᭹-Pitting᭹᭹᭺᭹᭺᭺᭾᭺-Selective᭹᭹᭺᭺Creep(Primary)[Note(4)]Erosion᭹᭹᭺᭾᭾Fatigue Cracks᭺᭹᭹᭾᭾᭹᭹Fretting(Heat Exchanger Tubing)᭾᭾᭾Hot Cracking᭾᭾᭾᭺᭾Hydrogen-Induced Cracking᭾᭾᭺᭾᭾Intergranular Stress-Corrosion Cracks᭺Stress-Corrosion Cracks(Transgranular)᭺᭾᭹᭺᭾᭾᭾Welding ImperfectionsBurn Through᭹᭹᭾᭺Cracks᭺᭹᭹᭾᭾᭹᭺᭹--`,,`,,,```,,,,``````,,`,,,``,-`-`,,`,,`,`,,`---Excessive/Inadequate Reinforcement᭹᭹᭾᭺᭺Inclusions(Slag/Tungsten)᭾᭾᭹᭾᭺᭺Incomplete Fusion᭾᭾᭾᭾᭹᭾᭾Incomplete Penetration᭾᭹᭹᭾᭹᭹᭾᭾Misalignment᭹᭹᭾Overlap᭾᭹᭹᭺᭺Porosity᭹᭹᭺᭹᭾᭺᭺Root Concavity᭹᭹᭾᭺᭺᭺Undercut᭹᭾᭾᭺᭹᭾᭺᭺Product Form ImperfectionsBursts(Forgings)᭺᭹᭹᭾᭾᭾᭾᭹Cold Shuts(Castings)᭺᭹᭹᭺᭹᭾᭾᭺Cracks(All Product Forms)᭺᭹᭹᭾᭾᭾᭺᭹Hot Tear(Castings)᭺᭹᭹᭾᭾᭾᭺᭺Inclusions(All Product Forms)᭾᭾᭹᭾᭺᭺Lamination(Plate,Pipe)᭺᭾᭾᭺᭹᭺᭹Laps(Forgings)᭺᭹᭹᭺᭾᭺᭺Porosity(Castings)᭹᭹᭺᭹᭺᭺᭺Seams(Bar,Pipe)᭺᭹᭹᭾᭺᭾᭾᭺Legend:AE–Acoustic Emission UTA–Ultrasonic Angle BeamET–Electromagnetic(Eddy Current)UTS–Ultrasonic Straight BeamMT–Magnetic Particle UTT–Ultrasonic Thickness MeasurementPT–Liquid Penetrant VT–VisualRT–Radiography᭹–All or most standard techniques will detect this imperfection under all or most conditions.᭾–One or more standard technique(s)will detect this imperfection under certain conditions.᭺–Special techniques,conditions,and/or personnel qualiﬁcations are required to detect this imperfection.GENERAL NOTE:Table A-110lists imperfections and NDE methods that are capable of detecting them.It must be kept in mind that this table is very general in nature.Many factors inﬂuence the detectability of imperfections.This table assumes that only qualiﬁed personnel are performing nondestructive examinations and good conditions exist to permit examination(good access,surface conditions,cleanliness,etc.).NOTES:(1)Methods capable of detecting imperfections that are open to the surface only.(2)Methods capable of detecting imperfections that are either open to the surface or slightly subsurface.(3)Methods capable of detecting imperfections that may be located anywhere within the examined volume.(4)Various NDE methods are capable of detecting tertiary(3rd stage)creep and some,particularly using special techniques,are capable ofdetecting secondary(2nd stage)creep.There are various descriptions/deﬁnitions for the stages of creep and a particular description/deﬁnition will not be applicable to all materials and product forms.。

ASHP

939Am J Health-Syst Pharm—Vol 61 May 1, 2004A S H P R E P O R T SThe following individuals are acknowledged for serving on the ex-pert panel that revised these guidelines: Diane M. Cappelletty,Pharm.D., Greg Gousse, M.S., FASHP, William R. Martin, M.S.,FASHP, Mary Lynn McPherson, Pharm.D., BCPS, CDE, FASHP,and Antonio Petitta, M.B.A. The following individuals are grate-fully acknowledged for reviewing and submitting comments on interim drafts of these guidelines: Patricia Grunwald, Pharm.D.,Thomas G. Hall, Pharm.D., FASHP, Maureen Knell, Pharm.D.,BCPS/Cardiology, Calvin H. Knowlton, Ph.D., M.Div., Kristen A.Perkerson, Pharm.D., David S. Roffman, Pharm.D., BCPS, Linda S. Tyler, Pharm.D., FASHP, Elizabeth Udeh, Pharm.D., Doug Weschules, Pharm.D., and Chris Zimmerman, Pharm.D.ASHP Guidelines on the Pharmacist’s Role in the Development, Implementation,and Assessment of Critical Pathways D EVELOPED BY THE ASHP C OUNCIL ON P ROFESSIONAL A FFAIRS AND A PPROVEDBY THE ASHP B OARD OF D IRECTORS ON A PRIL 15, 2004Am J Health-Syst Pharm. 2004; 61:939-45The bibliographic citation for this document is as follows:American Society of Health-System Pharmacists. ASHP guidelines on the pharmacist’s role in the development, implementation, and assessment of critical pathways. Am J Health-Syst Pharm. 2004; 61:939-45.Index terms: American Society of Health-System Pharmacists; Crit-ical pathways; Documentation; Drug use; Drugs; Economics;Guidelines; Pharmacists; Quality assurance; Toxicity Copyright 2004, American Society of Health-System Pharma-cists, Inc. All rights reserved. 1079-2082/04/0501-0939$06.00.Summary of guidelines. Because pharmacotherapy is a central component of many criti-cal pathways, pharmacists should take leadership roles in their development, implementa-tion, and assessment.Pharmacists can improve the development of critical pathways by ensuring the evidence-based selection of medications, establishing measures for monitoring patients for drug efficacy and adverse effects, and evaluating the proposed critical pathway for patient safety.Pharmacists can improve the implementation of critical pathways by documenting processes and outcomes, ensuring proper patient selection and medication use, monitor-ing patients for drug efficacy and adverse effects, and providing for continuity of care.Pharmacists can improve the assessment of critical pathways by measuring and analyz-ing processes and outcomes, disseminating the results of those analyses, and reviewing the critical pathways’ pharmacotherapy to keep pace with changes in best practices.PurposeThe purpose of these guidelines is(1) to describe the pharmacist’s rolein the development, implementa-tion, and assessment of critical path-ways (CPs) and (2) to help pharma-cists prepare for that responsibility.Because pharmacotherapy is a centralcomponent of many CPs, pharmacistsshould take leadership roles in thedevelopment, implementation, andassessment of CPs. By assuming lead-ership roles, pharmacists can help im-prove patient outcomes, contribute tocost-effective patient care, and pro-mote multidisciplinary approaches topatient care and performance im-provement. Although pharmacist in-volvement in the early stages of CPdevelopment is crucial to success, CPdevelopment is generally cyclical, and pharmacists should seek opportunities to become involved at any stage in the cycle of CP development, implemen-tation, and assessment.Background The development of CPs has been stimulated by the desire to im-prove patient outcomes by apply-ing evidence-based clinical practice guidelines; increased interest in mea-suring and improving the quality of health care, including continuous-quality-improvement (CQI) initia-tives; and managed care and other market-driven health care reforms.CPs can be defined as patient careASHP REPORTS Critical pathways 940Am J Health-Syst Pharm—Vol 61 May 1, 2004management plans that delineate key steps along an optimal treatment timeline to achieve a set of predeter-mined intermediate and ultimate goals for patients who have clearly defined diagnoses or require certain procedures.1 CPs have also been called care guides, clinical pathways,clinical care plans, and care maps.2CPs derive from the industrial engi-neering concept of critical paths 3 and were originally associated with inpa-tient acute care and used primarily by nurses. CPs have evolved to incor-porate the spectrum of patient care providers and settings and to include CQI concepts.4 CPs and similar tools are developed in many facilities for many purposes.5-20 Although dis-missed by some critics as “cookbook medicine,” CPs have in some cases been shown to improve patient out-comes 21-26 and to reduce health care expenses.27-36 ASHP believes that carefully developed and skillfully managed CPs can improve the care of patients across the spectrum of health-system settings, as well as im-prove the allocation of scarce health care resources.Typical CP process Each health system will use a process of CP development, imple-mentation, and assessment that meets its own needs within its own structure, culture, practice settings,and policies and procedures, but these processes do share common el-ements. Typically, once a disease or procedure is selected for CP develop-ment, a multidisciplinary team ana-lyzes its current management (in-cluding process variances, costs, and outcomes), evaluates the scientific literature, and develops a plan of care. The planned actions for each discipline on the health care team are mapped on a timeline for the specific disease or procedure.37 Pharmacists perform the following functions in a typical CP: oversee the selection of medications by using an evidence-based approach, develop the criteria for medication selection or dosages,monitor patients for drug efficacy and adverse effects (or establish pa-rameters for monitoring), and en-sure continuity of care across the health system. The following de-scribes common steps in the devel-opment, implementation, and as-sessment of CPs.31.Select diagnoses and procedures. Diag-noses and procedures selected for CP development usually include those with high process variability, high cost, high patient volume, and high risk. CPs can be developed for com-mon or specialized diagnoses (e.g.,myocardial infarction, diabetes melli-tus) or procedures (e.g., transurethralprostatectomy, coronary artery by-pass grafting), for diagnoses that are likely to cause changes in health sta-tus (e.g., uncontrolled asthma), and for diseases requiring complicated pharmacotherapeutic regimens (e.g.,AIDS). The criteria for selection should be developed with input from admin-istrative and clinical leaders to ensure institutionwide acceptance and should be based on scientific evidence.Guidelines of the American Society of Health-System Pharmacists (ASHP) represent a consensus of professional judgment, expert opinion, and documented evidence in an area of pharmacy practice. ASHP guidelines provide pharmacy practitioners with progressive, challenging, yet attainable best practices. All ASHP guideline documents are compiled annually in Best Practices for Health-System Pharmacy, which can be viewed on ASHP’s Web site (/bestpractices/index.cfm).ASHP members are encouraged to take an active role in developing ASHP policy.Members can propose topics for new documents, suggest modifications to current documents, or volunteer to be drafters or reviewers by visiting the ASHP Web site or by contacting ASHP at ppsad@.2.Appoint a development team. The de-velopment team should include key health care providers from all organi-zational components involved in the CP. The importance of a multidisci-plinary approach to CP development cannot be overemphasized. If possi-ble, consideration should be given to including a patient representative on the CP development team. Although insurance carriers and managed care providers may not have representa-tives on the development team, their protocols or guidelines should be evaluated by the team for inclusion in the CP as appropriate.3.Conduct a search of the scientific litera-ture. A literature and database search conducted early in the process will help