Revista Romn de

BOLI INFECIOASE
Volumul XI Nr. 3 Anul 2008
ISSN 1454-3389 COD CNCSIS 770
Redactor ef Ludovic Pun Redactor ef Adjunct Mircea Chiotan Secretar de Redacie Simin-Aysel Florescu

CONSILIUL TIINIFIC
Petre Calistru Bucureti Dumitru Crstina Cluj Emanoil Ceauu Bucureti Augustin Cupa Craiova Olga Dorob Bucureti Doinia Ispas Bucureti Vasile Luca Iai Lucian Negruiu Timioara Rodica Pascu Tg. Mure Ileana Rebedea Bucureti Sorin Rugin Constana Doina Stnescu Timioara Adrian Streinu-Cercel Bucureti Dumitru Suciu Sibiu

Secretariatul Redaciei: Simin-Aysel Florescu Clinica de Boli Infecioase i Tropicale Victor Babe, os. Mihai Bravu nr. 281, sector 3, Bucureti Telefon/Fax: 01 - 317 27 20

Editura Medical AMALTEA

Editori: Dr. M.C. Popescu Dr. Cristian Crstoiu Director executiv: George Stanca Redactori: Oana Cristina Plcint, Alina-Nicoleta Ilie Prepress: AMALTEA TehnoPlus Tehnoredactor: Gabriela Cpitnescu DTP: Petronella Andrei, Gabriela Cpitnescu Producie: Mihaela Conea Distribuie: Mihaela Stanca ________________ CONTACT: AMR@medica.ro ABONAMENTE: redactia@amaltea.ro TIPAR: EMPIRE Print RomExpo, Pavilion T, Bucureti tel.: 021 / 316 96 40, 031 / 405 99 99 email: office@empireprint.ro

Cuprins
1. Cuvnt ctre cititori ____________________________________________________________ 123 Ludovic Pun 2. Bolile infecioase, armele biologice, bioterorismul, provocare major a societii civile contemporane ____________________________________________________________ 125 Ludovic Pun 3. Influenza Activity ______________________________________________________________ 127 Department of Health and Human Services Center for Disease Control and Prevention 4. Bacterial Pneumonia and Pandemic Influenza Planning ____________________________ 133 Ravindra K. Gupta, Robert George, Jonathan S. Nguyen-Van-Tam 5. Deaths from Bacterial Pneumonia during 1918-19 Influenza Pandemic _______________ 139 John F. Brundage, G. Dennis Shankst 6. Environmental Contamination during Influenza A Virus (H5N1) Outbreaks, Cambodia, 2006 _______________________________________________________________ 146 Sirenda Vong, Sowath Ly, Sek Mardy, Davun Holl, Philippe Buchy 7. Highly Pathogenic Avian Influenza Virus (H5N1) in Experimentally Infected Adult Mute Swans _____________________________________________________________ 149 Donata Kalthoff, Angela Breithaupt, Jens P. Teifke, Anja Globig, Timm Harder, Thomas C. Mettenleiter, Martin Beer 8. Highly Pathogenic Avian Influenza Virus (H5N1) Isolated from Whooper Swans, Japan _____ 153 Yuko Uchida, Masaji Mase, Kumiko Yoneda, Atsumu Kimura, Tsuyoshi Obara, Seikou Kumagai, Takehiko Saito, Yu Yamamoto, Kikuyasu Nakamura, Kenji Tsukamoto, Shigeo Yamaguchi 9. Oseltamivir Prescribing in Pharmacy-Benefits Database, United States, 2004-2005 _____ 156 Justin R. Ortiz, Laurie Kamimoto, Ronald E. Aubert, Jianying Yao, David K. Shay, Joseph S. Bresee, Robert S. Epstein 10. Pandemic Influenza and Excess Intensive-Care Workload __________________________ 160 Raoul E. Nap, Maarten P.H.M. Andriessen, Nico E.L. Meessen, Dinis dos Reis Miranda Tjip S. van der Werf 11. Influenza Virus (H5N1) in Live Bird Markets and Food Markets, Thailand ____________ 168 Alongkorn Amonsin, Chuensakon Choatrakol, Jiradej Lapkuntod, Rachod Tantilertcharoen, Roongroje Thanawongnuwech, Sanipa Suradhat, Kamol Suwannakarn, Apiradee Theamboonlers, and Yong Poovorawan 12. Replacement of Sublineages of Avian Influenza (H5N1) by Reassortments, Sub-Saharan Africa ____________________________________________________________ 173 Ademola A. Owoade, Nancy A. Gerloff, Mariette F. Ducatez, Jolaoso O. Taiwo, Jacques R. Kremer, and Claude P. Muller 13. Bibliografie de consultat _______________________________________________________ 178

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CUVNT CTRE CITITORI
Foreword

Stimai cititori, n orientarea activitii editoriale, Consiliul tiinific al Revistei Romne de Boli Infecioase a aderat la punctul actual de vedere, care consider specialitatea de boli infecioase o specialitate clinic, independent, implicat n Clinica, Diagnosticul, Terapia i Profilaxia mbolnvirilor produse de ageni etiologici cunoscui sau n curs de identificare, patogenii la toate vrstele deopotriv i n evoluiile epidemiologice ale bolilor infecioase acute transmisibile n comunitile umane (focare infecioase, epidemii, pandemii). Acest mod de abordare a bolilor infecioase acute transmisibile a determinat cu necesitate, n condiiile Globalizrii* patologiei infecioase, elaborarea i aplicarea la nivel mondial, de ctre rile membre ale Organizaiei Mondiale a Sntii (OMS), (incluznd n program i rile care nu sunt nc membre OMS) a Regulamentului Sanitar Internaional, OMS 2005 (Revista Romn de Boli Infecioase, Regulamentul Sanitar Internaional Vol. XI, Nr. 1/2008), activitate programat s se desfoare pe durata a 5 ani, ncepnd cu anul 2005. Dinamica bolilor infecioase acute transmisibile la sfritul secolului XX i nceputul secolului al XXI-lea, severitatea evoluiilor naturale ale acestora, determinat de diversitatea fondului etiologic comun pe Tera i n Cosmos, att natural, ct i modificat intenionat Weaponizat ** a permis aprecierea ameninrii neconvenionale la adresa securitii naionale (John C. Ganon The Global Infectious Threat and Its Implications for Unites Statter NIE 99, 17 January, 2000). Atacul cu Spori de Bacillus anthracis, 11 Septembrie 2001, a reprezentat doar o dovad nedorit, dar, din pcate, nu unic.

