M olecular epidemiology of dengue
viruses in Brazil
Ep id e mio lo g ia mo le c ular d o s vírus
d e ng ue no Brasil
1 Laboratório d e Flavivíru s, Dep a rt a m en t o d e Virologia , In st it u t o Osw a ld o Cru z , Fu n d a çã o Osw a ld o Cru z , Av. Bra sil 4365, Rio d e Ja n eiro, RJ 21045- 900, Bra sil.
Rit a M a ria Rib eiro N ogu eira 1 M a riz e Pereira M ia gost ov ich 1 Herm a n n Gon ça lv es Sch a t z m a yr 1
Abst ract D en gu e v iru ses (DEN ) a re fou n d a s fou r a n t igen ica lly d ist in ct serot yp es d esign a t ed DEN - 1, 2, 3, a n d 4. La b ora t ory ev id en ce t h a t st ra in - in t ra t yp ica l v a ria t ion occu rs a m on g DEN v iru ses h a s b een d em on st ra t ed sin ce t h e 1970s, a lt h ou gh on ly w it h t h e a d v a n ces in m olecu la r t ech n ologies h a s it b een p ossib le t o d et erm in e t h e gen et ic va ria b ilit y of ea ch serot yp e. Gen ot yp ca l id en t ifica t ion h a s p rov en t o b e a u sefu l t ool for d et erm in in g t h e origin a n d sp rea d of ep i-d em ics a n i-d t o correla t e v iru len ce of st ra in s. In t h is rep ort w e p resen t t h e resu lt s of m olecu la r ep i d em i ologi ca l st u d i es w i t h t h e D EN - 1 a n d D EN - 2 v i ru ses t h a t ca u sed d en gu e ep i d em i cs i n Braz il d u rin g t h e last d ecad e.
Key words Den gu e Viru ses; Den gu e; Molecu lar Ep id em iology
Resumo Os v íru s d en gu e (DEN ) a p resen t a m p rop ried a d es a n t igên ica s d ist in t a s q u e ca ra ct eri-z a m q u a t ro sorot ip os d en om in a d os DEN - 1, 2, 3 e 4. Desd e a d éca d a d e 70, ev id ên cia s la b ora t o-ria is t êm d em on st ra d o a ocorrên cia d e v a o-ria çã o in t ra t íp ica en t re os v íru s DEN ; en t ret a n t o, so-m en t e coso-m o a v a n ço d a s so-m et od ologia s so-m olecu la res foi p ossív el est a b elecer v a ria n t es gen ét ica s p a ra ca d a sorot ip o. A id en t ifica çã o gen ot íp ica t em sid o u m a im p ort a n t e a bord a gem p a ra d et erm in ar a origeerm e a d isp ersão d e ep id eerm ias e p ara t en t ar est abelecer correlação d e v iru lên cia en t re a s v a ria n t es d os v íru s DEN . N est e t ra ba lh o, a p resen t a m os resu lt a d os obt id os a t ra v és d e est u d os d e ep id em iologia m olecu la r rea liz a d os com a m ost ra s d e v íru s DEN 1 e DEN 2, q u e ca u sa -ram ep id em ias n o Brasil, n a ú lt im a d écad a.
Th e d evelop m en t of n ew tech n iqu es in m olec-u la r b io lo gy h a s m ea n t a h olec-u ge step in va rio olec-u s fie ld s o f scie n ce a n d t e ch n o lo gy, in clu d in g th a t o f h ea lth . Th e p o ssib ility o f p recisely d e-term in in g th e com p osition of m icroorgan ism s’ gen o m es h a s crea ted n ew p ro sp ects fo r ep id em iological stu id ies, allowin g for th e m olecu -lar ch aracterization of circu latin g viral sam p les a n d kn owled ge o f th eir geo gra p h ic d istrib u -tion .