identify measures for assessing current processes and outcomes and will help ensure an evidence-based approach to the CP.4.Document current processes and out-comes. The current processes, costs,variances, and outcomes need to be documented, usually through flow charting of the current process, retro-spective chart review, and bench-marking. Benchmarking may be internal or external to the health sys-tem. Depending on the health sys-tem’s resources, benchmarking may rely on chart review or may use com-puterized databases that compare physicians’ use of resources, health-system costs, and outcomes for spe-cific diagnosis-related groups. This step identifies the health system’s practice and compares it with pub-lished clinical guidelines that are pref-erably consensus based (e.g.,guidelines from the Agency for Healthcare Research and Quality or the American Heart Association).The team developing the CP should use an evidence-based approach to identify, discuss, and resolve the gaps between clinical guidelines and cur-rent local practice. Input from the practitioners who will be involved in the CP is crucial to the success of this process evaluation and CP develop-ment in general.ASHP REPORTS Critical pathways5.Develop the CP.Multidisciplinary,standardized development of the CP ensures integration of care and elimi-nation of duplication and oversights.The CP should state its goals, define actions essential to achieving those goals, provide for patient education, outline assessment of patient safety, identify measures of conformance and outcomes, and describe required documentation. The actions and re-sources required for implementation should be discussed and agreed upon by all disciplines involved.a.Goals and outcomes•Define the specific goals or measurable outcomes of theCP (e.g., decreased length ofstay, decreased ventilator time,reduced overall patient cost,decreased pain scores, earlyambulation).•Select the areas of focus or cat-egories of actions essential toachieving the goals and out-comes. To ensure consistencyand continuity, these areas offocus should be standardizedfor all CPs developed within ahealth system; examples of fo-cus areas are treatments, medi-cations, and patient educationand counseling.•Determine the appropriate time frame. This will vary ac-cording to the disease or proce-dure being addressed and thepractice setting (e.g., emergen-cy room, ambulatory care clin-ic, acute care hospital). Thetime frame may be specified inminutes, hours, days, or phas-es. A workload assessment maybe required to develop appro-priate time frames.• D efine the activity for the focus area under the appropriatetime frame (e.g., “pharmacistprovides medication-use edu-cation and counseling on dis-charge day”).b.Patient education•Modify the CP, using lay terms in the patient’s primary lan-guage, to educate the patientabout activities to be performed,time frames, and expected out-comes. In some practice settings,the patient may be an activepartner in CP decision-makingand implementation.c.Patient safety•Identify potential risks to thepatient that may arise from useof the CP. For example, the CPcould be subjected to the insti-tution’s failure-mode and effectsanalysis, or the medicationsafety self-assessment tool of theInstitute for Safe MedicationPractices could be used to as-sess the safety of the practicesoutlined in the CP.38d.Monitoring•Identify measures of conform-ance and variance so that theCP and the resulting outcomescan be continuously improved.Variances are deviations fromthe CP that may be positive ornegative, avoidable or un-avoidable, consequential or in-consequential. Sources ofvariances include patient re-sponses to medications, physi-cian decisions, and systembreakdowns.e.Documentation•Develop a single, multidisci-plinary work sheet or other toolthat describes the CP’s actionsand time frames and providesspaces for documenting that ac-tions were performed.12 De-scribe how this tool will be usedand how data will be collected.6.Obtain approval for the CP and edu-cate participants. To ensure global ac-ceptance among all health careprovider groups, the CP should beapproved by appropriate committees,especially those of the medical staff.The impact of CP implementation onpractitioner workloads will requirecareful consideration, and depart-mental policies or procedures mayneed to be modified. The pharmacyand therapeutics (P&T) committeeshould review pharmacotherapeuticissues associated with the CP earlyenough during development thattherapeutic concerns can be ad-dressed as they arise rather than afterthe CP is implemented. After the CPis approved, all health care teammembers involved in the care of thepatients affected by the CP should beeducated about the anticipated out-comes, specific actions and timeframes, and professional responsibili-ties associated with the CP.7.Implement the CP.After the necessaryeducation and training of providers,the CP is available for use. Startingthe CP as a pilot project for a smallnumber of patients may provide valu-able initial assessments that will facili-tate wider implementation. Staffmembers should be designated toidentify patients suitable for the CPand to guide their enrollment and theCP’s use. Patients enrolled in a CP areassigned to a health care team whosemembers have specific responsibili-ties for actions and time frames.8.Assess the CP. Because not all conse-quences of a CP are foreseeable, CPsrequire periodic assessment. Assess-ments after a few days or weeks of usemay gauge only the feasibility of theCP and not its success in achievingdesired goals, but they should identifyunexpected problems that may re-quire modifications to the CP. Sur-veys of health care practitioners maybe useful in such initial assessments.Assessments after a few months of usemay provide an initial perspective onthe CP’s success but may not be suffi-cient to fully evaluate outcomes. As-sessments after longer periods shouldproduce evidence supporting theCP’s original goals. Regular analysisof the results and variances, as well asnew information (e.g., new indica-tions for medications) and technolo-gies (e.g., new pharmacotherapy),provides data for a root-cause analy-sis and continuous improvement ofthe CP.9.Disseminate the results of the assess-ment. The results of the assessment941Am J Health-Syst Pharm—Vol 61 May 1, 2004ASHP REPORTS Critical pathways 942Am J Health-Syst Pharm—Vol 61 May 1, 2004should be shared not just with health-system managers or members of the CP development or oversight com-mittees but with all staff involved in the CP. Widespread dissemination al-lows for more suggestions for im-provement from all members of the health care team and encourages ac-ceptance of any alterations in the CP required by the assessment. Publish-ing the results in a journal, sharing the experience with a practice net-work, or presenting the results at a meeting expands the general pool of knowledge concerning CPs.Pharmacist involvement These guidelines suggest actions to help prepare pharmacists and pharmacy departments for involve-ment in the development, imple-mentation, and assessment of CPs at various levels of care and in different practice settings. The applicability of these guidelines depends on a phar-macist’s or pharmacy department’s current level of involvement in pa-tient care and CPs. Pharmacists should focus on incorporating con-temporary pharmaceutical care principles (e.g., assessing medication orders, developing pharmacothera-peutic regimens and monitoring plans, educating and counseling pa-tients, calculating doses according to pharmacokinetic principles, con-ducting medication-use evaluations [MUEs], and managing anticoagula-tion therapy) in the development,implementation, and assessment of CPs.39-42Preparing for involvement in CP development. Pharmacists should learn about their health system’s ap-proach to CP development and as-sess their readiness for involvement.They should 1.Review the health system’s current strategic plan with respect to CPs. Be-cause CPs are inherently collaborative,pharmacists should try to understand this strategic plan from the perspective of other health care providers, seeking advice from them when necessary.When reviewing the strategic plan,pharmacists should also consider the needs of specific patient populations served by their cate the pharmacy staff on the purposes and processes of CP devel-opment and the contents of CPs. The patient care decisions required in CPs demand clinical knowledge, and the pharmacotherapy involved should be based on evidence in the scientific lit-erature. This clinical knowledge is fundamental to the CP. An effective contributor to the CP process needs to first acquire the clinical knowledgeon which the CP is based and then useCQI, teamwork, negotiation, and ad-ministrative skills to develop, imple-ment, and assess the CP.3.Discuss CP experiences with pharma-cy colleagues within and outside the health system. Pharmacists should create a forum within the health sys-tem for ongoing dialogue about CPs;for example, CPs could be made a regular agenda item for the P&T committee meeting and for other multidisciplinary clinical and depart-mental meetings.4.Monitor pharmacy, nursing, quality management, health-system, health care, and management literature for ideas on CP development, implemen-tation, and assessment. CPs from other institutions may be used as examples of and frameworks for mapping care, butthey should not be adopted directly,because acceptance and use are great-er when CPs are developed or adapted by their users.5.Identify opportunities for contribut-ing, through the provision of phar-maceutical care, to the health system’s patient care delivery and im-provement efforts.Initiating involvement. Pharma-cists should begin their involvement in the CP process in ways most ap-structure, culture, practice settings,and policies and procedures. In-volvement may vary substantially from one health system to another,but in general pharmacists should 1.Develop relationships with nursing,medical, dietary, laboratory, quality management, risk management, res-piratory care, and other personnel through routine meetings, nursing and medical forums, and other mul-tidisciplinary opportunities. These relationships should be used to pro-mote pharmacists’ contributions to collaborative patient care. The phar-macy department should support the use of CPs as an effective way to integrate and align services, processes,and costs.2.Support or initiate the implementa-tion of a multidisciplinary team for CP development and oversight and ensure that the pharmacy department and the P&T committee are repre-sented on the oversight committee.3.Seek leadership roles on the health system’s CP oversight committee and development teams but be will-ing to accept subordinate roles. For example, pharmacists should be will-ing to lead or assist with literature evaluation for the health system’s CP development teams.4.Identify qualities required for the pharmacist’s role and develop a con-sistent process for selecting the most appropriate pharmacists to partici-pate on the various CP development teams.5.Ensure the ongoing involvement of the P&T committee in the CP proc-ess. The P&T committee can facili-tate the process by •Reviewing and endorsing the pharmacotherapy proposed for inclusion in each CP.