Perspectiva evoluiei pandemice naturale a gripei A (H5N1) a impus: a. instituirea alertei epidemiologice, n ciuda numrului redus de cazuri umane (n jur de 100, localizate n marea majoritate n rile din Asia de Sud-Est); b. abordarea actual a manifestrilor clinice, diagnosticul i terapia cazurilor individuale de grip; c. precum i reactualizarea, compararea i valorificarea concluziilor rezultate din studiul actualizat al evoluiilor pandemiei de grip A (H5N1), 1918-1919 (articolele 3, 4, 5 din Sumar). Consiliul tiinific al Revistei Romne de Boli Infecioase a considerat oportun acordarea spaiului editorial al Nr. 4/2008, pentru informarea Corpului medical din Romnia, a Grupului Tehnic de specialiti (clinicieni n boli infecioase aduli i copii) epidemiologi, microbiologi cu datele i opiniile publicate n: Emerging Infectious Diseases, Vol. 14, Nr. 8, August, Nr. 9, Septembrie 2008 i Morbidity and Mortality Weekly Report, Iunie 27/2008, Vol. 57, Nr. 25. Ambele publicaii sunt de acord cu reproducerea integral (pag. 132; pag. 159). Publicarea sumarelor articolelor i n limba romn o considerm o facilitate pentru cititori. n legtur cu Gripa Pandemic A (H5N1) sau Gripa viral determinat de alt tip de virus, Consiliul tiinific consider c procednd neconvenional la ntocmirea sumarului Nr. 4/2008 al Revistei Romne de Boli Infecioase a dovedit flexibilitate n abordarea marilor probleme pe care le ridic morbiditatea prin boli infecioase i informarea specialitilor n secolul al XXI-lea. Dup ce va obine punctul de vedere al cititorilor n legtur cu iniiativa sa, Consiliul tiinific va continua s acorde spaiu editorial, de preferin

*) Concept universal, ce dorete a surprinde evoluiile mondiale naturale social-economice i politice dup cel de-al Doilea Rzboi Mondial

i al Rzboiului Rece i ncearc s codifice i s clasifice reaciile persoanelor, grupurilor de persoane, organizaii etc. care recurg inclusiv la violen n lupta pentru putere (James M. Lutz i Brenda Lutz n Global Terrorism Rontledge, 2004, Causes of Terrorism, pag. 17) **) Transformat prin procedee biotehnologice genetice specifice n arme biologice.

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n suplimente la numerele curente, lsnd liber spaiul editorial al revistei pentru publicarea lucrrilor cu caracter de originalitate individual sau a grupurilor de specialiti.

n acest sens, ar putea fi elaborate Sumare pentru: Infeciile cu genul Vibrio, Etiologia infecioas a bolilor cronice, Bioterorismul n Secolul al XXI-lea.

Redactor ef, Prof. Dr. Ludovic Pun

Adres de coresponden: Prof. Dr. Ludovic Pun, Spitalul Clinic de Boli Infecioase i Tropicale Dr. Victor Babe, os. Mihai Bravu, Nr. 281, Sector 3, Bucureti

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BOLILE INFECIOASE, ARMELE BIOLOGICE, BIOTERORISMUL, PROVOCARE MAJOR A SOCIETII CIVILE CONTEMPORANE
Infectious diseases, biological weapons, bioterrorism major provocation of contemporary civil society
Prof. Dr. Ludovic Pun Membru de onoare AOS

Infecia biologic este una dintre cele mai vechi lupte de pe planet, se poate spune un rzboi; originea celor mai multe microorganisme cauzatoare de boal este necunoscut. Cu toate acestea, cercetarea medical-arheologic scoate n eviden faptul c n viaa marin pietrificat, resturile animalelor prdtoare pentru om i mumiile conin rmie fosilizate ale microorganismelor. Istoria uman arat c fiecare secol are propriii si patogeni emergeni, respectiv boli de importan primordial (1). Dinamica epidemiologic a bolilor infecioase, utilizarea patogenilor ca arme biologice i bioterorismul reprezint asocierea generatoare a provocrii majore pentru Sntatea Public i Asistena Medical contemporan n domeniul bolilor infecioase. Evoluiile epidemiologice ale Sindromului Acut Respirator Sever (SARS), Gripei Aviare (H5N1), Atacului bioterorist cu spori de Bacillus anthracis, SUA 2001, i presiunea continu a ameninrilor cu atacuri bioteroriste, sunt concretizri convingtoare cu privire la dimensiunile provocrii pentru care Societatea civil, Sntatea Public i Asistena Medical, Structurile informative i de Aplicare a legii sunt investite s rspund. Formularea obiectivului actual al statelor care resping Terorismul n general, inclusiv Terorismul biologic: Prevenirea i Rspunsul Societii Civile la atacul bioterorist, exprim sintetic politica privind abordarea armelor biologice i bioterorismul. Bolile infecioase constituie expresia clinic a unui concept unitar (2) referitor la biologia relaiilor dualiste naturale sau provocate ntre dou entiti biologice vii, (caz unic ntre armele de

distrugere n mas: explozive, nucleare, chimice, biologice), relaii care definesc conceptul istoric, dinamic: Boli infecioase, arme biologice, bioterorism. Microorganismele unicelulare (virusuri, bacterii, parazii, fungi, prioni) i macroorganismele pluricelulare, structurate organic (oameni, animale, plante i alte structuri biologice vii) dezvolt relaii care se reflect n abordrile din Sntatea Public. Asistena Medical a populaiei i animalelor ocup un loc principal n cercetarea tiinific medical (uman i veterinar), n Biotiin i Biotehnologie. Conceptul Boli infecioase, arme biologice, bioterorism, concept unitar, dinamic, marcat de evoluia bolilor infecioase, istoricete constituit i extins la armele biologice produse prin tehnologie industrial specific, (dup descoperirea agenilor patogeni/etiologici ai bolilor infecioase n secolul al XIX-lea: Louis Pasteur, Robert Koch) este asociat cu bioterorismul (manifestare psihologic de mas), agroterorismul i cyberterorismul. Contemporaneitatea cu cyberterorismul (form a terorismului tehnic cu implicare asupra reelelor electronice informative, a tehnologiei electronice n general, de care beneficiaz i domeniul medical), biotiina i biotehnologia amplific major opional riscul bioterorismului n mileniul trei. De-a lungul istoriei, epidemiile (naturale) bolilor infecioase au provocat un numr cu mult mai mare de decese dect decesele nregistrate n rzboaie: aproape un sfert din populaia Europei (aproximativ 25 de milioane) au decedat n cursul epidemiei de pest (cium bubonic n secolul al XIV-lea (3); variola, rujeola, gripa, tifosul i pesta/ ciuma bubonic au omort peste 95% din populaia
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nativilor americani precolumbieni, iar n epidemia mondial de grip (H5N1) 1918-1919 au decedat peste 21 de milioane de oameni, n mare majoritate tineri, mai muli dect au fost omori prin armele de foc n cursul celui de-al Doilea Rzboi Mondial (3). Cu toate sucesele obinute n limitarea morbiditii prin boli infecioase, n SUA mor anual aproximativ 150.000 de oameni (3). n prezent, prevenirea epidemiilor majore de boli infecioase reprezint o provocare mai mare dect anterior, din cauza globalizrii, cltoriilor i comerului, marilor concentraii ale populaiilor, convieuirii n proximitatea animalelor, ale cror boli (unele!) pot fi transmise oamenilor (3). La ameninrile bolilor infecioase transmise natural se adaug ameninrile bioteroriste (2)! Una dintre particularitile armelor biologice const n faptul c acestea (armele biologice) fac parte dintre organismele vii, spre deosebire de alte arme cu extindere n mas (explozive, nucleare, chimice); armele biologice (forme vii de existen) sunt o prezen universal, n mediul fizic i biologic, pe Terra i n cosmos, fondul etiologic comun. n aceste condiii (existena dualist a formelor unicelulare i multicelulare de via), se creeaz n mod obligatoriu fie relaii echilibrate, fie dezechilibre biologice care implic sntatea i calitatea vieii, induc evoluii epidemiologie individuale i de grup, inclusiv bioterorismul. Relaiiie biologice ntre cele dou existene vii sunt concretizate prin manifestri clinice, epidemiologice, date i explorri de laborator, markeri ai evoluiei biologice, care, consemnate i sintetizate alturi de supravegherea clinic, se constituie n baza de date necesar managementului, dup caz, a unei epidemii naturale i/sau a unui atac bioterorist. Un raport, declasificat recent n Statele Unite, elaborat de National Intelligence Council pentru