Th e m o lecu la r ep id em io lo gy o f d en gu e viru ses (DEN) h as b een u sed to d eterm in e th e origin of th e viru ses th at h ave cau sed ou tbreaks an d ep id em ics, esp ecially in th e attem p t to es-ta b lish a co rrela tio n b etween th e viru len ce o f sam p les an d th e im p act of th ese viru ses on th e p op u lation .
Th e DEN viru ses b elon g to th e Flavivirid ae
fa m ily ( Westa wa y et a l., 1985) a n d th e Fla -v i-v iru sgen u s, wh ich in clu d es 68 sp ecies in 8
serologicallly related grou p s (4 tran sm itted b y m o sq u ito es, 2 b y ticks, a n d 2 still with o u t kn own vecto rs) a n d a gro u p o f vir u ses th a t is n ot classified with in th ese serogrou p s by n eu -tra liza tio n , in clu d in g th e yellow fever viru s. Th ey d isp lay d ifferen t an tigen ic p rop erties th at ch aracterize th e 4 serotyp es called th e DEN-1, DEN-2, DEN-3, an d DEN-4 viruses (Sabin , 1952; Ha m m o n et a l., 1960). Th ey a re sp h erica l, en -velo p ed viru ses, with a d ia m eter o f a p p roxi-m a tely 40-50 n roxi-m a n d a gen o roxi-m e co n sistin g o f sin gle-stra n d RNA with a p o sitive p o la rity o f so m e 11 kb. Th e vira l RNA is su rro u n d ed by a n u cleo ca p sid with a n ico sa h ed ra l sym m etr y, m a d e u p o f a sin gle so -ca lled C p ro tein , su r-rou n d ed by a d ou b le lip id layer associated with m em b ran e (M) an d en velop e (E) p rotein s. Pro-tein E is th e p rin cip al stru ctu ral p roPro-tein an d is d irectly related to im m u n ity an d p rob ab ly th e viru len ce o f th e sa m p les. Th ese viru ses h a ve seven oth er n on -stru ctu ral p rotein s th at are re-lated to viral rep lication (Brin ton , 1986; Deu b el et al., 1993).
Bro a d d isp ersa l o f th e m o sq u ito vecto r
Aed es a egyp t im a d e d en gu e th e m o st im p o
r-ta n t h u m a n a rb oviru s d isea se in th e wo rld , with a p p roxim a tely 2.5 b illio n in d ivid u a ls ex-p osed to risk of in fection in som e 100 cou n tries with trop ical an d su b trop ical clim ates (Kn u d -sen , 1996).
In fection with an y of th e serotyp es lead s to a feb rile illn ess kn own as d en gu e fever (classic d en gu e). Th e severe form , ch aracterized by th e a p p ea ra n ce o f h em o rrh a ge a n d / o r h yp ov-o lem ic sh ov-o ck, is ter m ed h em ov-o rrh a gic d en gu e (DH F) o r d en gu e sh o ck syn d ro m e (DSS) a n d o ccu rs in so m e 0.5% o f ca ses (OMS, 1987). World wid e estim ates su ggest 100 m illion cases
of d en gu e an d h u n d red s of th ou san d s of DHF p er year, d ep en d in g on ep id em ic activity. From 1981 to 1997, 24 cou n tries in th e Am erica s re-p o rted la b o ra to r y-co n firm ed ca ses o f DH F (Gu b ler, 1998). Th e ca se fa ta lity ra te for DHF/ DSS in th e Am erica s is 1.4%, a lth o u gh wid e va ria tio n (1 to 11.9%) h a s b een rep o rted fro m o n e co u n tr y to a n o th er (Pin h eiro & Ch u it, 1998).
In fection with on e serotyp e con fers p artial a n d tem p o ra r y p ro tectio n a ga in st th e o th er serotyp es, an d secon d ary in fection is p ossib le a fter a rela tively sh o rt p erio d o f tim e (OMS, 1987).