•Establishing a standing P&T sub-committee or liaison position to assist CP development teams. The subcommittee or liaison would have the opportunity to educate the CP team about the formu-lary process, the process for MUE, the appropriate use of re-stricted medications, and other critical medication-use issues.ASHP REPORTS Critical pathways•Publishing CPs and informationabout the health system’s experi-ences with CPs in the P&T com-mittee newsletter.•Developing the drug therapy por-tion of the CP assessment into anMUE.6.Emphasize to pharmacists, otherhealth care providers, and health-system administrators pharmacists’responsibility for implementing CP steps that involve pharmacothera-peutic regimens and monitoring, medication distribution, and patient education and counseling.7.Initiate, after appropriate approvals,the development of the pharmaco-therapeutic components of CPs. 8.Offer to evaluate and adapt the phar-macotherapeutic components of ex-isting protocols and guidelines for CPs under development. Maintain a pharmacotherapy database to facili-tate CP updates when new medica-tions and pharmacotherapeutic alternatives become available.9.Develop patient education andcounseling materials for the pharma-cotherapeutic components of CPs and develop plans for pharmacists or other members of the health care team to provide education and counseling to patients.10.Advocate the development and use ofpreprinted medication orders (hard copy or electronic) and consistent use of terminology (e.g., generic drug names, decimals and units of mea-surement, and standardized terms and abbreviations) within the CP. 11.Be proactive in anticipating alterna-tive processes or drug regimens that may be required in unusual circum-stances, such as drug shortages.Maintaining involvement. Phar-macists’ continued involvement in CPs will depend on their ability to demonstrate their contributions to patient care delivery and improve-ment to the CP oversight committee and development teams and to the health system’s administration. To accomplish this, pharmacists can 1.Monitor the literature of pharmacy,nursing, quality management, healthsystems, health care, and manage-ment for ideas on CP development,implementation, and assessment.2.Identify new areas for CP develop-ment on the basis of medication-usedata (e.g., medication error data andMUE findings).3.Incorporate CPs into the pharmacydepartment’s culture by building re-sponsibilities and performance ex-pectations for CP development,implementation, and assessment intopharmacists’ job descriptions.4.Identify and train pharmacy staff ontheir roles and responsibilities for im-plementing the pharmaceutical carecomponents of CPs.5.Provide objective clinical input that isbased on scientific evidence.6.Ensure consistent use of terminology(e.g., generic drug names, decimalsand units of measurement, and stan-dardized terms and abbreviations),rational medication use, and appro-priate monitoring. This could bedone by the P&T committee or by apharmacist who coordinates and re-views the pharmacotherapeutic ef-forts of all CPs.7.Maintain good working relationshipswith CP development teams. Thepharmacist member should be confi-dent, assertive, cooperative, and ef-fective in communicating with otherhealth care providers.8.Develop and maintain clinical andmanagement skills through ongoingself-education.Contributing to the CQI aspectsof CPs. The pharmacist should en-sure that the pharmaceutical care ac-tions of the CP contribute to patientsatisfaction, desired clinical out-comes, and financial goals by1.Monitoring the literature for best-practice results relating to specificdisease states (as defined by federaldiagnosis-related-group classifica-tion) and comparing these resultswith the health system’s experience.2.Ensuring that an internal system ofCP tracking and therapeutic review isin place for the rapid insertion ofnew, more effective therapies into theCP and that CPs are integrated intothe institution’s CQI processes.3.Assisting the development of patientsatisfaction surveys.4.Monitoring the results of the CPs andusing them to perform MUEs.42 Sincepatients enrolled in CPs are receivingpredetermined pharmacotherapeuticregimens and monitoring, this is anexcellent opportunity to performdisease- and outcome-orientedMUEs. The pharmacist should reviewthe variances and outcomes associat-ed with the CP, determine the effectsof pharmacotherapy, and use thisanalysis to modify the CP. The phar-macotherapeutic component must bespecific enough (e.g., specifying themedication and dosage) that its influ-ence on the outcomes can be deter-mined with confidence.5.Ensuring that CPs are updated whenthere are changes in institutionalpractices (e.g., formulary changes) orwhen external practices change (e.g.,guidelines are revised, dosage ormonitoring recommendations arerevised).Ensuring the continuity of a CP.Many CPs require continuity of careacross various levels of care and prac-tice settings. Such CPs should specifyreferral patterns among the levels ofcare and practice settings. To help ac-complish this, pharmacists can1.Ensure that members of all organiza-tional components are included inthe development and assessment ofthe CP as appropriate for the particu-lar disease or procedure. Includedmight be personnel in the emergencydepartment, the operating room, theintensive care unit, the step-downunit, the general nursing unit, the re-habilitation unit, the long-term-carefacility, the ambulatory care clinic,the laboratory, and the home careservice.943Am J Health-Syst Pharm—Vol 61 May 1, 2004ASHP REPORTS Critical pathways 944Am J Health-Syst Pharm—Vol 61 May 1, 20042.Develop relationships with ambula-tory care, home care, and long-term-care pharmacists and other health care providers to foster the seamless provision of pharmaceutical care by inviting pharmacist members of managed care organizations to partic-ipate in CP development, exchanging CPs with the managed care organiza-tions, and creating work teams to en-sure the continuity of anize interdisciplinary sharing of information (e.g., recent laboratory test results) and documentation that are useful to all health care providers.4.Develop a plan to communicate and monitor both internal and external CPs so that pharmacists have a full understanding of the CP process and can evaluate and adjust the CP as nec-essary when new therapeutic modali-ties emerge. Optimize this by using electronic and Internet technology to rapidly insert new pharmacotherapies into CPs when appropriate and eval-uate which pharmacotherapies are included in which CPs to ensure con-sistency in medication management.5.Initiate dialogue among pharmacists to ensure the continuity of the indi-vidual patient’s CP. For example,when a patient is admitted, the hospi-tal pharmacist should, if necessary and with the patient’s permission,contact the patient’s community pharmacist or managed care compa-ny to obtain information as allowed under local, state, and federal laws (e.g., the Health Insurance Portability and Accessibility Act). The pharma-cist should document an accurate medication history (prescription and nonprescription products, dietary supplements, and home remedies),the patient’s history of adverse drug reactions and allergies, the patient’s medication-taking behaviors (adher-ence, health beliefs, and social and cul-tural issues), and any self-monitoring.This does not obviate the require-ment for the pharmacist to establish a professional relationship with the pa-tient and to obtain information di-rectly. When the patient is dis-charged, the hospital pharmacist could (in accordance with the institu-tion’s policies and procedures and with the patient’s permission) pre-pare an original discharge summary or append to the physician’s dis-charge summary any necessary notes regarding medications. An appropri-ate member of the discharge team should forward the discharge sum-mary to relevant health care providers(e.g., physicians, home care nurses,and community or home care phar-macists). The summary should in-clude the patient’s chief complaint,the patient’s height and weight, a his-tory of the present illness (including the hospital course), pregnancy and lactation status, prescription and nonprescription medications on ad-mission and at discharge, patient education and counseling, the monitoring plan (including patient self-monitoring and a plan for long-term monitoring of the pa-tient’s pharmacotherapeutic regi-men), and a person to contact if the patient has questions. The pharmacistshould check any discharge prescrip-tions against the indications listed in the discharge summary.The pharmacist’s responsibility Carefully developed and skillfully managed CPs can improve the quali-ty of care and the allocation of health care resources. Because pharmaco-therapy is a central component of many CPs, pharmacists can help im-prove patient outcomes, contribute to cost-effective patient care, and promote team approaches to patient care and performance improvement by incorporating contemporary pharmaceutical care principles, ac-tivities, and services into the devel-opment, implementation, and as-sessment of CPs. Health systems will develop CPs in ways that are most appropriate to their patient popula-tions, organizational structure, cul-ture, and environment, so it is the responsibility of health-system phar-macists to identify opportunities to become involved in and improve the CPs in their institutions.References 1.Darer J, Pronovost P, Bass E. Use and evaluation of critical pathways in hospi-tals . Eff Clin Pract. 