Central Intelligence Agency (CIA), concluzioneaz c: Bolile infecioase nu sunt numai o problem de sntate public, ci i una de securitate naional (4), populaia Statelor Unite este vulnerabil att la bolile infecioase emergente, ct i reemergente (4). n anul 1993, U.S. Congresionat Office of Techonology estima c diseminarea a 100 de kilograme, spori de Antrax, asupra aezrii umane Washington DC, ar putea produce ntre 130.000 3 milioane de decese, i ar putea fi considerate o arm la fel de letal ca i o bomba cu hidrogen (3). Accidentul Sewerdlowsk (URSS, 1979) cu spori de Bacillus anthracis insuficient mediatizat, i mai ales atacul bioterorist cu spori de Bacillus anthracis, SUA 2001, ca i ameninrile teroriste cu recurgerea la armele biologice se constituie n dovezi clare pentru susinerea existenei provocrii majore actuale pe care bolile infecioase (n evoluia natural), armele biologice (evoluie provocat) i bioterorismul (manifestri pshiologice de mas) le dezvolt la adresa societii civile. Informaiile difuzate pe Internet cu privire la fabricarea armelor biologice constituie o surs tehnic de sprijin pentru cei ce provoac sau se pregtesc s practice bioterorismul. Este necesar ca guvernele, prin structurile de Sntate Public, mpreun cu Structurile informative i a celor de Aplicare a legii s reacioneze tehnic, prompt i inteligent n eventualitatea suspiciunii unei epidemii, indiferent dac aceast epidemie este natural sau provocat, disjuncie foarte greu de realizat n primele ore de la debut, altfel hotrtoare, n legtur cu evoluiile ulterioare ale epidemiei. Exerciiile Dark Winter (Variola) (2) i Topoff (ciuma) (3), dou exerciii efectuate n SUA, atrag atenia asupra necesitii amplificrii activitii de prevenire i rspuns la atacul bioterorist asupra societii civile n ntregul su.

BIBLIOGRAFIE
1. History of the development and use of weapons. CDR Aylen M.Marty. Clinics in Laboratory Medicine, Vol. 21, No.3, September 2001, 421-423. Ludovic Pun Boli fnfecioase, Arme Biologice, Bioterorism. Ed. Amaltea, Bucureti 2003. Laurence O. Gostin et al The State Emergency Health Power Act for and Response to Bioterorism and Naturally Occuring (infectious Diseases JAMA, August 7, 2002, Vol. 2B8, No. 5, p. 622-625 John C. Cavon The Global Infectious Diseases Threat and its Implications for the Planing Unites States NIE, 99-17D January 2000 Jamis M. Hughes Center for Disease Control and Prevention Atlanta Georgia USA, The Emerging Threat Bioterrorism http;// www.c.d.c. govfricidod/EiDA/ol.15, no.4 hughost btm. (5)

4. 5.

2. 3.

Adres de coresponden: Prof. Dr. Ludovic Pun, Spitalul Clinic de Boli Infecioase i Tropicale Dr. Victor Babe, os. Mihai Bravu, Nr. 281, Sector 3, Bucureti

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INFLUENZA ACTIVITY UNITED STATES AND WORLDWIDE, 2007-08 SEASON*
Abstract

* Articol preluat din: Morbidity and Mortality Weekly Report. www.cdc.gov/mmwr. Vol. 57, No. 25, June 2008

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REZUMAT
n cursul sezonului gripal 2007-2008, activitatea gripei a atins punctul culminant n Statele Unite la mijlocul lunii februarie i a fost asociat cu o mortalitate i o frecven mare a internrilor n spital a copiilor ntre 0-4 ani, n comparaie cu cele trei sezoane anterioare. n Statele Unite, gripa A (H1N1) a predominat la nceputul sezonului; virusurile grupei A (H3N2) i-au intensificat circulaia n luna ianuarie i au predominat peste tot, n timp ce gripa cu virusurile A (H1N1), A (H3N2) i B circulau la nivel mondial. Gripa cu virusul A (H1N1) a fost mult mai frecvent raportat n Canada, Europa i Africa, iar gripa cu virusul B a fost predominant n rile Asiei. Acest raport sintetizeaz activitatea gripal n Statele Unite i la nivel mondial n cursul sezonului gripal 2007-2008 (30 septembrie 2007 - 17 mai 2008).

The Morbidity and Mortality Weekly Report (MMWR) Series is prepared by the Centers for Disease Control and Prevention (CDC) and is available free of charge in electronic format. To recive an electronic copy each week, send an email message to listserv@listserv.cdc.gav. The body content should read SUBscribe mmurtoc. Electronic copy also is available from CDCs Internet server at http://www.cdc.glov/mmwr or from CDCs file transfer protocol server at ftp:// ftp.cdc.govpublications/mmwr. Paper copy subscriptions are available through the Superimendent of Documents, U.S. Government Printing Office, Washintong, DC 20402; telephone 202-512-1800. Data in the weekly MMWR are provisional, based on weekly reports to CDC by state health departments. The reporting week concludes at clos of bussines on Friday; compiled data on a national basis are officially released to the public on the following Friday. Data are complied in the National Center for Public Health Informatics,

Division of Integrated Surveillance System. Address all inquiries about the MMWR Series, including material to be considered for publication, to Editor, MMWR Series. Mailstop E-90, CDC, 1600 Clifron Rd., N.E., Adanta, GA 30333 or to mmwrq@cdc.gov. All materia in the MMWR Series is in the public domain and may be used and reprinted without permission; citation as to source, however, is appreciated. Use of trade names and commercial sources is for identification only and does not imply endorsement by the U.S. Department of Health and Human Services. References to non-CDC sites on the Internet are provided as a service to MMWR teaders and do not constitute or imply endorsement of these organizations or their programs by CDC or the U.S. Department of Health and Human Services. CDC is not responsable for the content of these sires. URI, addresse listed in MMWR were current as of the date of publication.

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*

Abstract

* Articol preluat din: Emerging Infectious Disease. www.cdc.gov/eid. Vol. 14, No. 8, August 2008

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REZUMAT
La nivel mondial, planificarea n gripa pandemic evolueaz bine; medicamentele antivirale i vaccinurile domin agenda terapeutic. Au fost depuse mai puine eforturi pentru planificarea i stocarea medicaiei antibacteriene, util n pneumoniile bacteriene secundare, asociere agravant n evoluia gripei i cauza direct a decesului n epidemiile i pandemiile anterioare. n pandemii, medicaia antimicrobian se impune pentru a se obine un beneficiu substanial n sntatea public. Vaccinarea cu vaccinurile pneumococice polizaharidice i conjugate este considerat ca parte a strategiei pandemice. Multe dintre decesele prin grip pot s apar n rile n curs de dezvoltare, n care inhibitorii de neuraminudaz i vaccinurile nu sunt nici la ndemn i nici disponibile. Astfel, n comparaie cu rile industrializate, beneficiul tratrii complicaiilor bacteriene n rile n curs de dezvoltare ar putea fi mult mai mare.