Prior in fection with a given serotyp e is con -sid ered an im p ortan t risk factor for th e d evel-op m en t of DHF/ DSS, alth ou gh th e occu rren ce o f p rim a ry DH F/ DSS ca ses su ggests th a t vira l viru len ce m ay also b e resp on sib le for th e vari-a tion in th e d isevari-a se’s clin icvari-a l exp ression (Hvari-a lstead , 1970; Rosen , 1977; Th ein et al., 1997). In -d ivi-d u al, ep i-d em iological, an -d viral risk factors a re cu rren tly b ein g exp lored in rela tion to th e d evelop m en t of DHF/ DSS (Kou ri et al., 1987).
Antigenic and genetic diversity of dengue viruses
In tratyp ical variation in th e d en gu e viru ses was in itia lly stu d ied by m ea n s of serologica l tech n iq u es th a t d em o n stra ted a n tigen ic a n d b io -lo gica l d ifferen ces b etween sa m p les fro m th e sam e serotyp e (McClou d et al., 1971; Ru ssel & McCown , 1972).
Oth er m eth od ologies, like an tigen ic an aly-sis u sin g a p a n el o f m o n o clo n a l a n tib o d ies (Mon ath et al., 1986), cDNA-RNA h yb rid ization (Blo ck et a l, 1984; Blo ck, 1985), h yb rid iza tio n u sin g syn th etic p ep tid es (Kersch en er et a l., 1986), an d restriction en d on u clease an alysis of RTPCR products (Vorn dam et al., 1994), dem on -stra ted th e a n tigen ic a n d gen etic va ria b ility am on g th e d en gu e viru ses.
(1990), ch a ra cterized five gen o m ic gro u p s fo r th e 1 viru ses an d five grou p s for th e DEN-2 viru ses. Co m p lete seq u en cin g o f th e gen e cod in g for p rotein E in th e DEN-2 viru s (Lewis et a l., 1993) esta b lish ed five su b typ es co rre-sp on d in g essen tially to th ose su ggested by Ri-co-Hesse (1990). Partial sequ en cin g of d ifferen t sa m p les o f DEN3 a n d DEN4 vir u s d em o n -stra ted th e existen ce o f fo u r a n d two gen etic su b typ es, resp ectively (La n cio tti et a l., 1994; 1997).
Dengue viruses in Brazil
Rein festation of Brazil by Aed es aegyp tiin 1977, th e DEN-1 viru s p a n d em ic, a n d th e in trod u c-tio n o f th e DEN-4 viru s in to th e Am erica s m arked th e rein trod u ction of th e DEN viru ses in to Brazil. In 1981, th e first sam p les of DEN-1 an d DEN-4 viru ses were isolated in an ou tbreak in Bo a Vista , Ro ra im a (Osa n a i et a l., 1983). However, it wa s o n ly fro m 1986 o n wa rd th a t d en gu e b eca m e a n a tio n wid e p u b lic h ea lth p rob lem , with th e in trod u ction of DEN-1 viru s in to th e Sta te o f Rio d e Ja n eiro a n d its su b se -q u en t sp rea d to va rio u s o th er Sta tes o f th e co u n tr y (Sch a tzm a yr et a l., 1986; Figu eired o, 1996).
Th e situ ation was aggravated in 1990 by th e in tro d u ctio n o f DEN-2 vir u s in to th e Sta te o f Rio d e Jan eiro (Nogu eira et al., 1990). Difficu lty in im p lem en tin g a n effective n a tion wid e vec-to r co n tro l p ro gra m resu lted in th e ra p id sp rea d o f th e viru s a n d co n seq u en tly th e o c-cu rren ce o f ep id em ics in va rio u s Sta tes. Cu r-ren tly, 22 of th e 26 Brazilian States h ave rep ort-ed d en gu e ep id em ics, totalin g som e 1,513,784 ca ses; fro m its in tro d u ctio n in 1986 u n til th e 35th ep id em iological week of 1999, sim u ltan e-ou s circu lation of th e DEN-1 an d DEN-2 viru s-es wa s co n firm ed b y la b o ra to r y a n a lysis with vira l iso la tio n in 16 Sta tes. In 1998, Bra zil a c-cou n ted for som e 85% of th e d en gu e cases re-p orted in th e Am ericas.