2002; 5:114-9.2.Lumsdon K, Hagland M. Mapping care.Hosp Health Netw. 1993; 67:34-40.3.Coffey RJ, Richards JS, Remmert CS et al.An introduction to critical paths. Qual Manag Health Care. 1992; 1:45-54.4.Jaggers LD. Differentiation of critical pathways from other health care manage-ment tools. Am J Health-Syst Pharm.1996; 53:311-3.5.Koch KE. Opportunities for pharmaceu-tical care with critical pathways. Top Hosp Pharm Manag. 1995; 14:1-7.6.Stevenson LL. Critical pathway experi-ence at Sarasota Memorial Hospital. Am J Health-Syst Pharm. 1995; 52:1071-3.7.Gousse GC, Rousseau MR. Critical path-ways at Hartford Hospital. Am J Health-Syst Pharm. 1995; 52:1060-3.8.Shane R, Vinson B. Use of critical path-ways and indicators in pharmacy prac-tice. Top Hosp Pharm Manag. 1995; 14:55-67.9.Gouveia WA, Massaro FJ. Critical path-way experience at New England Medical Center. Am J Health-Syst Pharm. 1995;52:1068-70.10.Saltiel E. Critical pathway experience at Cedars-Sinai Medical Center. Am J Health-Syst Pharm. 1995; 52:1063-8.11.Nelson SP. Critical pathways at University of Iowa Hospitals and Clinics. Am J Health-Syst Pharm. 1995; 52:1058-60.12.Marrie TJ, Lau CY, Wheeler SL et al. A controlled trial of a critical pathway for treatment of community-acquired pneu-monia. CAPITAL Study munity-Acquired Pneumonia Inter-vention Trial Assessing Levofloxacin.JAMA . 2000; 283:749-55.13.Cannon CP, Hand MH, Bahr R et al.Critical pathways for management of pa-tients with acute coronary syndromes: an assessment by the National Heart Attack Alert Program. Am Heart J. 2002; 143:777-89.14.Jones S. A clinical pathway for pediatric gastroenteritis. Gastroenterol Nurs . 2003;26:7-18.15.Nierman DM. A structure of care for the chronically critically ill. Crit Care Clin.2002; 18:477-91.16.Kercsmar CM, Myers TR. Clinical path-ways in treatment of asthma. Curr Opin Allergy Clin Immunol. 2002; 2:183-7.17.Mavroukakis SA, Muehlbauer PM, White RL Jr et al. Clinical pathways for manag-ing patients receiving interleukin 2. Clin J Oncol Nurs. 2001; 5:207-17.18.Zevola DR, Raffa M, Brown K. Using clinical pathways in patients undergoing cardiac valve surgery. Crit Care Nurse .2002; 22:31-9,44-50.19.Rymer MM, Summers D, Soper P. Devel-opment of clinical pathways for stroke。

SAE J 434-2004 (R) AUTOMOTIVE DUCTILE (NODULAR) IRON CASTINGS

SURFACEVEHICLESTANDARD SAE Technical Standards Board Rules provide that: “This report is published by SAE to advance the state of technical and engineering sciences. The use of this report isentirely voluntary, and its applicability and suitability for any particular use, including any patent infringement arising therefrom, is the sole responsibility of the user.”SAE reviews each technical report at least every five years at which time it may be reaffirmed, revised, or cancelled. SAE invites your written comments and suggestions. Copyright © 2004 SAE InternationalAll rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of SAE.TO PLACE A DOCUMENT ORDER: Tel: 877-606-7323 (inside USA and Canada)Tel: 724-776-4970 (outside USA)4.HardnessThe hardness ranges shown in Table 1 are for guidelines only.4.1The area or areas on the castings where hardness is to be checked should be established byagreement between manufacturer and purchaser.4.2The foundry shall exercise the necessary controls and inspection techniques to insure compliancewith the typical hardness range. Brinell hardness shall be determined according to ASTM E 10,Standard Test Method for Brinell Hardness of Metallic Materials, after sufficient material has been removed from the casting surface to insure representative hardness readings. The 10mm tungsten ball and 3000kg load shall be used unless otherwise agreed upon.5.Heat Treatment5.1The heat treatment of castings and test specimens produced to this standard in order to meethardness or other mechanical property requirements, for thermal stress relief or for other purposes is permissible only with the express approval of the casting purchaser.5.2Purchaser approval may be blanket or may be issued on a case-by-case basis, as agreed.5.3Unless otherwise agreed, the heat treat cycles employed; times, temperatures, rates, quenchants,etc. shall be at the option of the manufacturer.5.4Unless otherwise agreed, any separately cast test specimens or any removed from the castings,must accompany the castings they represent as part of the same heat treatment lot(s) or besubjected to the same thermal cycle as the castings they represent, in all respects.6.MicrostructureThe graphite component of the microstructure shall consist of at least 80% spheroidal graphite conforming to Types I and II (per ASTM A247) in Fig. 1 (nodularity rating chart developed by the Ductile I ron Society may be used as a guide). The matrix microstructure shall consist of ferrite, ferrite and pearlite, pearlite, tempered pearlite, or tempered martensite, or a combination of these. The microstructure shall be substantially free of primary cementite. The microstructure shall be substantially free of undesirable carbides, the details of which are agreed upon between the casting manufacturer and the purchaser. Examples of matrix microstructures for each grade are shown in Fig. 2.FIGURE 1—CLASSIFICATION OF GRAPHITE SHAPE IN CAST IRONS (FROM ASTM A 247)D400D450FIGURE 2—TYPICAL MATRIX MICROSTRUCTURES(PHOTOS COURTESY OF CLIMAX RESEARCH SERVICES)D500D550FIGURE 2—TYPICAL MATRIX MICROSTRUCTURES (CONTINUED)(PHOTOS COURTESY OF CLIMAX RESEARCH SERVICES)D700D800FIGURE 2—TYPICAL MATRIX MICROSTRUCTURES (CONTINUED)(PHOTOS COURTESY OF CLIMAX RESEARCH SERVICES)7.Quality AssuranceIt is the responsibility of the manufacturer to demonstrate process capability. The specimen(s) used to do so shall be of a configuration and from a location agreed upon between the manufacturer and the purchaser. Sampling plans shall be agreed upon between the manufacturer and purchaser. The manufacturer shall employ adequate controls to ensure that the parts conform to the agreed upon requirements.8.General8.1Castings furnished to this standard shall be representative of good foundry practice and shallconform to dimensions and tolerances specified on the casting drawing.8.2Minor imperfections usually not associated with the structural functioning may occur in castings.These imperfections are often repairable; however, repairs should be made only in areas and by methods approved by the purchaser.8.3Purchaser and manufacturer may agree to additional casting requirements, such as manufactureridentification, other casting information, and special testing. These should appear as additionalproduct requirements on the casting drawing.9.Notes9.1Marginal IndiciaThe change bar (l) located in the left margin is for the convenience of the user in locating areas where technical revisions have been made to the previous issue of the report. An (R) symbol to the left of the document title indicates a complete revision of the report.PREPARED BY THE SAE AUTOMOTIVE IRON & STEEL CASTINGS COMMITTEEA.3.5Typical microstructures of the grades of ductile iron are as follows:D400 (D4018) is a ferritic ductile iron most commonly made by annealing. The annealing time and temperature cycle is such that primary carbides, if present in the as-cast structure, are decomposed, and the resulting matrix is ferritic.D450 (D4512) is ferritic ductile iron supplied either as-cast or heat-treated. The matrix, is predominantly ferrite, but this grade can contain pearlite, depending on section size.D500 (D5006) is ferritic-pearlite ductile iron supplied either as-cast or heat-treated. The matrix, is essentially pearlite. This grade may contain substantially more ferrite than Grade D550.D550 (D5504) is pearlitic-ferritic ductile iron supplied either as-cast or heat-treated. The matrix, is essentially pearlite. This grade may contain less ferrite than D500.D700 (D7003) is either as-cast or air quenched to a specified hardness range. The resulting matrix is pearlite.D800 is either as-cast or air or liquid quenched and tempered to a specified hardness range.The resulting matrix is pearlite or tempered martensite.DQ&T is a liquid quenched and tempered grade. The resulting matrix is tempered martensite.A.4C asting Mechanical PropertiesA.4.1The mechanical properties are shown in Table 1. Since properties may vary with location in agiven c asting, the suitability of a particular grade for an intended use is best determined bylaboratory or service t ests.A.4.2The mechanical properties will vary with microstructure that, especially in the as-cast condition, isdependent upon section size as well as chemical composition and some foundry processes.A.4.3For optimum mechanical properties in the quenched and tempered grade, section size forunalloyed iron should generally not exceed 19mm (3/4 inch) to insure uniform, thoroughhardened structure.A.4.4 A young's Modulus value of 166GPa (24Mpsi) is typically found for ductile iron when measuredper ASTM E111, Standard Test Method for Young's Modulus, Tangent Modulus and ChordModulus. Ductile Iron components used for high strain rate applications should use a reducedvalue of the modulus stated above (some designers have used up to 15% less than the modulus stated above).A.4.5T ypical un-notched Charpy impact energy properties are shown in Table A2. These typicalresults are from low residual element content iron (especially phosphorus). Impact values areaffected by microstructure and section size.- 11 -RationaleNot Applicable.Relationship of SAE Standard to ISO StandardNot Applicable.ApplicationThis SAE standard covers the minimum mechanical properties measured on separately cast test pieces of varying thickness and microstructural requirements for ductile iron castings used in automotive and allied industries. Castings may be specified in the as-cast or heat-treated condition. If castings are heat-treated, prior approval from the customer is required.The appendix provides general information on chemical composition, microstructure and casting mechanical properties, as well as other information for particular service conditions.In this standard SI units are primary and in-lb units are derived.Reference SectionASTM E10—Standard Test Method for Brinell Hardness of Metallic MaterialsASTM E23—Standard Test Methods for Notched Bar Impact Testing of Metallic MaterialsASTM E111—Standard Test Method for Young's Modulus, Tangent Modulus and Chord ModulusASTM A247—Standard Test Method for Evaluation the Microstructure of Graphite in Iron CastingsASTM A536—Standard Specification for Ductile Iron CastingsSTP-455—Gray, Ductile, and Malleable Iron Castings Current Capabilities (out-of-print)Metals Handbook, Vol. 1,2 and 5, 8th Edition, ASM International, Materials Park, OH (out-of-print)Gray and Ductile Iron Castings Handbook, Gray and Ductile Iron Founder's Society, Cleveland, OHH.D. Angus, Physical Engineering Properties of Cast ron, British Cast ron Research Association,Birmingham, EnglandDeveloped by the SAE Automotive Iron & Steel Castings Committee。