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Abstract

* Articol preluat din: Emerging Infectious Diseases. www.cdc.gov/eid. Vol. 14, No. 8, August 2008

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REZUMAT
Decesele din timpul pandemiei de grip 1918-1919 au fost atribuite unei sue hipervirulente de grip (virus). Pregtirile pentru pandemia urmtoare se concentreaz aproape n exclusivitate pe prevenirea prin vaccin ca msur preventiv i tratament antiviral pentru noua su de virus gripal. Cu toate acestea, noi emitem ipoteza c infeciile cu su pandemic de virus provoac o boal autolimitant n general (rar fatal), boal care favorizeaz sue colonizante de bacterii, capabile s produc pandemii severe asociate cu mortalitate ridicat. Aceast ipotez secvenial este concordant cu pandemia1917-1918, cu opinia experilor contemporani i cunotinele curente cu privire la efectele fiziopatologice ale virusurilor gripale i interaciunile acestora cu bacteriile respiratorii. Aceast ipotez sugereaz oportuniti de prevenire i tratament n cursul urmtoarei pandemii (cu vaccinuri antimicrobiene i medicamente antimicrobiene), n special dac vaccinul pandemic specific de su nu este disponibil sau este inaccesibil pentru cei izolai, mulimi sau populaii fr sau cu puin asisten medical.

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Abstract

* Articol preluat din: Emerging Infectious Disease. www.cdc.gov/eid. Vol. 14, No. 8, August 2008

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REZUMAT
Pentru a determina riscul potenial pentru transmiterea de la psri la om a gripei A (A5H1), n cursul epidemiei printre psrile de curte n mediul rural din Cambodgia, am colectat mostre din mediul nconjurtor. RNA-ul viral a fost detectat n 27 (35%) din 77 de mostre de noroi, ap din eleteu, plante de ap i cruste din sol. Rezultatele noastre descurajeaz nevoia de dezinfecie regulat n ariile cu psri.

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Abstract

* Articol preluat din: Emerging Infectious Diseases. www.cdc.gov/eid. Vol. 14, No. 8, August 2008

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REZUMAT
Lebedele adulte sntoase au fost infectate experimental cu virusul gripal aviar nalt patogen A /Cygnus cygnus/ Germany/R 65/ 2006, subtipul H5N1. Psrile naive imunologic au murit, n timp ce acelea cu anticorpi naturali virus specifici preexisteni au devenit infectate asimptomatic i purttoare de virus. Lebedele adulte sunt nalt susceptibile, execret virus i pot fi protejate clinic prin imunitatea obinut anterior.

Abstract

* Articol preluat din: Emerging Infectious Diseases. www.cdc.gov/eid. Vol. 14, No. 8, August 2008

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REZUMAT
n data de 21 aprilie 2008, patru lebede iptoare au fost gsite moarte pe Lacul Towada, Prefectura Akito, Japonia. A fost izolat din specimenele recoltate de la psrile afectate virusul gripal nalt patogen A, subtipul H5N1. Gena hemaglutininei (HA) ale izolatelor aparine cladei 2.3.2., din arborele filogenetic HA.

Abstract

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REZUMAT
Noi am revzut informaiile din baza de date ale managerilor beneficiari ntre 2004 i 2005, perioad cu activitate gripal mic. Am calculat rata de prescripii ale Oseltamivirului pentru cei nscrii. Rata prescrierilor a crescut semnificativ de la 27,3%/100.000 n 2004, la 134/100.000 n 2005 (p<0,05), ceea ce sugereaz c a aprut stocarea Oseltamivirului.

All material published in Emerging Infectious Diseases is in the public domain and may be used reprinted without special permission; proper citation, however, is required.

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*

Abstract

* Articol preluat din: Emerging Infectious Diseases. www.cdc.gov/eid. Vol. 14, No. 10, October 2008

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REZUMAT
n rile de jos, Olanda, majoritatea pregtirilor planificate pentru o epidemie sau pandemie constau n meninerea serviciilor publice eseniale, de exemplu poliia, departamentul pompierilor, personal organizat din cadrul armatei i muncitorii sanitari. Noi asigurm estimrile pentru vrful cererii pentru muncitorii sanitari, apreciem absenteismul printre muncitorii din sntate i folosim estimrile din modelele epidemiologice publicate n relaie cu impactul pe vrful valului capacitii facilitilor de ngrijiri de sntate i unitile de terapie intensiv (ICUS). Folosind scenarii variate publicate, noi estimm eforturile lor n creterea disponibilitii ngrijitorilor de sntate pentru sarcinile unei pandemii. Noi artm c n vrful epidemiei tuturor pacienilor care necesit internri n spital i n unitile de terapie intensiv li se poate asigura internarea, inclusiv a acelora care sufer de alte maladii dect gripa sau complicaiile acesteia. Pentru aceast difereniere riguroas sunt eseniale: ierarhizarea clar a managementului, comunicarea/informarea neechivoc i disciplinarea; noi recomandm informarea i instruirea muncitorilor din sntate, alii dect cei care lucreaz n departamentele de terapie intensiv despre sarcinile din unitile de terapie intensiv.

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INFLUENZA VIRUS (H5N1) IN LIVE BIRD MARKETS AND FOOD MARKETS, THAILAND*
Alongkorn Amonsin, Chuensakon Choatrakol, Jiradej Lapkuntod, Rachod Tantilertcharoen, Roongroje Thanawongnuwech, Sanipa Suradhat, Kamol Suwannakarn, Apiradee Theamboonlers, and Yong Poovorawan

ABSTRACT
A surveillance program for influenza A viruses (H5N1) was conducted in live bird and food markets in central Thailand during July 2006August 2007. Twelve subtype H5N1 viruses were isolated. The subtype H5N1 viruses circulating in the markets were genetically related to those that circulated in Thailand during 20042005.

In Thailand, from 2004 through 2008, 6 major outbreaks of avian influenza occurred (January March 2004, JulyOctober 2004, October December 2005, JanuaryMarch 2006, NovemberMarch 2007, and January 2008). We report on a 14-month avian influenza surveillance program and its finding of influenza virus (H5N1) in live bird and food markets in Thailand. The Study From July 2006 through August 2007, an influenza (H5N1) surveillance program was conducted in live bird and food markets in 10 provinces of central Thailand (Figure 1). Cloacal swabs (n = 381) were sampled from live chickens, ducks, pigeons, and house sparrows. Visceral organs and bird meats (n = 549) were collected from carcasses of chickens, ducks, quails, water cocks, water hens, swamp hens, crakes, parakeets, and moor hens at local food markets (Tables 1, 2). An average of 4 markets (range 16) were visited each month, and 18 samples were collected from each market. All samples were from backyard animals or local meat birds. None were from birds from standard farming systems with high biosecurity. The viruses were propagated by embryonated egg inoculation ( 1). Allantoic fluids were tested

for influenza subtype H5N1 by hemagglutination (HA). Multiplex reverse transcriptionPCR (RTPCR) was performed to amplify H5, neuraminidase (N) 1, and matrix (M) genes from HA-positive samples (2 ). Influenza virus (H5N1) was identified in 12 of 930 samples tested. In November 2006, a total of 5 samples with influenza virus (H5N1) were isolated from 1 healthy chicken and 4 visceral organs obtained from 1 live bird market (chicken) and 3 different food markets (moor hen, water cock, and quail). In December 2006, a total of 5 samples with influenza virus (H5N1) were isolated from 5 visceral organs (quail, water cock) from 1 food market. In January 2007, a total of 2 samples with influenza virus (H5N1) were isolated from 2 healthy ducks obtained from 1 live bird market. In the study, 7 isolates were sequenced for whole genome analysis, and the remaining 5 samples were sequenced for H5 and N1 genes. The respective viruses were designated as A/moorhen/ Thailand/CU-317/2006 (GenBank accession nos. EU616825EU616826), A/moorhen/Thailand/ CU-318/2006 (EU616827EU616828), A/ watercock/Thailand/CU-319/2006 (EU616829 EU616830), A/quail/Thailand/CU-320/2006 (EU616831EU616832), A/chicken/Thailand/ CU-321/2006 (EU616833EU616834), A/quail/

