BODY OF REPORT. SEAT0 Medic Study No. 4. Serologic Response to Tha.i Hemorrha.gic Fever Virus Infection. Project No.
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1 BODY OF REPORT SEAT Medic Study No. 4 Project No. 3A 2561 A 811 Tsk 1: Serologic Response to Th.i Hemorrh.gic Fever Virus Infection Mi1it.q Medicl Reserch Prgr.m S. E. Asi. Mi1it.r~ Medic.1 Rese.rch Prgr.m S. E. Asi. Militry Medic 1 Reserch Progrm SEAS LA (Thi lnd) Reporting Ins t.ll.tion: US Army-SEAT Medic 1 Reserch Lbortory APO Sn Frncisco Division of Medicl Reserch Lbortories Dep.rtment of Virology Period Covered by Report: 1 April 1964 to 31 M.rch 1965 Princip.1 1nvestig.tor: Associte Investigtor: Reports Control Symbol: Security Clssifiction: M.jor Scott B. Hlsted, MC Dr. Suchind. Udoms.kdi MEDDH-288 UNCLASSIFIED Objective: To study the serologic response of hum.ns.nd lbortory.nim.1~ to viruses ssoci.ted with Th.i hemorrh.gic fever. Techniques employed include the hemgglutintion-inhibition, complement-fix,tion.nd the neutrliztion test. Description: Dt presented.re concerned with the following.spects of dengue nd chikunguny, serologic response: 1. Primry nd secondry type.ntibody response following clinicl hemorrhgic fever; 1 2. The development nd loss of HI nd CF ntibody in cses of hemorrhgic fever followed for 7 months; 3. The est.blishment of serologic criteri, for recent dengue virus infection for
2 FIGURE 1. ARRAY OF TITERS IN 4 6 REPRESENTATIVE HEMORRHAGIC FEVER CONVALESCENT SERA COMPARING DENGUE 1 WlTH TH SMAN CF ANTIGENS, BANGKOK PATIENTS, 1962 FIGURE 2. ARRAY OF TITERS IN 46 REPRESENTATIVE HEMORRHAGIC FEVER CONVALESCENT SERA COMPARING DENGUE 2 WlTH TH 36 CF ANTIGENS, BANGKOK PATIENTS, DENGUE 1 DENGUE 2 < TH SMAN < I CF TITER * * EXPRESSED AS RECIPROCAL OF SERUM DILUTION < * EXPRESSED AS RECIPROCAL OF SERUM DILUTION
3 residents of Thilnd; 4. Antibody response in we.nling mice to 2 or 3 dengue virus exposures. Acute.nd conv.lescent phse ser, hve collected from 4 groups of p.tients:.. Hos pit.lized hemorrh.gic fever b. P.tients hospitlized with febrile disese other thm hemorrhgic fever c. Out-ptients with miscell.neous febrile syndromes d. Surgic 1 ptients. Acute ph.se p1sm.s were collected gener.11~ before the sixth d.y fter onset of illness. Two weeks l,ter. conv.lescent serum w.s obt.ined. Acetone-ether extrcted hem.gglutintion.nd complement-fixtion.ntigens were used. All hem.gglutintion-inhibition tests were done in microvolumes using Microtiter equipment. The overnight hem.gg1utin.tion-inhibition test,s described by Cl.rke.nd Csls w,s employed. Ser. were tested ginst 4-8 hemgglutin.tion units. Known positive nd negtive hum.n ser. were used in.11 tests. The complement-fixtion (CF) test ws done in microvolumes. Antigens.nd complement titrtions were done in K.hn tubes. Two units of.ntigen.nd two exct units of complement.nd three units of.nti-sheep hemolysin were used. Complement, serum nd sline controls were m.int.ined for e.ch test.s well s known positive ser. which were const.ntly retested over, period of 2 ye.rs. Progress: Selection of serologic.ntiqens. Numerous studies hve been undert.ken to determine the number of distinctly different dengue virus ntigens which could provide useful serologic informtion. In one such study, 46 hemorrhgic fever p.tients from whom 3 or more specimens were vilble were tested.ginst high mouse p.ssge prototype CF dengue 1-4 nd TH-36.nd TH-Smn. Figure 1 nd 2 show tht for this p.rticuh.r group of ser the correl.tion of nt'ibody titers between dengue 1.nd TH-Sm.n or between dengue 2.nd TH-36 is good. The ntigenic similirity or dissimili.rity of dengue 1 with TH-Sm.n.nd dengue 2 with TH-36 were studied further by-ex,mining conv.lescent ser, from ptients showing rel.tively specific CF.ntibody response (prim.ry infection?)who cquired hemorrhgic fever in sm.11 communities of R.yong.nd Ubol. In R.yong, eleven type 2 viruses were recovered from Aedes.eqypti. In Ubol, twelve type 1 viruses were recovered from thirty four ser, tested. Distribution of.ntibody in convlescent ser, me.sured by type 1 nd TH-Sm,n.ntigens or type 2.nd TH-36.ntigens re indic.ted in Figure 3,nd 4. These studies hve been extended to sever.1 hundred other hum.n nd.nim.1 ser., ll with essentilly the sme result. In our experience results with dengue 1 or TH-Sm.n nd dengue 2 or TH-36 did not