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(Copyright ownership is indicated in the notice in the lower right-hand corner of theﬁrst page of each article.)Mathematics of Computation is published quarterly by the American Mathematical Society at201Charles Street,Providence,RI02904-2294USA.Periodicals postage is paid at Providence,Rhode Island.Postmaster:Send address changes to Mathematics of Computation,American Mathematical Society,201Charles Street,Providence,RI02904-2294USA.c 2004by the American Mathematical Society.All rights reserved.This journal is indexed in Mathematical Reviews,Zentralblatt MATH,Science CitationIndex ,Science Citation Index TM–Expanded,ISI Alerting Services SM,CompuMathCitation Index ,and Current Contents /Physical,Chemical&Earth Sciences.The paper used in this book is acid-free and falls within the∞guidelines established to ensure permanence and durability.10987654321090807060504MATHEMATICS OF COMPUTATIONCONTENTSVol.73,No.247July2004 Susanne C.Brenner,Convergence of nonconforming V-cycle and F-cycle multigrid algorithms for second order elliptic boundary value problems1041 Susanne C.Brenner,Korn’s inequalities for piecewise H1vectorﬁelds..1067 Jun Zhao,Analysis ofﬁnite element approximation for time-dependent Maxwell problems (1089)W.Dahmen,B.Faermann,I.G.Graham,W.Hackbusch,and S.A.Sauter,Inverse inequalities on non-quasi-uniform meshes andapplication to the mortar element method (1107)Jinchao Xu and Zhimin Zhang,Analysis of recovery type a posteriori error estimators for mildly structured grids (1139)C.Carstensen,Allﬁrst-order averaging techniques for a posterioriﬁniteelement error control on unstructured grids are eﬃcient and reliable..1153 Zhiming Chen and Jia Feng,An adaptiveﬁnite element algorithm with reliable and eﬃcient error control for linear parabolic problems (1167)Alan Demlow,Piecewise linearﬁnite element methods are not localized.1195 Bruno Despres,Lax theorem andﬁnite volume schemes (1203)K.H.Karlsen,C.Klingenberg,and N.H.Risebro,A relaxation scheme for conservation laws with a discontinuous coeﬃcient (1235)Mao Ye,Existence and asymptotic stability of relaxation discrete shock proﬁles (1261)Ivan P.Gavrilyuk,Wolfgang Hackbusch,and Boris N.Khoromskij, Data-sparse approximation to the operator-valued functions of ellipticoperator (1297)Mohamed El-Gamel,John R.Cannon,and Ahmed I.Zayed,Sinc-Galerkin method for solving linear sixth-order boundary-value problems1325 D.P.Dryanov,M.A.Qazi,and Q.I.Rahman,Local behaviour ofpolynomials (1345)Allal Guessab and Gerhard Schmeisser,Convexity results and sharp error estimates in approximate multivariate integration (1365)J.M.Pe˜n a,A stable test to check if a matrix is a nonsingular M-matrix1385 Carlos M.Mora,Numerical simulation of stochastic evolution equations associated to quantum Markov semigroups (1393)Bernard Deconinck,Matthias Heil,Alexander Bobenko,Mark van Hoeij,and Marcus Schmies,Computing Riemann theta functions1417 Richard P.Groenewegen,Bounds for computing the tame kernel (1443)Nils Bruin,Visualising Sha[2]in Abelian surfaces (1459)Arjeh M.Cohen,Scott H.Murray,and D.E.Taylor,Computing in groups of Lie type (1477)Joachim von zur Gathen and Michael N¨o cker,Computing special powers inﬁniteﬁelds (1499)David S.Dummit,Brett A.Tangedal,and Paul B.van Wamelen, Stark’s conjecture over complex cubic numberﬁelds (1525)Felix Lazebnik and Andrew Thomason,Orthomorphisms and the construction of projective planes (1547)Pedro Berrizbeitia and T.G.Berry,Biquadratic reciprocity and a Lucasian primality test (1559)Marc Del´e glise,Pierre Dusart,and Xavier-Fran¸c ois Roblot,Counting primes in residue classes (1565)Reviews and Descriptions of Tables and Books (1577)J.C.Mason and D.C.Handscomb4,Stanley Osher and Ron Fedkiw5,M.D.Buhmann6,I.Shparlinski7,R.A.Mollin8No microﬁche supplement in this issueEditorial InformationInformation on the backlog for this journal can be found on the AMS website starting from /mcom.In an eﬀort to make articles available as quickly as possible,articles are posted to the AMS website individually after proof is returned from authors and before appearing in an issue.A Consent to Publish and Copyright Agreement is required before a paper will be published in this journal.After a paper is accepted for publication,the Providence oﬃce will send out a Consent to Publish and Copyright Agreement to all authors of the paper. 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B.WAHLBIN,Center for Applied Mathematics,657Frank H.T.Rhodes Hall, Cornell University,Ithaca,NY14853-3801USA;E-mail:awahlbin@ JOSEPH D.WARD,Department of Mathematics,Texas A&M University,College Station,TX77843-3368USA;E-mail:jward@Board of Associate EditorsRANDOLPH E.BANK,Department of Mathematics,University of California San Diego,C-012,La Jolla,CA92093-0001USA;E-mail:reb@ CHRISTINE BERNARDI,Laboratoire d’Analyse Numerique,C.N.R.S.et Universit´e Pierre et Marie Curie,B.C.187,4place Jussieu,75252Paris Cedex05,France;E-mail: bernardi@ann.jussieu.frPETER B.BORWEIN,Department of Mathematics and Statistics,Simon Fraser Uni-versity,Burnaby,BC,Canada V6T1Z2;E-mail:pborwein@cecm.sfu.ca DAVID W.BOYD,Department of Mathematics,University of British Columbia,Van-couver,BC Canada V6T1Z2;E-mail:boyd@math.ubc.caSUSANNE C.BRENNER,Department of Mathematics,University of South Carolina, Columbia,SC29208USA;E-mail:brenner@RICHARD P.BRENT,Oxford University Computing Laboratory,Wolfson Building, Parks Road,Oxford OX13QD,England;E-mail:Richard.Brent@ CARSTEN CARSTENSEN,Humboldt University of Berlin,Institute of Mathematics, Sitz:Rudower Chaussee25,Unter den Linden6,D-10099Berlin,Germany;E-mail: mathcomp@math.hu-berlin.deARJEH M.COHEN,Faculteit Wiskunde en Informatica,TU Eindhoven,Postbus513, 5600MB Eindhoven,Netherlands;E-mail:amc@win.tue.nlRONALD F.A.COOLS,Department of Computer Science,Katholieke Universiteit Leuven,Celestijnenlaan200A,B-3001Heverlee,Belgium;E-mail:ronald.cools@cs. kuleuven.ac.beHOWARD ELMAN,Department of Computer Science,University of Maryland,College Park,MD20742-0001USA;E-mail:elman@RICHARD S.FALK,Department of Mathematics,Rutgers University,Hill Center,110 Frelinghuysen Road,Piscataway,NJ08854-8019USA;E-mail:falk@ DANIEL W.LOZIER,National Institute of Standards and Technology,100Bureau Drive,Mail Stop8910,Gaithersburg,MD20899-8910USA;E-mail:dlozier@ ZHI-QUAN LUO,Department of Electrical and Computer Engineering,McMaster Uni-versity,Room CRL/225,Hamilton,ON Canada L8S4K1;E-mail:luozq@mcmail.cis. mcmaster.caHARALD NIEDERREITER,Department of Mathematics,National University of Sin-gapore,2Science Drive2,Singapore117543,Republic of Singapore;E-mail:nied@math. .sgRICARDO H.NOCHETTO,Department of Mathematics,University of Mary-land,Mathematics Building084,College Park,MD20742-0001USA;E-mail: rhn@STANLEY OSHER,Department of Mathematics,University of California,P.O.Box 951555,Los Angeles,CA90095-1555USA;E-mail:sjo@ HAESUN PARK,Department of Computer Science,University of Minnesota,4-192 EE/CS,200Union Street,Minneapolis,MN55455USA;E-mail:hpark@ JOSEPH E.PASCIAK,Department of Mathematics,Texas A&M University,507B Blocker Hall,MS3368,College Station,TX77843USA;E-mail:pasciak@ LOTHAR REICHEL,Department of Mathematics&Computer Science,Kent State University,P.O.Box5190,Kent,OH44242-0001USA;E-mail:reichel@ IGOR E.SHPARLINSKI,Department of Computing,Macquarie University,Sydney, New South Wales2109,Australia;E-mail:igor@.auFRANK STENGER,School of Computing,University of Utah,Salt Lake City,UT 84112-1102USA;E-mail:stenger@DENIS TALAY,INRIA,2004Route des Lucioles,BP93,06902Sophia Antipolis Cedex,France;E-mail:talay@sophia.inria.frNICO M.TEMME,Centrum voor Wiskunde en Informatica,P.O.Box94079,1090-GB Amsterdam,Netherlands;E-mail:nicot@cwi.nlHUGH C.WILLIAMS,Department of Mathematics and Statistics,University of Cal-gary,Calgary AB,Canada T2N1N4;E-mail:williams@math.ucalgary.ca JINCHAO XU,Department of Mathematics,Pennsylvania State University,McAllister Building,University Park,PA16802-6401USA;E-mail:xu@(Continued from back cover)Joachim von zur Gathen and Michael N¨o cker,Computing special powers inﬁniteﬁelds (1499)David S.Dummit,Brett A.Tangedal,and Paul B.van Wamelen, Stark’s conjecture over complex cubic numberﬁelds (1525)Felix Lazebnik and Andrew Thomason,Orthomorphisms and the construction of projective planes (1547)Pedro Berrizbeitia and T.G.Berry,Biquadratic reciprocity and a Lucasian primality test (1559)Marc Del´e glise,Pierre Dusart,and Xavier-Fran¸c ois Roblot,Counting primes in residue classes (1565)Reviews and Descriptions of Tables and Books (1577)J.C.Mason and D.C.Handscomb4,Stanley Osher and Ron Fedkiw5,M.D.Buhmann6,I.Shparlinski7,R.A.Mollin8No microﬁche supplement in this issue0025-5718(200407)73:247;1-V。

V308研究

Christophe Tournigand, Thierry André, Emmanuel Achille, Gérard Lledo, Michel Flesh, Dominique Mery-Mignard, Emmanuel Quinaux, Corinne Couteau, Marc Buyse, Gérard Ganem, Bruno Landi, Philippe Colin, Christophe Louvet, and Aimery de Gramont
ABSTRACT
Purpose In metastatic colorectal cancer, phase III studies have demonstrated the superiority of ﬂuorouracil (FU) with leucovorin (LV) in combination with irinotecan or oxaliplatin over FU ϩ LV alone. This phase III study investigated two sequences: folinic acid, FU, and irinotecan (FOLFIRI) followed by folinic acid, FU, and oxaliplatin (FOLFOX6; arm A), and FOLFOX6 followed by FOLFIRI (arm B).
Results Median survival was 21.5 months in 109 patients allocated to FOLFIRI then FOLFOX6 versus 20.6 months in 111 patients allocated to FOLFOX6 then FOLFIRI (P ϭ .99). Median second progression-free survival (PFS) was 14.2 months in arm A versus 10.9 in arm B (P ϭ .64). In ﬁrst-line therapy, FOLFIRI achieved 56% response rate (RR) and 8.5 months median PFS, versus FOLFOX6 which achieved 54% RR and 8.0 months median PFS (P ϭ .26). Second-line FOLFIRI achieved 4% RR and 2.5 months median PFS, versus FOLFOX6 which achieved 15% RR and 4.2 months PFS. In ﬁrst-line therapy, National Cancer Institute Common Toxicity Criteria grade 3/4 mucositis, nausea/vomiting, and grade 2 alopecia were more frequent with FOLFIRI, and grade 3/4 neutropenia and neurosensory toxicity were more frequent with FOLFOX6.