M olecular epidemiology of dengue viruses in Brazil
Th e growin g activity of DEN viru ses in Brazil in th e 1980s led to th e estab lish m en t of a Nation -a l Den gu e Di-a gn o sis Netwo rk (Sch -a tzm -a yr et al., 1996), with th e im p lem en tation of p rim ary d iagn ostic m eth od s, in clu d in g th e sp ecific an -tib o d y ca p tu re im m u n o en zym a tic test (Ma c-Elisa), rou tin e serology, an d th e u se of Aed es al-bop ictu s clon e C6/ 36 cell lin e an d m on oclon al a n tib o d ies fo r vira l iso la tio n (Iga ra sh i, 1978; Gu b ler et al., 1984).
Wid esp rea d a p p lica tio n o f th ese m eth o d -o l-o gies, u sin g sera fr-o m p a tien ts, p r-o d u ced h u n d red s of sam p les of th e DEN-1 an d DEN-2 viru ses, th e on ly on es circu latin g in th e cou n try in th e la st 12 yea rs (Mia go stovich et a l., 1993; Nogu eira et al., 1993; 1999). Du rin g th is p eriod , th e Flaviviru s Lab oratory of th e Oswald o Cru z In stitu te, th ro u gh a m o lecu la r ep id em io lo gy p rogram , estab lish ed d ifferen t m eth od ologies for gen etic an alysis of th ese sam p les.
An alysis was in itially p erform ed on gen om e fragm en ts from DEN-1 an d DEN-2 viru ses iso-la ted in th e Sta te o f Rio d e Ja n eiro, u sin g re-striction en d on u clease on th e Hae III en zym e. Th is in vestiga tio n wa s d irectly a p p lica b le to th e d eterm in ation of circu latin g viru ses, id en -tifyin g th e Ca rib b ea n a n d Ja m a ica gen o typ es for th e DEN-1 an d DEN-2 viru ses, resp ectively (Vorn d am et al., 1994). Th ese resu lts were con -firm ed by th e p artial seq u en cin g of a fragm en t of th e gen e cod in g for th e en velop e (E) p rotein , b etween n u cleotid es (n ts) 85 an d 282, after exten sion an d am p lification by reverse tran scrip -tion followed by th e p olym erase ch ain reac-tion (RT-PCR) (Deu b el et a l., 1993; Ch u n gu e et a l., 1995). Com p arison of th e DEN-2 viru ses isolat-ed in Rio d e Ja n eiro (two sa m p les o b ta in isolat-ed from classic d en gu e cases an d on e sam p le iso la ted fro m a fa ta l ca se) sh owed th e sa m e se -q u en ce of n u cleotid es, h en ce with n o id en tifi-ca tio n o f m a rkers fo r viru len ce in th e regio n stu d ied (Deu b el et al., 1993).
A secon d p h ase in clu d ed th e sequ en cin g of th e region en com p assed b etween n ts 1685 an d 2504 in th e p ro tein E gen e fro m sa m p les o f DEN-2 viru s iso la ted in th e Sta tes o f Rio d e Jan eiro (RJ), Ceará (CE), Bah ia (BA), an d Alago-a s (AL), in o rd er to in vestigAlago-a te th e gen o typ es circu la tin g in Bra zil in 1990-1995. An a lysis o f th ese resu lts, sh own in Figu re 1, con firm ed th e origin of th e DEN-2 viru ses circu latin g in Brazil a n d id en tified th e p resen ce of on ly on e gen o -typ e ( Ja m a ica ) a s o f 1995, d em o n stra tin g th e sp read of th is viru s from Rio d e Jan eiro to oth er States. Th ere was also great u n iform ity am on g o u r sa m p les, wh ich u n d er wen t few m o d ifica -tion s over th e cou rse of th e years in wh ich th ey circu lated in th e cou n try. Th e sam p les isolated in Rio d e Jan eiro in 1995 were th e on ly on es to p resen t a la rger n u m b er o f a ltera tio n s in th e n u cleic a cid b a ses, reflectin g th e evo lu tio n o f th e DEN-2 viru ses sin ce th eir in trod u ction in to th e State (Miagostovich et al., 1998).
affect-in g va rio u s a re a s o f t h e Ca rib b e a n affect-in 1981. Th is con firm s th at th e DEN1 an d DEN2 sam -p les circu la tin g in Bra zil o rigin a ted in th e Carib b ean , reach in g th e cou n try th rou gh in fe-cted p erson s or vectors (Rico-Hesse, 1990).