Author affiliation: Chulalongkorn University, Pathumwan, Bangkok, Thailand * Articol preluat din: Emerging Infectious Diseases. www.cdc.gov/eid. Vol. 14, No. 11, November 2008

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333/2006 (EU616875EU616882), A/watercock/ Thailand/CU-334/2006 (EU61683EU616890), A/duck/Thailand/CU-328/2007 (EU616835 EU616842), and A/duck/Thailand/CU-329/2007 (EU616843EU616850). To analyze the isolates, nucleotide sequences were compared with those of influenza subtype H5N1 viruses in Thailand, Peoples Republic of China, Vietnam, Indonesia, Lao, Myanmar, and Cambodia. The sequences were aligned by using the DNASTAR program ( 3 ) to elucidate and compare the genetic changes. Phylogenetic analysis was conducted by applying the PAUP program ( 4) with the neighbor-joining algorithm and using branch swapping and bootstrap analysis with 1,000 replicates. Conclusions In the course of the 14-month surveillance program, we isolated influenza virus (H5N1) from 12 samples from live birds and from bird meats obtained from the markets. Bird meats were the source of 9 virus-containing samples (5 quail, 2 moor hens, and 2 water cocks), which indicates a risk for influenza virus (H5N1) contamination in bird meats, especially quail. In addition, 3 highly pathogenic avian influenza viruses were isolated from healthy live poultry (1 chicken and 2 ducks). However, the samples that contained influenza virus subtype H5N1 were detected only during the 3-month winter season (NovemberJanuary). A possible explanation for virus contamination in live bird and food markets may be animal movement from outbreak areas to the markets as

Figure 1 A) Poultry at live bird market; B) house sparrows at live bird market; C) chicken meat at food market; D) moor hen meat at food market.

Thailand/CU-330/2006 (EU616851EU616858), A/quail/Thailand/CU-331/2006 (EU616859 EU616866), A/quail/Thailand/CU-332/2006 (EU616867EU616874), A/quail/Thailand/CU-

Table 1 Test results for samples collected during influenza virus (H5N1) surveillance program in live bird and food markets, by collection date, central Thailand

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Table 2 Test results for samples collected during the influenza virus (H5N1) surveillance program in live bird and food markets, by bird species, central Thailand

well as an attempt to sell infected (dead or dying) birds, especially quail, as bird meat. In addition, most animals or meats in the markets came from backyard farms, where they were in unavoidably close contact with wild birds. Phylogenetic analysis of the virus HA and NA genes indicated that all 12 subtype H5N1 isolates were part of the Vietnam and Thailand lineage (clade 1). The viruses were closely related to those investigated in Thailand (20042005) as well as

to other subtype H5N1 isolates in clade 1. In contrast, they differed from influenza subtype H5N1 viruses of the south China and Indonesia lineages (clade 2) (Figure 2). In this study, we did not discern any Thailand isolates closely related to the south China lineage, as previously established in Lao and Cambodia (5). Phylogenetic analysis of 6 remaining genes showed them to be also closely related to the Vietnam and Thailand isolates.

Figure 2 Phylogenetic analysis of the hemagglutinin (A) and neuraminidase genes (B) of influenza virus (H5N1) isolates. Phylogenetic trees were generated by using the PAUP computer program (4) and applying the neighbor-joining algorithm with branch swapping and bootstrap analysis with 1,000 replicates. The trees were rooted to A/goose/China/ Guangdong/1/96 (H5N1).

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Analysis of the deduced amino acid sequences of the HA and NA proteins indicated that the viruses had characteristics of highly pathogenic avian influenza. The HA cleavage site consists of multiple basic amino acids RERRRKKR (in 1 isolate, CU-329, REKRRKKR). All influenza subtype viruses harbor Glu-222 and Gly-224 at the receptor binding site, indicating preferential binding to the avian receptor SA--2, 3-Gal. In addition, the virus sequences contain 7 glycosylation sites as previously identified in most isolates from Thailand (6 ). A glycosylation site adjacent to receptor binding sites may help increase virus infectivity in host cells (7). In some isolates, polymorphisms of amino acids related to antigenic properties of the viruses at position V86A, L138Q, and K140N were observed. All 12 subtype H5N1 viruses had a 20-aa deletion in the NA protein, typical for the NA stalk region of recent subtype H5N1 isolates (20032007) ( 8,9 ). None of the subtype H5N1 isolates had any amino acids indicating oseltamivir resistance at the crucial positions 119 (E), 275 (H), 293 (R), and 295 (N) of the NA protein. In summary, the 12 viruses isolated from this study were similar to the viruses from other sources in Thailand, which indicates that the viruses are endemic to Thailand, are circulating in the country, and can be found in any exposed species. The route of influenza virus (H5N1) introduction into the markets remains to be established. We suspect that this contamination might have occurred as a consequence of animal movement from outbreak areas or from viruscontaminated cages, trucks, and equipment.

Unfortunately, the original sources of animals in the markets could not be identified because birds from different sources were housed in 1 or several cages. Fortunately, no human infection was found during 20072008 in those provinces where the viruses were isolated. It has been known that live bird and wet markets play a major role in facilitating emergence or reemergence of influenza and some other respiratory diseases ( 1012). Monitoring of live bird and food markets as an early warning system should be implemented in Asian countries where such markets are still commonplace, and routine surveillance of these markets should be conducted year-round. In addition, raw bird meats should be handled with caution, and consumption of raw bird meats should be avoided. Increased public awareness about the risks for influenza virus (H5N1) in association with live bird and food markets will help prevent and control subtype H5N1 infection in humans. Acknowledgment We thank Petra Hirsch for reviewing the manuscript. This study was supported by grants from the National Research Council of Thailand and Chulalongkorn University, Rachadaphiset Shomphod Endowment Fund (Chulalongkorn University) for fiscal year 2006, and the Thailand Research Fund, Senior Research Scholar. Dr Amonsin is an associate professor in Veterinary Public Health at Chulalongkorn University, Bangkok. His research interests include molecular epidemiology of emerging diseases.