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J OURNAL OF C LINICAL M ICROBIOLOGY,Aug.2004,p.3406–3414Vol.42,No.8 0095-1137/04/$08.00ϩ0DOI:10.1128/JCM.42.8.3406–3414.2004Copyright©2004,American Society for Microbiology.All Rights Reserved.High Genetic Diversity of the Attachment(G)Protein ofHuman MetapneumovirusNobuhisa Ishiguro,1Takashi Ebihara,1Rika Endo,1Xiaoming Ma,1Hideaki Kikuta,1*Hiroaki Ishiko,2and Kunihiko Kobayashi1Department of Pediatrics,Hokkaido University Graduate School of Medicine,Sapporo,1and Departmentof Infectious Diseases,Mitsubishi Kagaku Bio-Clinical Laboratories,Tokyo,2JapanReceived21February2004/Returned for modiﬁcation7May2004/Accepted10May2004Complete genes encoding the predicted nucleoprotein(N),phosphoprotein(P),matrix protein(M),fusionprotein(F),M2-1protein,M2-2protein,small hydrophobic protein(SH),and attachmentprotein(G)of sevennewly isolated human metapneumoviruses(hMPVs)were analyzed and compared with previously publisheddata for hMPV genes.Phylogenetic analysis of the nucleotide sequences indicated that there were two geneticgroups,tentatively named groups1and2,similar to the grouping of human respiratory syncytial virus.Although the predicted amino acid sequences of N,P,M,F,and M2were highly conserved between the twogroups(amino acid identities,96%for N,85%for P,97%for M,94%for F,95%for M2-1,and90%for M2-2),the amino acid identities of the SH and G proteins were low(SH,58%;G,33%).Furthermore,each group couldbe subdivided into two subgroups by phylogenetic analysis,tentatively named subgroups1A and1B andsubgroups2A and2B.The predicted amino acid sequences of G within members of each subgroup were highlyconserved(amino acid identities,88%for group1A,93%for group1B,and96%for group2B).The G of hMPVis thought to be the major antigenic determinant and to play an important role in the production of neutral-izing antibodies.Clariﬁcation of the antigenic diversity of G is important for epidemiological analysis and forestablishment of strategies to prevent hMPV infection.Human metapneumovirus(hMPV),ﬁrst isolated in The Netherlands in2001,is a member of the genus Metapneumo-virus of the subfamily Pneumovirinae of the family Paramyxo-viridae(37).hMPV causes upper respiratory tract infections andﬂu-like illnesses(5,32);but it is also associated with lower respiratory tract infections,such as wheezing bronchitis,bron-chitis,bronchiolitis,and pneumonia,in very young children, elderly individuals,and immunocompromised patients(7,10, 11,15,30).The genomic organization of hMPV is3Ј-N-P-M-F-M2-SH-G-L-5Ј(36,37).The M2gene contains two open reading frames(ORFs),M2-1and M2-2.Although the predicted hMPV proteins have not yet been identiﬁed or functionally analyzed,hMPV is thought to encode the following nine pro-teins:N,the nucleocapsid RNA binding protein;P,the nucleo-capsid phosphoprotein;M,the nonglycosylated matrix protein; F,the fusion glycoprotein;M2-1,the transcription elongation factor;M2-2,the RNA synthesis regulatory factor;SH,the small hydrophobic surface protein;G,the major attachment protein;and L,the major polymerase subunit(4,36,37). Pneumoviruses encode two major surface glycoproteins(gly-coproteins F and G).F promotes fusion of the viral and cell membranes,allowing penetration of the viral ribonucleopro-tein into the cell cytoplasm(39),and G mediates virus binding to the cell receptor(24).G is known to be the most variable protein in human respiratory syncytial virus(hRSV)and avian pneumovirus(APV)(8,22).In both viruses there are at least two distinct groups.For hRSV G,there is53%identity in amino acid sequences between groups A and B(19),whereas there is only38%identity in the amino acid sequences between types A and B for APV G(20).Neutralizing antibodies against F and G are important for protection against hRSV infection. The antibody against F protects animals from viruses of both hRSV group A and hRSV group B(17,18,28).G induces an antibody response protective only against infections caused by one hRSV group(19,40).The sequences of several hMPV genes(the genes for N,P, M,F,and L)and gene fragments have been determined,and the results of phylogenetic analysis have shown that hMPV can be divided into two major groups(2,3,5,6,9,25,29,38). However,sequence data for G are limited(4,37).In the present study,we determined the nucleotide sequences of pu-tative ORFs for N,P,M,F,M2-1,M2-2,SH,and G of seven newly isolated hMPVs and compared them with the previously published sequences for hMPV.MATERIALS AND METHODSVirus propagation.Seven hMPV isolates(9)were propagated in LLC-MK2 cells in vitro.The isolates were from nasopharyngeal swab samples of seven Japanese patients(age range,8months to3years)with lower respiratory tract infections obtained during the winter and spring of2003-2004in Sapporo,Japan. The cells were cultured in Eagle’s minimum essential medium for3to4weeks, with the medium changed weekly.Trypsin(Sigma-Aldrich)was added to the medium at a concentration of1␮g/ml.RNA extraction,cDNA synthesis,RT-PCR,and sequencing.Virus RNA was extracted from inoculated LLC-MK2cells by the RNAzol B(TEL-TEST,Inc., Friendswood,Tex.)method,according to the protocol of the manufacturer. Approximately1␮g of each RNA sample was incubated in a solution containing 100ng of random hexadeoxynucleotides and200U of Moloney murine leukemia virus reverse transcriptase(First-Strand cDNA Synthesis kit;Amersham Phar-macia Biotech,Piscataway,N.J.)in aﬁnal volume of15␮l at37°C for1h to synthesize cDNA.The cDNA(0.3␮l)was subjected to reverse transcription-*Corresponding author.Mailing address:Department of Pediatrics, Hokkaido University Graduate School of Medicine,N-15,W-7,Kita-ku,Sapporo060-8638,Japan.Phone:81-11-706-5954.Fax:81-11-706-7898.E-mail:hide-ki@med.hokudai.ac.jp.3406 at Penn State Univ on April 14, 2008 Downloaded fromFIG.1.Phylogenetic analysis of hMPV isolates.The putative ORFs for N,P,M,F,M2-1,M2-2,SH,and G were analyzed.The corresponding gene sequences from APVC were also analyzed.Bootstrap proportions were plotted at the main internal branches of the phylogram to show support values.Note that the scale of each tree was different.Phylogenetic analysis was performed by use of the neighbor-joining method of the MEGA program.V OL .42,2004HIGH GENETIC DIVERSITY OF hMPV G PROTEIN 3407at Penn State Univ on April 14, 2008 Downloaded fromPCR (RT-PCR).The RT-PCR mixture consisted of 100␮mol of each deoxyri-bonucleotide,1.0U of Ampli Taq Gold,50mmol of potassium chloride per liter,10mmol of Tris-HCl (pH 8.3)per liter,1.5mmol of magnesium chloride per liter,0.01%(wt/vol)of gelatin,10pmol of each primer,and cDNA in a volume of 25␮l.The PCR conditions were as follows:94°C for 10min,followed by 35cycles of 94°C for 1min,50°C for 2min,and 72°C for 3min.The PCR products were sequenced directly by using a BigDye Dye terminator cycle sequencing ready reaction kit (Perkin-Elmer Applied Biosystems,Tokyo,Japan)with an ABI PRISM 310Genetic Analyzer (Perkin-Elmer Applied Biosystems).Strategy for sequencing.Speci ﬁc primers were designed on the basis of the total sequence of hMPV strain 00-1and the partial sequence of isolate JPS02-76,which were obtained previously (data not shown).Fragments ranging from 1,000to 3,000bp were generated by RT-PCR.The PCR product was sequenced directly by the genome-walking method.The sequences were subsequently con-ﬁrmed by generating overlapping RT-PCR fragments.The genomes of hMPV isolates were sequenced from the start of the gene for N to the noncoding sequences between the genes for G and L.Nucleotide sequences.The GenBank database nucleotide sequence accession numbers for the pneumovirus strains and proteins used in this study are AF371337for strain hMPV 00-1,AY297749for strain hMPV CAN97-83,AY297748for strain hMPV CAN98-75,AF176590for the APV type C (APVC)gene for N,AF176591for the APVC gene for P,AF262571for the APVC gene for M,AY579780for the APVC gene for F,AF176592for the APVC gene for M2,APN457967for the APVC gene for SH,and APN457967for the APVC gene for G.Analysis of nucleotide and amino acid sequences.Sequences were aligned by use of the CLUSTAL W program (35).The transmembrane region was predicted by the method of Stoffel et al.(33),available online at the Swiss Institute for Experimental Cancer Research (/software/TMPRED _form.html).ORFs were predicted by using ORF ﬁnder software (http://www /gorf/gorf.html).O-linked glycosylation and N-linked glycosyl-ation were predicted by using NetOGlyc2.0(14)and NetNGlyc1.0(13)software,respectively;all software was obtained from the Center for Biological Sequence Analysis,Technical University of Denmark,Lyngby,Denmark (http://www.cbs .dtu.dk/services/).Local similarities between nucleotide and amino acid se-quences were studied by use of the BLAST 2algorithm (1).Phylogeny.The nucleotide sequences of the genes for N,P,M,F,M2-1,M2-2,SH,and G were assembled by using CLUSTAL W software.Phylogenetic trees were generated by the neighbor-joining method with the MEGA program (21).Nucleotide sequence accession numbers.The hMPV sequences presented in this paper have been deposited in GenBank under accession numbers AY530089to AY530095for strains JPS02-76,JPS03-176,JPS03-178,JPS03-180,JPS03-187,JPS03-194,and JPS03-240,respectively.RESULTSPhylogenetic analysis.Phylogenetic analyses were based on the putative ORFs for N,P,M,F,M2-1,M2-2,SH,and G of seven hMPV isolates,three published full-length hMPV se-quences (for isolates 00-1,CAN97-83,and CAN98-75),and the APVC sequence (Fig.1).Ten hMPV strains were divided into two major genetic groups that were previously tentatively named groups 1and 2(2,5).The hMPV Dutch isolate char-acterized by van den Hoogen et al.