Th e Ja m a ica gen o typ e rep resen ts sa m p les wit h a h igh p o t e n t ia l fo r ca u sin g se ve re d is-ease, esp ecially in areas wh ere th e DEN-1 an d DEN-4 viru ses circu lated p reviou sly (Vorn d am
et al., 1994). Its in trod u ction in to th e Am ericas re su lt e d in b o t h a n in cre a se in t h e se ve re fo r m s o f t h e d ise a se a n d va rio u s e p id e m ics ( Ve n e zu e la , 1989; Bra zil, 1990-91) in wh ich t h e re wa s a sign ifica n t n u m b e r o f DH F/ DSS ca se s. Un t il t h e 1980s, ca se s o f DH F/ DSS in t h e Am e r ica s we re sp o ra d ic a n d a sso cia t e d with th e Pu erto Rico gen otyp e, isolated for th e first tim e in 1953 in Trin id a d a n d resp o n sib le Ne w G uine a C – 44 Sri Lanka – 68 Sri Lanka – 69 Philip p ine s – 83
Taiwan – 87 Thailand – 58 Thailand – 64 Thailand – 80
Malasya – 86 Jamaic a – 83 Brazil – 90 39122 – RJ 1990
40247 – RJ 1990 40678 – RJ 1990 48576 – CE 1994 48577 – CE 1994
48586 – CE 1994 48643 – BA 1994 48844 – BA 1994 49255 – RJ 1995
49367 – RJ 1995 H140700 – AL 1991 H140749 – AL 1991 Ind o ne sia – 76
So malia – 84 Burkina Faso – 83 Se yc he lle s – 77 Sri Lanka – 82
Sri Lanka – 85 Sri Lanka – 89 Sri Lanka – 90 Trinid ad – 53
Ind ia – 57 Pue rto Ric o – 69 To ng a – 74 De ng ue 3 Fig ure 1
Phylo g ram g e ne rate d b y p artial analysis o f the nuc le ic ac id se q ue nc e fro m a frag me nt o f g e ne E fro m 12 Brazilian samp le s o f DEN-2 virus and 24 o b taine d fro m G e ne b ank.
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Acknowledgments
Th e au th ors th an k Con selh o Nacion al d e Desen volvi-m e n to Cie n tífico e Te cn o ló gico (CNPq ), Fu n d a çã o Oswa ld o Cru z (FIOCRUZ), Coord en a çã o d o Sistem a Nacion al d e Lab oratórios d e Saú d e Pú b lica d o Min is-tério d a Saú d e, In tern ation al Develop m en t Research Ce n tre / Ca n a d a , a n d Fu n d a çã o Ba n co d o Bra sil fo r th e ir fin a n cia l su p p o rt a n d th e Ce n te rs fo r Dise a se Co n tro l a n d Pre ve n tio n (CDC) in Pu e rto Rico a n d Fo rt Co llin s, USA, a n d th e Pa steu r In stitu te in Pa ris fo r th e ir re se a rch co lla b o ra tio n in th e m o le cu la r ch aracterization of sam p les.
fo r e xt e n sive e p id e m ics in t h e 1960s a n d 70s (Gu b ler, 1992).
Th e gen o m ic a n a lysis recen tly p erfo rm ed by Rico-Hesse et al. (1997) p roved th e existen ce o f a n ew va ria n t o f th e DEN-2 viru s in th e Am ericas, related to sam p les circu latin g in Asia a n d h igh ly viru len t wh en in vo lved in sec-on d ary in fectisec-on s. Circu latisec-on of th is varian t in 1994, 1995, a n d 1996, esp ecia lly in Ven ezu ela , resu lted in a la rge n u m b er o f DH F/ DSS ca ses an d h igh case fatality.