REFERENCES
1. 2. World Organisation for Animal Health Manual of standards for diagnostic tests and vaccines, 4th ed. Paris: The Organisation; 2000. Payungporn S, Phakdeewirot P, Chutinimitkul S, Theamboonlers A, Keawcharoen J, Oraveerakul K, et al Single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) for influenza A virus subtype H5N1 detection. Viral Immunol. 2004;17:58893. PubMed DOI Burland TG DNASTARs Lasergene sequence analysis software. Methods Mol Biol. 2000;132:7191. Swofford DL. PAUP Phylogenetic analysis using parsimony (*and other methods), version 4. Sunderland (MA): Sinauer Associates; 2002. Boltz DA, Douangngeun B, Sinthasak S, Phommachanh P, Rolston S, Chen H, et al H5N1 influenza viruses in Lao Peoples Democratic Republic. Emerg Infect Dis. 2006;12:15935. Amonsin A, Chutinimitkul S, Pariyothorn N, Songserm T, Damrongwantanapokin S, Puranaveja S, et al Genetic characterization of influenza A viruses (H5N1) isolated from 3rd wave of Thailand AI outbreaks. Virus Res. 2006;122:1949. PubMed DOI 7. Matrosovich M, Zhou N, Kawaoka Y, Webster R The surface glycoproteins of H5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties. J Virol. 1999;73:114655. 8. Obenauer JC, Denson J, Mehta PK, Su X, Mukatira S, Finkelstein DB, et al Large-scale sequence analysis of avian influenza isolates. Science. 2006;311:157680. PubMed DOI 9. Viseshakul N, Thanawongnuwech R, Amonsin A, Suradhat S, Payungporn S, Keawchareon J, et al The genome sequence analysis of H5N1 avian influenza A virus isolated from the outbreak among poultry populations in Thailand. Virology. 2004;328:16976. PubMed DOI 10. Liu M, He S, Walker D, Zhou N, Perez DR, Mo B, et al The influenza virus gene pool in a poultry market in south central China. Virology. 2003;305:26775. PubMed DOI

3. 4.

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Wang M, Di B, Zhou DH, Zheng BJ, Jing H, Lin YP, et al Food markets with live birds as source of avian influenza. Emerg Infect Dis. 2006;12:17735. 12. Webster RG Wet marketsa continuing source of severe acute respiratory syndrome and influenza? Lancet. 2004;363:2346. PubMed DOI

Address for correspondence: Yong Poovorawan, Center of Excellence in Viral Hepatitis Research, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand; email: yong.p@chula.ac.th

REZUMAT
Un program de Supraveghere pentru Virusul Gripal A (H5N1) a fost efectuat la psrile vii i pieele alimentare din centrul Tailandei n intervalul iulie 2006 - august 2007. Au fost izolate 12 subtipuri de virusuri H5N1. Subtipul de virus H5N1 care circula n piee era nrudit genetic cu cel care a circulat n Tailanda n cursul anilor 2004-2005.

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REPLACEMENT OF SUBLINEAGES OF AVIAN INFLUENZA (H5N1) BY REASSORTMENTS, SUB-SAHARAN AFRICA*
Ademola A. Owoade,1 Nancy A. Gerloff,1 Mariette F. Ducatez,2 Jolaoso O. Taiwo, Jacques R. Kremer, and Claude P . Muller

ABSTRACT
Eight new full-length sequences from highly pathogenic avian influenza viruses (H5N1) from 4 states in southwest Nigeria were analyzed. All gene sequences were more closely related to the first strains found in Nigeria in 2006 than to any strain found outside the country. Six viruses had evolved by at least 3 reassortment events (ACHA/NS, ACNS) from previously identified sublineages A (EMA 2) and C (EMA 1). Our results suggest that highly pathogenic avian influenza viruses (H5N1) initially imported into Nigeria in 2006 have been gradually replaced by various reassortments. In all reassortants, nonstructural genes were derived from sublineage C with 2 characteristic amino acids (compared with sublineage A). If the high prevalence of reassortants was typical for West Africa in 2007, the absence of such reassortants anywhere else suggests that reintroductions of influenza A (H5N1) from Africa into Eurasia must be rare.

Highly pathogenic avian influenza (HPAI) virus subtype H5N1 in Africa was first reported from northern Nigeria in February 2006. Phylogenetic analysis of the complete genome showed that these viruses were clearly distinct from the 2 lineages that were found during the same period in southwestern Nigeria ( 1,2 ). The 3 sublineages (referred to as A, B, and C), 2 of which emerged from a common node, had evolved from subtype H5N1 strains that were originally found around Qinghai Lake in 2005. These strains clustered with viruses isolated from 2006 from southern Russia, Europe, and the Middle East (clade 2.2, www.who.int/csr/disease/influenza/tree_large.pdf) but not with the strains prevalent in southeast Asia ( 3 ). The timeline, the observed influenza A (H5N1) substitution rates in Africa, and the phylogenetic relationship suggested that the sublineages were independently introduced into the country (1,2 ). These sublineages were later found throughout Africa with a distinct geographic distribution (2,4). Sublineage A was also found in Niger and Togo (hemagglutinin [HA] sequence); sublineage B was detected in Egypt and in a human

patient in Djibouti (partial HA sequence), and sublineage C was found in Burkina Faso, Sudan, Cte dIvoire, Ghana (HA and neuraminidase [NA] sequences) (5) and Cameroon (NA sequence) ( 6). Sublineage A strains were also referred to as EMA 2, and both sublineages B and C belong to EMA 1 ( 3). In 2006, one strain with reassorted genes was reported among 35 full-length sequences of the EuropeanMiddle EasternAfrican lineage (14). We describe new HPAI (H5N1) strains collected in southwestern Nigeria during the second half of 2007, most of which were different reassortants of sublineages A and C. Materials and Methods Cloacal swabs were obtained from 8 chicken farms in Lagos (1), Ogun (5), Oyo (1) and Ekiti (1) States from June through November 2007. RNA extraction from cloacal swabs, reverse transcriptionPCR amplification, and gene sequencing were conducted as described (1 ). For most viruses, complete sequences were obtained for all gene segments. Kimura distances were calculated on the basis of complete or partial gene

Author affiliations: University of Ibadan, Ibadan, Nigeria (A.A. Owoade); National Public Health Laboratory, Luxembourg (N.A. Gerloff, M.F. Ducatez, J.R. Kremer, C.P. Muller); and Ogun State Ministry of Agriculture, Abeokuta, Nigeria (J.O. Taiwo) 1These authors contributed equally to this article. 2Current affiliation: St. Jude Childrens Research Hospital, Memphis, Tennessee, USA. * Articol preluat din: Emerging Infectious Diseases. www.cdc.gov/eid. Vol. 14, No. 11, November 2008

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was calculated by using the maximum likelihood method implemented in PAUP 4.0 (7). The substitution model was obtained by using MODELTEST (8). Bootstrap values (%) were calculated with the maximum-likelihood method with 1,000 replications and are indicated on key nodes. Scale bars represent 1% of nucleotide changes between close relatives. A/ duck/Anyang/AVL-1/2001 was used as an outgroup.

sequences by including the maximum sequence length available from all strains included in the comparison. Phylogenetic trees were calculated by using PAUP version 4.0 beta 10 ( 7 ) with the maximum-likelihood method. The best model was determined by using MODELTEST ( 8 ). The sequences have been submitted to GenBank with the accession nos. FM160635FM160642 and FM164800FM164855. Results
Reassortants

All genes of A/chicken/NIE/EKI15/2007 and A/chicken/NIE/OYO14/2007 clustered phylogenetically with sublineage A strains (Figures 1, 2).