4 FIGURE 3. ARRAY OF TITERS IN 23 REPRESENTATIVE CONVALESCENT SERA FROM HEMORRHAGIC FEVER PATIENTS IN UBOL, THAILAND. DENGUE 1 (HAWAID COMPARED WITH TH SMAN CF ANTIGENS. FIGURE 4. ARRAY OF TITERS IN 1 REPRESENTATIVE CONVALESCENT SERA FROM HEMORRHAGIC FEVER PATIENTS IN RAYONG, THAILAND. DENGUE 2 (NEW GUINEA C) COMPARED WITH TH 36 ANTIGENS. 3 DENGUE 1 ~ 6 L s L 3 U LC o 4 J LC m 3 3 J s m 1 Y s 2 2,, $ 1 7 s DENGUE 2 ( < J s 2 2 f 7 s U 6 L LC 5 m 1 m - 4 o 4 C J 2 m J 2 3 s ; r 2 < * EXPRESSED AS RECIPROCAL OF SERUM DILUTION ( * EXPRESSED AS RECIPROCAL OF SERUM DILUTION
5 FIGURE 5. SCATTER DIAGRAM OF HI ANTIBODY IN ACUTE AND CONVALESCENT SERA OF CHILDREN -1 AND 1-2 YEARS OLD WITH CLINICAL HEMORR- HAGIC FEVER AND SEROLOGICALLY CONFIRMED DENGUE INFECTION. HI TITER yer HI TITER I I DAYS AFTERONSET 1-2 yers U I I3 I I DAYS AFTER ONSET differ enough to wrr.nt inclusion of the TH.ntigens in serologic tests. HI nd CF ntibody response followinq denque hemorrh.qic fever in residents of Bnqkok.. Multivlent HI nd CF response. Forty six hemorrhgic fever ptients with 3 or more serum specimens were selected for intensive study. The ge nd sex distribution of these ptients resembles the distribution of hospitlized hemorrhgic fever except tht children of less thn 1 yer were not included. HI titer results using d 1-4, TH-Smn, TH-36 nd JE ntigens were nerly the sme; the ntibody response to vrious CF ntigens is predominntly multivlent. Antibody titers in convlescent serum to dengue 3 nd 4 CF ntigens were lower thn dengue 1 nd 2 titers. Only rrely did monovlent CF ntibody response occur from which it might be possible to predict the infecting dengue virus type. For the ser studied, it ppers tht use of dengue 1 ntigen for either HI or CF resulted in greter number of "positive" dignoses of dengue infection thn with use of other ntigens. Whether is is becuse dengue 1 is more ntibody vid or tht greter number of Bngkok ptients hd previous dengue 1 ntigenic exposure is not known. b. Antibody response.nd.ge. P.ired ser, hve been obtined from 22 children under the ge of 1 with clinicl di.gnosis of Th.i hemorrhgic fever. The HI,ntibody response in this group is contrsted with the response in 2 1 children between 1 nd 2 ye.rs of.ge in Figure 5. The mrked difference in response is unmist.ke,ble. The response of children under the.ge of one ppers to resemble
6 FIGURE 6. GEOMETRIC MEAN TITERS OF HI AND CF ANTIBODY IN ACUTE AND CONVALESCENT SERA OBTAINED UP TO 7 MONTHS FROM 46 PATIENTS WITH CLINICAL HEMORRHAGIC FEVER BANGKOK, 1962 the.ntibody response of C.ucsi.ns, i. e., response to initi.1 infection. Most cute phse ser,.r e HI.ntibody negtive.nd re followed by. r.ther low ntibody response in most 1 ye.r old children with hemorrhgic fever. Most ntibody responses in children over 12 months.ppe.r to be of the second.ry type. Antibody surveys of the Bngkok popul.tion in the s.me ye.r showed th.t not more th.n 15% of children t 1-2 ye.rs of ge hve dengue HI ntibody. If the observed.ntibody response of 1 yer old children is the secondry type then preceeding dengue infection in 1 ye.r old children is consider.bly more frequent in HF c,ses thn in the normls. This would suggest tht hemorrh.gic fever is. dise.se following sec ond.ry dengue exposure. c. Development nd dur.tion of HI.nd CF.ntibody followinq denque hemorrhsic fever. Figure 6 shows the geometric me.n titers of dengue HI nd CF.ntibody in cute nd conv.lescent ser obt.ined over, period of 7 months in 46 ptients with hospitlized hemorrh.gic fever. A very r.pid development of HI nd CF.ntibody ch.r.cterizes the.ver.ge p.tientts response to infection during the first 7-1 d.ys of fever. Three months fter infection, geometric me.n HI titers h.ve f.llen to 1:768, while CF titers.re.t 1:35.2. These d.t. ppe.r to h.ve some v.lue for dting dengue infection in,n verge B.ngkok resident. In this lbor,tory, HI titers of 1:64 or higher in conv.lescent ser, of p.tients with dise.se resembling Th.i hemorrhgic fever re considered s presumptive evidence of recent dengue infection. The frequency of HI.ntibody titers of 1:649 or higher in 13 surgic.1 ptients without recent febrile illness studied during.nd fter seson.1 dengue virus dissemin.tion in 1962 w.s 3.8%. i