In Bra zil, in fection s by DEN-2 viru ses p re-sen ted d ifferen t clin ical p attern s, p rin cip ally in rela tio n to th e severity o f th e d isea se. In re-gion s wh ere th e DEN-2 viru s accou n ted for p ri-m a ry typ e in fection s, a s in th e Sta tes of Ba h ia a n d Esp írito Sa n to (No gu eira et a l., 1995), th e clin ica l p ictu re wa s typ ica l o f cla ssic d en gu e, with freq u en t exa n th em , p ru ritis, a n d few se-vere cases. However, in oth er States, wh ere th e DEN-2 viru s circu la ted a fter exten sive ep i-d em ics cau sei-d b y DEN-1, as in Rio i-d e Jan eiro, Ceará, Pern am b u co, an d Rio Gran d e d o Norte, an in crease in th e n u m b er of severe cases was o b served . Begin n in g with th e in tro d u ctio n o f DEN-2 viru s in 1990, a to ta l o f 754 DH F/ DSS ca ses were rep o rted , with 34 d ea th s, a s o f th e 39th ep id em io lo gica l week, a n d it is b elieved th a t th ere m a y h a ve m o re ca ses th a t were n o t officially con firm ed (Nogu eira et al., 1990; Za-gn e et al., 1994; Sou za et al., 1995; Vascon celos et al., 1995).
Th e a b ove-m en tio n ed d a ta d em o n stra te th e im p o rta n ce o f o n -go in g m o n ito r in g o f sa m p les o f d en gu e viru ses circu la tin g in th e cou n try in ord er to id en tify th e en tr y of a n ew
serotyp e or gen otyp e a n d a p oten tia l a ssocia-tion b etween gen otyp e an d viru len ce, in ad d i-tion to d efin in g th e p attern of en d em icity in a given a rea . Th e in crea se in a ir tra vel h a s fo s-tered a sp atial sh iftin g of in d ivid u als d u rin g th e virem ic p h a se, fa cilita tin g th e in trod u ction of n ew viru ses an d / or gen otyp es. Molecu lar ch ar-acterization can id en tify th e sim u ltan eou s cir-cu lation of an tigen ically sim ilar (b u t gen etical-ly d istin ct) sa m p les a n d fo llow th eir sp rea d sep arately.
Kn owled ge o f sa m p les circu la tin g in a re-gion also h as im p lication s for th e p oten tial in-tro d u ctio n o f va ccin es, m a kin g it p o ssib le to eva lu a te th e gen o m ic rela tio n s b etween th e va ccin a l viru s a n d th ese sa m p les. Th e sa m e is tru e fo r th e d evelo p m en t o f sero lo gica l d ia gn o stic tests, wh ich sh o u ld ign clu d e vira l sa m -p les id en tica l o r clo se to th o se circu la tin g in th e region .
Th rou gh gen om ic an alysis, it is p ossib le to recon stitu te th e p robable evolu tion of th e viru s. Fo r th e DEN viru ses, va ria n ts were estim a ted fo r th e su b typ es th a t em erged in th e la st 200 yea rs a n d were rela ted to p o p u la tio n growth an d con dition s of ecological im balan ce (Zan ot-to et al., 1996).
Given th e lim ited o p tio n s fo r th e p reven -tion an d con trol of d en gu e ep id em ics, d u e n ot o n ly to th e la ck o f a tetra va len t va ccin e fo r large-scale ap p lication b u t also th e d ifficu lties in erad icatin g th e m osq u ito vector, m olecu lar ep id em io lo gy h a s b eco m e a n in d isp en sa b le tool for id en tifyin g m ore viru len t gen etic vari-a n ts wh ich cvari-a n p o ten tivari-a lly cvari-a u se m o re severe form s of th e d isease.
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