(37)(isolate 00-1)belongs to group 1.The results of our phylogenetic analysis are con-sistent with data previously reported for the partial or com-plete sequences of the genes for N,P,M,F,and L of hMPV isolates from The Netherlands (37),Canada (2,5,31),Ger-many (38),Italy (25),and Japan (9).Furthermore,seven hMPV strains in group 1were subdivided into two subgroups,tentatively named subgroup 1A (strains 00-1and JPS03-180)and subgroup 1B (strains CAN97-83,JPS03-176,JPS03-178,JPS03-187,and JPS03-240).Three hMPV strains in group 2were also subdivided into two subgroups,tentatively named subgroup 2A (strain CAN98-75)and subgroup 2B (strains JPS02-76and JPS03-194).ORFs and proteins of hMPV isolates.Table 1shows the lengths of the putative ORFs for N,P,M,F,M2-1,M2-2,SH,T A B L E 1.L e n g t h s o f p u t a t i v e O R F s a n d p r e d i c t e d a m i n o a c i d s o f h M P V i s o l a t e sG r o u p ,s u b g r o u p ,a n d i s o l a t eNPMFM 2-1M 2-2S HGn t (a a )aM M bn t (a a )M Mn t (a a )M Mn t (a a )M Mn t (a a )M Mn t (a a )M Mn t (a a )M Mn t (a a )M MG r o u p 1S u b g r o u p 1A 00-11,185(394)43.5885(294)32.5765(254)27.61,620(539)58.5564(187)21.2216(71)8.2552(183)20.9711(236)25.8J P S 03-1801,185(394)43.5885(294)32.6765(254)27.61,620(539)58.5564(187)21.2216(71)8.2552(183)21.1711(236)25.7S u b g r o u p 1B C A N 97-831,185(394)43.5885(294)32.7765(254)27.61,620(539)58.5564(187)21.2216(71)8.2540(179)20.6660(219)23.7J P S 03-1761,185(394)43.5885(294)32.7765(254)27.61,620(539)58.5564(187)21.3216(71)8.2552(183)21.1660(219)23.8J P S 03-1781,185(394)43.5885(294)32.7765(254)27.61,620(539)58.5564(187)21.3216(71)8.2552(183)21.1660(219)23.8J P S 03-1871,185(394)43.5885(294)32.7765(254)27.61,620(539)58.6564(187)21.3216(71)8.1552(183)21.1660(219)23.8J P S 03-2401,185(394)43.5885(294)32.7765(254)27.61,620(539)58.6564(187)21.3216(71)8.2552(183)21.1660(219)23.7G r o u p 2S u b g r o u p 2A ,C A N 98-751,185(394)43.6885(294)32.5765(254)27.61,620(539)58.4564(187)21.2216(71)8.2534(177)20.4711(236)25.5S u b g r o u p 2B J P S 02-761,185(394)43.6885(294)32.5765(254)27.61,620(539)58.5564(187)21.2216(71)8.2534(177)20.4696(231)25.4J P S 03-1941,185(394)43.6885(294)32.4765(254)27.61,620(539)58.5564(187)21.2216(71)8.2534(177)20.3696(231)25.3an t ,l e n g t h o f p u t a t i v e O R F (i n b a s e p a i r s ).;a a ,l e n g t h o f p r e d i c t e d a m i n o a c i d s e q u e n c e .bM M ,e s t i m a t e d m o l e c u l a r m a s s (i n k i l o d a l t o n s )c a l c u l a t e d f r o m t h e s e q u e n c e s .3408ISHIGURO ET AL.J.C LIN .M ICROBIOL .at Penn State Univ on April 14, 2008 Downloaded fromand G and the predicted amino acid sequences of isolates 00-1,CAN97-83,and CAN98-75and seven hMPV isolates.The cal-culated molecular masses of the predicted proteins are also shown.The lengths of the coding regions of the putative genes for N,P,M,F,M2-1,and M2-2and the predicted amino acid sequences were consistent between isolates (N,1,185bp and 394amino acids [aa];P,885bp and 294aa;M,765bp and 254aa;F,1620bp and 539aa;M2-1,564bp and 187aa;and M2-2,216bp and 71aa).The lengths of the coding region of the putative gene for the SH protein and the predicted amino acid sequence were different for strains in groups 1and 2(group 1,552bp and 183aa;group 2,534bp and 177aa)except for isolate CAN97-83(540bp and 179aa),which had a stop codon (TAA)at position 541from the start of the gene for SH.The lengths of the coding region of the putative gene for G and predicted amino acid sequences were different for strains in subgroups 1A,1B,2A,and 2B (subgroup 1A,711bp and 236aa;subgroup 1B,660bp and 219aa;subgroup 2A,711bp and 236aa;and subgroup 2B,696bp and 231aa).Table 2shows the levels of identity of the nucleotide and amino acid sequences of the various hMPV ORFs between and within groups.Between two groups,the ORFs for N,P,M,M2-1,and M2-2shared identities of 81to 86%,on average,whereas the ORFs for the three surface proteins (F,SH,and G)were more divergent (F,60%;SH,67%;G,58%).The predicted amino acid sequences of N,P,M,M2-1,and M2-2were highly (85to 97%)conserved between two groups,as expected.While the predicted amino acid sequences of SH and G were divergent between two groups (58and 33%,respec-tively),as expected,those of F were surprisingly well conserved (94%).Within each group,the ORFs for N,P,M,M2-1,and M2-2shared high degrees of identity,on average,95to 97%;and the predicted amino acid sequences of these proteins were highly conserved (97to 99%)(Table 2).The ORFs for F and SH were well conserved within the two groups (F,92and 87%,respec-tively;SH,94and 91%,respectively),and the predicted amino acid sequences of F and SH were also well conserved (F,98%for both groups;SH,92and 88%,respectively).The ORFs for G were moderately identical within groups (82and 85%,re-spectively),and the predicted amino acid sequences of G were marginally less conserved (78and 75%,respectively).Tables 3and 4show the levels of identity of the nucleotide and amino acid sequences of various hMPV ORFs between and within subgroups.The ORFs and the predicted amino acid sequences of G were highly identical within members of each subgroup (nucleotide identities,92%for subgroup 1A,96%for subgroup 1B,and 97%for subgroup 2B;amino acid iden-tities,88%for subgroup 1A;93%for subgroup 1B,and 96%for subgroup 2B).SH.SH is predicted to be a type II glycoprotein that is inserted in the plasma membrane by a hydrophobic signal-anchor sequence located near its amino terminus (Fig.2).The predicted length of SH in group 1isolates except for strain CAN97-83was 183amino acids,as stated above (Table 1).The predicted length of SH in group 2isolates was 177amino acids.The predicted extracellular domain had two to four potential motifs for N-linked glycosylation and two to ﬁve potential sitesTABLE 2.Levels of identity of nucleotide and amino acid sequences within and between two hMPV groupsProtein%Nucleotide sequence identitywithin group a %Nucleotide sequence identity between groups a%Amino acid sequence identitywithin group a %Amino acid sequence identity between groups aGroup 1Group 2Group 1Group 2N 94–100(96)95–99(96)86–87(86)99–100(99)98–100(99)95–96(96)P 92–100(95)98–99(95)80–82(81)94–99(97)95–99(97)84–86(85)M 94–99(97)95–99(96)84–86(85)98–100(99)99–100(99)96–97(97)F 85–100(92)81–98(87)55–61(60)98–99(98)98–99(98)93–94(94)M2-194–99(97)94–99(96)86–87(86)96–100(98)97–100(98)94–95(95)M2-295–100(97)95–100(97)85–87(86)95–100(97)97–100(98)88–90(90)SH 90–99(94)88–98(91)66–69(67)83–99(92)81–98(88)54–60(58)G75–99(82)78–97(85)56–60(58)61–99(78)63–96(75)31–35(33)aValues are ranges (average)of identity obtained for comparison of strains within each group and between groups.TABLE 3.Levels of identity of nucleotide and amino acid sequences within and between subgroups 1A and 1BProtein%Nucleotide sequence identitywithin subgroup %Nucleotide sequence identity between subgroups1A and 1B a%Amino acid sequence identitywithin subgroup %Amino acid sequence identity between subgroups1A and 1B aSubgroup 1ASubgroup 1B aSubgroup 1ASubgroup 1B aN 9997–100(99)93–94(94)9999–100(99)99–100(99)P 9796–100(98)92–93(92)9898–100(99)95–96(95)M 9997–100(99)94–95(94)10099–100(99)99(99)F 9696–100(99)84–86(85)9998–99(99)98(98)M2-19998–100(99)93–94(94)9998–100(99)96–97(97)M2-29997–100(99)95–96(95)10097–100(99)95–97(96)SH 9895–100(98)90–91(91)9794–100(97)83–97(86)G9292–100(96)75–78(76)8886–100(93)60–65(62)aValues are ranges (average)of identity obtained for comparison of strains within and between subgroups 1A and 1B.V OL .42,2004HIGH GENETIC DIVERSITY OF hMPV G PROTEIN3409at Penn State Univ on April 14, 2008 Downloaded fromfor O-linked glycosylation (14)(Fig.2).Although the NYT sequence in the CAN98-75isolate was a consensus N-linked glycosylation site,it is unlikely to be glycosylated within the membrane.The transmembrane domain might be shifted in this sequence,or this site might not be glycosylated.The pre-dicted SH sequences contained 9or 10cysteine residues,which were mostly in the extracellular domain,and 9of these were conserved among all strains.G.G is also predicted to be a type II glycoprotein (Fig.3).The predicted lengths of G in subgroup 1A,1B,2A,and 2B isolates were 236,219,236,and 231amino acids,respectively.The predicted extracellular domain had one to four potential motifs for N-linked glycosylation and more than 40potential sites for O-linked glycosylation (Fig.3).The NAS and NAT N-linked glycosylation sequences within the intracellular do-main were potential sites but are unlikely to be used.G con-tained one or two cysteine residues,one of which was con-served in the intracellular domain.In group 2isolates,G had two cysteine residues,one in the intracellular domain and the other in the extracellular domain;but those in group 1isolates had only one residue.Noncoding sequences.hMPV contains a 3Јleader region,followed by nine putative genes and a 5Јtrailer region (4,22,36).The transcriptional control sequences are conserved at the beginning (gene-start)and end (gene-end)of each gene.In-tergenic sequences are located between the gene boundaries.Figure 4shows the putative gene-start,gene-end,and inter-genic sequences of the seven new hMPV isolates and three strains described previously.The overall gene-start signals (G GGAC/UAAA/GU)for the genes for P,M,F M2,SH,and G identi ﬁed in the three strains described previously were well conserved in the seven new isolates (4,36).As described be-fore (4,36),there was an additional ATG codon upstream of the gene-start motif of the gene for SH (Fig.4).The putative gene-end signals (AGUU/AA/UnnA/UA4-7where n is any nucleotide)of the genes for N,P,M,F,M2,SH,and G were also conserved in the seven new isolates as well as the three published strains of hMPV (Fig.4).Strains JPS03-194(group 2)and CAN97-83(group 1)had one A-to-U nu-cleotide substitution in the poly(A)tail.The lengths of the putative intergenic sequences of the hMPV gene varied (Fig.4).The intergenic regions of theN-P,parison of the predicted amino acid sequences of SH of hMPV isolates.The predicted amino acid sequence of SH of hMPV with cysteine residues is shown in boldface type,potential N-linked glycosylation sites are shaded in gray,and potential O-glycosylation sites are underlined.Dashes indicate gaps introduced to maximize the alignment or to denote the absence of corresponding amino acids.The asterisks underneath each alignment denote amino acid identity among all sequences.Proposed intracellular,transmembrane,and extracellular domains are indicated above the sequences.3410ISHIGURO ET AL.J.C