The Kimura distances between the genes of these viruses were 0.4%1.4%. Among all subtype H5N1 virus sequences published in the Influenza Sequence Database ( 5 ), NIE/EKI15/2007 and NIE/OYO14/2007 gene sequences were most closely related to those found throughout 2006 and 2007 in Nigeria. Thus, these viruses have most probably evolved from a sublineage A virus initially imported into the country in 2006. This finding is also corroborated by published substitution rates from Africa (2 ). Five viruses had HA and nonstructural (NS) genes grouping with sublineage C virus genes, whereas the other gene segments were most closely related to sublineage A viruses (e.g., A/ chicken/NIE/OG2/2007 and OG5/2007, Figures 1, 2). These viruses evolved by reassortment from sublineages A and C viruses (AC HA/NS reassortment, Figure 2). Another virus (A/chicken/NIE/LAG6/2007) also showed evidence of reassortment between

Figure 1 Phylogeny of hemagglutinin (A) and neuraminidase (B) genes from 8 HPAI (H5N1) viruses collected in Nigeria during the second half of 2007 (p), in comparison with previously identified sublineage A (EMA 2), sublineage B and C (EMA 1), and (EMA 3) strains (1,3 ). The tree

Figure 2 Schematic presentation of sublineage Aderived highly pathogenic avian influenza viruses (H5N1) and reassortants of sublineage A and sublineage C derived viruses identified in Nigeria in 2007. The reassortant reported from Salzberg and others in 2007 (3) is also shown. Sublineage Aderived gene segments are shown in blue; sublineage Cderived gene segments are shown in red. Gene segments are represented in the following order (from top): PB2, PB1, PA, HA, NP, NA, M, NS.

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sublineage A and sublineage C. However, in this virus only the NS gene belonged to sublineage C (Figures 1, 2). The other 7 gene segments of A/ chicken/NIE/LAG6/2007 were derived from sublineage A (ACNS reassortant). Reassortments between Reassortants Four of the ACHA/NS reassortants (A/chicken/ NIE/OG2/2007, A/chicken/NIE/OG4/2007, A/ chicken/NIE/OG10/2007, and A/chicken/NIE/ OG11/2007), all of which were from Ogun State, had similar sequences in all genes (Kimura distances 0%0.7 %). The ACNS reassortant A/ chicken/NIE/LAG6/2007, obtained from a chicken farm in Lagos State, diverged by 0.9 % in the complete NS gene (derived from C lineage) and by 0.7% to 1.4 % in sublineage Arelated gene segments from the latter 4 ACHA/NS reassortants. Some gene segments of the ACHA/NS reassortant A/chicken/NIE/OG5/2007 were most closely related to the other 4 AC HA/NS reassortants, whereas, other gene segments were closer to the AC NS reassortant A/chicken/NIE/LAG6/2007. Matrix protein, HA, NS, NA, and nucleocapsid protein (NP) genes of NIE/OG5/2007 showed a maximal Kimura distance of only <0.4% to ACHA/ NS reassortant genes but a distance of 0.6%1.5% to the ACNS reassortant (A/chicken/NIE/LAG6/ 2007). In contrast, RNA polymerase B protein (PB2), PB1, and PA genes were more closely related to the ACNS reassortant (maximum Kimura distance: <0.6%) than to ACHA/NS reassortants (minimum Kimura distance for the different genes: 0.7%0.8%). For instance, A/chicken/NIE/OG5/ 2007 differed by 12 nucleotides in the PA gene from the most closely related ACHA/NS reassortant (A/chicken/NIE/OG2/2007) but by only 1 nucleotide from the ACNS reassortant (A/chicken/ NIE/LAG6/2007). On the other hand, A/chicken/ NIE/OG5/2007 had 15 nucleotides in the NP gene different from the A/chicken/NIE/LAG6/2007 but only 1 nucleotide difference compared with the closest AC HA/NS reassortant (A/chicken/NIE/ OG11/2007) (Figure 2). This finding strongly suggests that A/chicken/NIE/OG5/2007 is the result of an additional reassortment event involving an exchange of genes between the ACHA/NS and AC NS reassorted viruses. Mutations The amino acid sequences of the HA cleavage site (PQGERRRKKRG) of the strains described here are identical to those of all HPAI (H5N1)

strains reported from West Africa. All viruses had identical amino acids in all positions of the HA protein that are associated with preferential binding to 2,3-linked sialic acid ( 9,10 ) as described ( 2). As for all HPAI (H5N1) strains from Africa, the above viruses had the virulence marker lysine (K) in position 627 of PB2 associated with accelerated viral replication, reduced host defense, higher mortality rate in mice (11), and a wider host range of subtype H5N1 strains (12 ). None of the known markers in the matrix 2 gene associated with resistance to amantadine (13) and in the NA gene associated with resistance to oseltamivir (H274Y) (14 ) were detected. Discussion Gene sequences of all 8 HPAI viruses (H5N1) described here were more closely related to sublineages A or C strains found in Nigeria than to any other published H5N1 virus subtypes. In particular, they were more closely related to the first strains found in Nigeria in the beginning of 2006 than to any strains found outside the country. Thus, the viruses detected in southwestern Nigeria during the second half of 2007 probably evolved from the first viruses brought into the country in early 2006 ( 1), suggesting that HPAI (H5N1) has continuously circulated and is endemic to Nigeria. Sublineage A viruses have continued to circulate in Nigeria, whereas sublineage B was found only once on 1 farm (SO layer farm, Lagos, January 2006), and sublineage C viruses were no longer detected in 2007. Sublineage A viruses have been detected in northeastern Nigeria in February 2007 ( 15) and in 2 states of southwestern Nigeria during the last quarter of 2007 (A/chicken/NIE/EKI15/ 2007 and A/chicken/NIE/OYO14/2007). Sublineages B and C viruses may have been eliminated in Nigeria by effective countermeasures. All ACHA/NS described here were obtained from chicken flocks in Ogun State from June through August 2007. These results are similar to those found in the beginning of 2007 in other states of Nigeria ( 15). In addition, we identified an ACNS reassortant in Lagos State (A/chicken/NIE/LAG6/ 2007) distinct from the latter strain. At least 2 distinct reassortment events were necessary to generate sublineages A and C reassortants AC HA/ NS and AC NS, which probably had occurred already in 2006, as suggested by the conspicuous absence of sublineage C in 2007. Although it is obviously more difficult to demonstrate reassortment events between genetically similar viruses,

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the asymmetry in gene divergence of A/chicken/ NIE/OG5/2007 compared with the other AC HA/NS and AC NS reassortants suggests that additional reassortment events have taken place. In 2006, only 1 reassorted strain was found among 35 EuropeanMiddle Eastern African strains, including 19 viruses reported from Nigeria, belonging to 3 parent sublineages ( 14 ). In the beginning of 2007, 10 of 12 from northern, southern, and central states all belonged to the same AC HA/NS reassortants (15), distinct from the ACPB1/ HA/NP/NS reassortant detected in 2006 (3). Similar reassortants were also found in other regions of subSaharan Africa (unpub. data). During the second half of 2007, we found 6 reassortants including 3 distinct reassortants among 8 strains collected from 8 farms located in 4 contiguous Federal States of Nigeria (Figure 2). These results suggest that reassortants have largely replaced the initial sublineages from which they were derived and that reassortments are pervasive. This finding confirms that reassortments between subtype H5N1 viruses occur frequently when different strains cocirculate in the same region ( 16) and is of particular concern if the increasing prevalence is the result of adaptation to the African environment. Although segments of the replication complex (PB1, PB2, PA, and NP) may reassort individually without affecting viral fitness ( 16), there seems to be a coordinated evolution of the HA and NA genes (17). In all but 1 of the Nigerian reassortants, HA and NA genes originated from different sublineages (C and A), suggesting compatibility between phenotypes of both sublineages. All reassortants from Nigeria included sublineage C derived NS genes, which may suggest a higher fitness of these viruses. Sublineage Cderived NS1 and NS2 sequences from all Nigerian reassortants and 11 unpublished sequences from AC HA/NS reassortants identified in other sub-Saharan regions showed 2 amino acids (NS1 V194 and NS2 R34), which were never identified in sublineage A viruses. It has been shown that modifications in the NS proteins, including amino acids adjacent to V194, may modulate the virulence of HPAI (H5N1) (18 ,19 ). Alternatively, the observation that all reassortants in West Africa have sublineage Cderived NS genes may suggest a better adaptation to the African environment of viruses that came from the cold temperatures of central Asia. Thus, the influence of differences in ecology between Africa and Eurasia on viral selection and dynamics deserves further attention.

Although no reassortments have been reported among clade 2.2 viruses (www.who.int/csr/ disease/influenza/tree_large.pdf) in Central Asia, Europe, and the Middle East since their emergence from Qinghai Lake region in 2005, reassortments of these viruses seem to be rampant in sub-Saharan Africa, where they have become the critical determinant of genetic diversity of HPAI (H5N1). Because of low prevalence, mainly in wild birds, clade 2.2 viruses have few opportunities to reassort in Eurasia. In contrast, opportunities to reassort seem to be frequent in sub-Saharan Africa because of great difficulties in setting up a sensitive surveillance system in a complex socioeconomic environment, where backyard farms and large commercial farms with variable biosafety levels coexist, and where culling may threaten the livelihood and survival of the farm. If the high prevalence of reassortants was typical for West Africa in 2007, the absence of such reassortants anywhere else suggests that reintroductions of subtype H5N1 from Western Africa into Eurasia must be rare. Moreover, all HPAI (H5N1) strains from Nigeria in 2007 were more similar to those found in Nigeria in 2006 than to even the closest relative from Europe in 2007 (Hungary). Although subtype H5N1 has been found in wild birds from Africa, such as vultures ( 4 ), HPAI (H5N1) has so far not been reported in long-distance migrating birds in West Africa. Thus, the exchange of subtype H5N1 between Eurasia and Africa seems to be a rare event, which in 2006 may have been triggered by unusual bird migration as a result of the central Asian cold spell. The biological significance of reassortments between genetically similar viruses may be arguable, but the frequency of reassortment events is an important marker of virus endemicity in a region. Moreover, endemicity of HPAI (H5N1) and a high propensity of reassorting in a region where seasonal influenza is unchecked are essential ingredients of the anticipated pandemic. Acknowledgments We thank the Department of Veterinary Pathology of the University of Ibadan for access to samples from Ekiti and Oyo States, and Emilie Charpentier, Aurlie Sausy, and Sbastian de Landtsheer for their technical help. This study was supported by University of Ibadan Senate Research Grant (SRG/FVM/2006/ 10A) and Bourse Formation Recherche fellowship

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of the Ministry of Research and Higher Education, Luxembourg. The official designation of sublineages A, B, and C can be found in the WHO HPAI (H5N1) clade nomenclature update, to be published soon (www.who.int).

Dr Owoade is a senior lecturer, poultry disease specialist, and consultant to the Veterinary Teaching Hospital of the University of Ibadan, Nigeria. His main field of research is the molecular epidemiology of avian viruses in sub-Saharan Africa.

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REZUMAT
Au fost analizate nou secvene de mrime natural de la virusul gripar aviar nalt patogen (H5N1) din patru state din sud-vestul Nigeriei. n toate secvenele genice au fost mult mai strns legate, surs gsit n Nigeria n 2006, dect de orice alt surs din afara rii. ase virusuri au evoluat la cel puin trei evenimente reassortment (reaezri) (ACHANSIACNS) de la identificarea anterioar a sublineajului A(EMA2) i C(EMA1). Rezultatele noastre sugereaz c virusul aviar nalt patogen (H5N1) importat n Nigeria, n 2006, a fost treptat nlocuit prin variate reaezri. n toate reaezrile genele nestructurale au derivat din sublineajul C cu doi aminoacizi caracteristici (n comparaie cu sublineajul A). Dac prevalena nalt a reasorbanilor a fost tipic pentru Africa de Vest, n 2007, absena acestor reabsorbani oriunde n alt fel sugereaz c reintroducerea gripei A (H5N1) din Africa n Europa trebuie s fie rar.

13
BIBLIOGRAFIE DE CONSULTAT
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1. Current Concept, Update on Avian Infeluenza A (H5N1) - Virus Infection in Humans writing Committee of the Serconds World Health Organization Consultation on Clinical Aspects of Human Infection with Avian Influenza A (H5N1) Virus*. N.Engl. J.Med. 358; 3 www.NEJM.ORG., January, 2008 2. Avian Influenza an Omnipresent Pandemic Threat Daniel R.Perez,PhDt, Erin M.Sorrall, Msc* and Ruber Odonis DVM, PhDt. Supplement Article The Pediatric Infectious Diseases Journal, Vol.24,No.11, November 2005, S 208-216. Review Article. 3. Preparing avian influenza Richard A.Martinello. Currentt opinion in Pediatrics 2007, 19: 64-7 4. Review Article. The Critically ill Avian Influenza A (H5N1) Patient Yaseen Arabi, MD, FCCP; Charles D.Comersall, BSc, MD,FCCP, Bruc R.Boyton, MD, MPH, FAAP; Ziad A.Mernish, MD, FRCPC, FACP, FIDSA. Crit. Care Med. 2007, Vol.35, No.5, pag. 1397-1403 5. A Rationale for Using Steroides in the Treatment of Severe Cases of H5N1 Avian Influenza Marisa J.Carter Journal of Medical Microbiology (2007) 56, pag. 875-883 6. MMWR, Morbidity and Mortality Weekly Report www.cdc.gov/mmwr, Recommendation and Report, August 8, 2008, Vol.57, No.RR-7. Prevention and Control of Influenza Recommendations of the Advisory Committee on Immunization Practices (ACIP), 2008. Department of Health and Human Services Centers for Disease Control and Prevention Contents. Centers for Disease Control and Prevention: Julie L. Gerberding, MD, MPH, Director Tanja Popovic, MD, PhD, Chiev Sicence Officer James W. Stephens, PhD, Associate Director for Science Steven L. Solomon, MD, Director National Center for Health Marketing Editorial and Production Staff: Frederic E. Shaw, MD, JD, Editor, MMWR Series Susan F. Davis, MD (Acting) Managing Editor, MMWR Series David C. Johnoson (Acting) Lead Technical Writer-Editor David C. Johnoson, Project Editor Peter M. Jenkins, (Acting) Lead Visual Information Specialist Lynda G. Cupell, Malbea A. LaPete, Visual Information Specialists Quang M. Doan, MBA, Erica R. Shaver, Information Technology Specialists Editorial Board: William L. Roper, MD, MPH; Chapel Hill, NC, Chairman; Virginia A. Caire, MD, Indianapolis, IN; David W. Fleming, MD, Seattle, WA; William E. Halperin, MD, DrPH, MPH, Newark, NJ; Margaret A. Hamburg, MD, Washington, DC; King K. Holmes, MD, PhD, Seattle, WA; Deborah Holtzman, PhD, Atlanta, GA; John K. Iglehart, Berhesda, MD; Dennis G. Maki, MD, Madison, WI; Sue Mallonee, MPH, Oklahoma City, OK; Stanley A. Plotkin, MD, Doylestown, PA; Patricia Quinlisk, MD, MPH, Des Moines, IA; Patrick L. Remington, MD, MPH, Madison, WI; Barbara K. Rimer, DrPH, Chapel Hill, NC; John V. Rullan, MD, MPH, San Juan, PR; Anne Schuchat, MD, Atlanta, GA; Dixie E. Snider, MD, MPH, Atlanta, GA; John W. Ward, MD, Atlanta, GA

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