LIN .M ICROBIOL .at Penn State Univ on April 14, 2008 Downloaded fromP-M,M-F,M2-SH,and SH-G boundaries were 2to 3,8,30to 34,10to 13,and 115to 126bp,respectively.The length of the F-M2boundary differed in both the genetic groups and the subgroups.The F-M2boundaries in subgroup 1A isolates (iso-lates 00-1and JPS03-180)were 41and 67bp,respectively,and those in subgroup 1B isolates (isolates CAN97-83,JPS03-176,JPS03-178,JPS03-187,and JPS03-240)and group 2isolates (isolates CAN98-75,JPS02-76,and JPS03-194)were 13to 20and 1bp,respectively.DISCUSSIONIn the present study,the complete predicted gene sequences of N,P,M,F,M2-1,M2-2,SH,and G of seven newly isolated hMPV isolates were determined and compared with the se-quences of three hMPV genomes published previously.Phylo-genetic analyses indicated that hMPV isolates could be divided into two major genetic groups,tentatively named groups 1and 2(Fig.1),which is consistent with the partial or complete sequences of the genes for N,P,M,F,and L reported previ-ously (2,5,9,25,31,37,38).Each group could be further divided into two subgroups,tentatively named subgroups 1A and 1B and subgroups 2A and 2B (Fig.1).The other published sequences of the genes for N,P,M,and F (GenBank accession numbers AY145242to AY145301,AY256863to AY256867,AY321506,AY321507,AY355328,AY355335)were classi ﬁed into four subgroups (data not shown).In a previous phyloge-netic analysis based on partial F sequences (80bp)(9),57hMPV isolates were classi ﬁed into two distinct genetic groups (groups 1and 2)and also,potentially,into four subgroups.However,due to the small number of hMPV isolates,we can-not conclude that all hMPV isolates can be clustered into one of the four subgroups.The ﬁndings for additional sequences from around the world may require modi ﬁcation of the classi-ﬁcation,but so far,the classi ﬁcation seems to be appropriate.The most interesting observation in this study was that pu-tative ORFs for G in hMPVs were classi ﬁed into four sub-groups by phylogenetic analysis,as were other putative ORFs for N,P,M,F,M2,and SH,and that the predicted G amino acid sequences within each subgroup were highly conserved (Tables 3and 4).The numbers of amino acids in the ORFs within the subgroups were also identical.In hRSV,the num-bers of amino acids for G ranged from 289to 299,but the numbers for G in groups A and B are not the same (8,12,26,27,34).The nine partial sequences of hMPV G available in GenBank (accession numbers AY327802to AY327810)be-parison of the predicted amino acid sequences of G of hMPV isolates.The predicted amino acid sequence of G of hMPV with cysteine residues is shown in boldface type,potential N-linked glycosylation sites are shaded in gray,and potential O-glycosylation sites are underlined.Dashes indicate gaps introduced to maximize the alignment or to denote the absence of corresponding amino acids.The asterisks underneath each alignment denote amino acid identity among all sequences.Proposed intracellular,transmembrane,and extracellular domains are indicated above the sequences.The square indicates the positions of conserved amino acid residues.V OL .42,2004HIGH GENETIC DIVERSITY OF hMPV G PROTEIN 3411at Penn State Univ on April 14, 2008 Downloaded fromF IG .4.P r e d i c t e d g e n e b o u n d a r i e s ,c i s -a c t i n g g e n e -s t a r t a n d g e n e -e n d s i g n a l s ,a n d i n t e r g e n i c r e g i o n s o f h M P V i s o l a t e s .T h e a l i g n m e n t s s h o w t h e N -P ,P -M ,M -F ,F -M 2,M 2-SH ,S H -G ,a n d G b o u n d a r i e s .C o n s e r v e d s e q u e n c e m o t i f s a t t h e e n d a n d b e g i n n i n g o f e a c h g e n e a r e i n d i c a t e d b y b o l d u p p e r c a s e l e t t e r s ,a n d a c o n s e n s u s s e q u e n c e i s g i v e n b e l o w .T r a n s l a t i o n a l s t o p a n d s t a r t c o d o n s a r e u n d e r l i n e d .3412ISHIGURO ET AL.J.C LIN .M ICROBIOL .at Penn State Univ on April 14, 2008 Downloaded fromlonged to subgroup1A,and81%amino acid identity was detected among the G sequences from subgroup1A(data not shown).Considering that the attachment proteins of para-myxoviruses are one of the major protective antigens(8,22), immunological pressure might function to cause the genetic diversity of hMPV G in the extracellular domain(Fig.3).The subgroups of hMPV isolates might be useful not only for in-vestigation of the antigenic variability of hMPV but also for the study of the neutralizing antibodies against homologous or heterologous subgroups of hMPV.In hRSV,although the ex-tracellular domain of G is variable between groups A and B,a strictly conserved13-amino-acid region in the central extracel-lular domain is noted(19).This conserved domain contains four closely spaced,exactly conserved cysteine residues and coincides with a tight turn in the predicted structure(23).In contrast,the predicted sequence of the G of hMPV lacked both the conserved13-amino-acid region and the four cysteine residues(Fig.3).Instead,hMPV G had a relatively well con-served region from residues92to103,in which50%(6of12) of the amino acids were identical(Fig.3,square).This part might be involved in the attachment of G to cellular receptors. Cysteine residues of the predicted sequence of the SH of hMPV were well conserved,and this might be helpful in sta-bilizing the structure of SH.The sequences of the ORFs of three putative surface pro-teins(F,SH,and G)were divergent between the two groups (Table2).The predicted amino acid sequences of F were well conserved between the two groups,but the sequences of SH and G were less well conserved(Table2).Nucleotide substi-tutions in the SH extracellular domain and the G extracellular domain were closely associated with amino acid substitutions (SH,89.1%;G,91.6%);the rate of amino acid substitutions was low(10.6%)for F(data not shown).This observation is consistent with previously reportedﬁndings for the G of hMPV (4)and the G of hRSV(16).The putative gene-start signals(GGGAC/UAAA/GU)for the genes for P,M,F M2,SH,and G and the putative gene-end signals(AGUU/AA/UnnA/UA4-7)for the genes for N,P,M, F,M2,SH,and G identiﬁed in the three published strains were well conserved in the seven newly isolated hMPVs(Fig.4)(4, 36).Those motifs were similar to those of hRSV(gene-start, GGGGCAAAUU/A;gene-end,AGUU/AAnU/AU/AA/UA AAA)(8).Further study is needed to determine whether those motifs function as gene-start and gene-end signals.In conclusion,genes encoding the putative N,P,M,F,M2-1, M2-2,SH,and G of seven newly isolated hMPVs were ana-lyzed.Phylogenetic analysis of the nucleotide sequences indi-cated that there were two genetic groups(groups1and2), which were further subdivided into two subgroups,tentatively named subgroups1A and1B and subgroups2A and2B.The predicted amino acid sequences of G within members of each subgroup were highly conserved.The G of hMPV is thought to be the major antigenic determinant and to play an important role in the production of neutralizing antibodies.Clariﬁcation of the antigenic diversity of G is important for epidemiological analysis and for the establishment of strategies to prevent hMPV infections.ACKNOWLEDGMENTSThis study was partially supported by a grant-in-aid for exploratory research(grant14657179[2002])from the Ministry of Education, Culture,Sports,Science and Technology of Japan and by a grant-in-aid for the21st century Center of Excellence for Zoonosis Control from the Ministry of Education,Culture,Sports,Science and Technology of Japan.We thank Stewart Chisholm for proofreading the manuscript.REFERENCES1.Altschul,S.F.,T.L.Madden,A.A.Schaffer,J.Zhang,Z.Zhang,ler,and D.J.Lipman.1997.Gapped BLAST and PSI-BLAST:a new generation of protein database search programs.Nucleic Acids Res.25:3389–3402. 2.Bastien,N.,S.Normand,T.Taylor,D.Ward,T.C.Peret,G.Boivin,L.J.Anderson,and Y.Li.2003.Sequence analysis of the N,P,M and F genes of Canadian human metapneumovirus strains.Virus Res.93:51–62.3.Bastien,N.,D.Ward,P.Van Caeseele,K.Brandt,S.H.Lee,G.McNabb,B.Klisko,E.Chan,and Y.Li.2003.Human metapneumovirus infection in the Canadian population.J.Clin.Microbiol.41:4642–4646.4.Biacchesi,S.,M.H.Skiadopoulos,G.Boivin,C.T.Hanson,B.R.Murphy,P.L.Collins,and U.J.Buchholz.2003.Genetic diversity between human metapneumovirus subgroups.Virology315:1–9.5.Boivin,G.,Y.Abed,G.Pelletier,L.Ruel,D.Moisan,S.Cote,T.C.Peret,D.D.Erdman,and L.J.Anderson.2002.Virological features and clinicalmanifestations associated with human metapneumovirus:a new paramyxo-virus responsible for acute respiratory-tract infections in all age groups.J.Infect.Dis.186:1330–1334.6.Boivin,G.,G.De Serres,S.Cote,R.Gilca,Y.Abed,L.Rochette,M.G.Bergeron,and P.Dery.2003.Human metapneumovirus infections in hospi-talized children.Emerg.Infect.Dis.9:634–640.7.Cane,P.A.,B.G.van den Hoogen,S.Chakrabarti,C.D.Fegan,and A.D.Osterhaus.2003.Human metapneumovirus in a haematopoietic stem cell transplant recipient with fatal lower respiratory tract disease.Bone Marrow Transplant.31:309–310.8.Collins,P.L.,R.M.Chanock,and B.R.Murphy.2001.Respiratory syncytialvirus,vol.2,4th ed.Lippincott Williams&Wilkins,Philadelphia,Pa.9.Ebihara,T.,R.Endo,H.Kikuta,N.Ishiguro,H.Ishiko,M.Hara,Y.Taka-hashi,and K.Kobayashi.2004.Human metapneumovirus infection in Jap-anese children.J.Clin.Microbiol.42:126–132.TABLE4.Levels of identity of nucleotide and amino acid sequences within and between subgroups2A and2BProtein%Nucleotide sequence identitywithin subgroup%Nucleotide sequenceidentity betweensubgroups2A and2B a%Amino acid sequence identitywithin subgroup%Amino acid sequenceidentity between subgroups2A and2B a Subgroup2A Subgroup2B Subgroup2A Subgroup2BN NA b9995(95)NA10098(98)P NA9993–94(94)NA9995–96(95)M NA9995(95)NA10099(99)F NA9881–82(82)NA9998(98)M2-1NA10094(94)NA10097(97)M2-2NA10095(95)NA10097(97)SH NA9888(88)NA9882–83(82)G NA9778–79(79)NA9664–66(65)a Values are ranges(average)of identity obtained for comparison of strains within and between subgroups2A and2B.b NA,not applicable because only a single isolate was tested.V OL.42,2004HIGH GENETIC DIVERSITY OF hMPV G PROTEIN3413at Penn State Univ on April 14, 2008 Downloaded from。