7 Tble 1 COMPLEMENT-FIXATION TEST OF HYPERIMMUNE SERUM PREPARED TO ISOLATES (MULTIPLE STIMULI). Reciprocl of CF ntibody titer vs. Bngkok virus 2 units of indicted prototype dengue ntigen hyperimmune Dl D2 D3 D4 TH-36 TH-Smn Homologous serum virus BKM /3 2 BKM /32 BKM /12 8 * = Signifies reciprocl of highest dilution of serum which gives 2+ or more fixtion of complement. ** = No fixtion t 1:4 dilution of serum. *** = The numertor represents the mximum dilution of serum fixing complement in the presence of ntigen nd the denomintor the mximum dilution of n ntigen fixing complement in the presence of ntiserum. Tble 2 COMPLEMENT-FIXATION TEST USING IMMUNE SERUM PREPARED BY TWO INOCULATIONS OF INDICATED VIRUSES Reciprocl of ntibody titer vs. indicted 2 units of prototype dengue ntigen Dl D2 D3 D4 TH-36 TH-Smn BKM BKM BKM * No fixtion t 1:4 serum dilution. A35
8 Antibody response of we.nling mice to densue immuniz.tion. Technique 1: Fresh 1% suspensions of mouse brin in s.line were inoculted into 2 1 dy old mice intr.peritone lly (.2 ml..mounts). After five injections,t one week interv.1~, tri.1 bleedings were m.de to determine whether stisfctory.ntibody titer h.d been obt.ined. Technique 2: Three to four week old mice were inoculted IP with.3 ml of 1% fresh mouse br.in s.line suspension. Two weeks 1.ter mice were inocul.ted with.o3 cc of of mouse br,in suspension intrcerebrlly. (IC). A tril bleeding ws performed ten d.ys lter. If it reveled no s.tisf.ctory titer,.3 cc of 1-I virus suspension ws injected into mice intrcerebr,lly. Tri,l bleeding ws m.de 2 weeks lter. Results of CF tests of.ntiserum prep,red by technique 1.re shown in Tble 1. Mrked heterologous crossing is obvious. When fewer inocultions of serum were mde, more specific results shown in T.ble 2 were obtined. Extensive studies of dengue mouse immune ser prepred to B.ngkok dengue viruses h.ve been mde using denguel- TH-Sm.n nd dengue 2-TH-36.ntigens. Correl.tions.re shown in Tbles 3.nd 4. D.t, suggest the ntigenic homogeneity of the dengue 1 -TH-Sm.n nd dengue 2 -TH-3 6 groups respectively. Summry nd Conclusions : Studies during the report period h.ve shown tht following HF infection cused by dengue viruses the HI,ntibody response-is prompt.nd of gret m.gnitude. The men HI titer of 31 ser, obtined 8-14 dys fter onset of fever w.s 1:4,9 6. Beginning t 1-3 months.fter infection HI ntibody begins to decline nd 7 months.fter infection me.n v.lues.re below 1:32. CF ntibody fter dengue infection follows,n.lmost identic.1 p,ttern except tht vlues re pproxim.tely 1-1 fold lower. Serologic response to chikunguny. virus is quite different. HI.ntibody develops less r.pidly thn following dengue infection with ver.qe of 1:9 chieved 15-3 d.ys fter infection. Development of complement-fix.tion ntibody is definitely delyed; most ser. do not show.ny CF.ctivity until 15-3 d.ys.fter infection. Thus, ser hving high HI ntibody but low or no CF.ntibody to chikunguny, suggest recent infection ntibody titers in hum.n serum me.sured with dengue 1.nd 2.ntigens were similir to titers obt.ined with TH-Sm.n.nd TH-36 ntigens, respectively. By the HI test the infecting dengue virus could not be determined since ntibody titers to.11 dengue types.nd J.pnese encephlitis virus were nerly iderrtic.1. In B.ngkok.re., CF results were only slightly more specific. The.ver.ge p.tient developed CF.ntibody of,bout the s.me titer to 2 or more dengue viruses.nd J.p.nese enceph.litis virus. We.nling mice inoculted IC.nd IP with 2 or 3 injections of low mouse p.s s.ge dengue viruses developed low titered CF.ntibody which w.s dengue type specific. CF titers to dengue 1.nd 2.ntigens were the s.me.s to TH-Sm.n nd TH-3 6.ntigens, respectively.
9 Tble 3 CORRELATION TABLE SHOWING CF ANTIBODY TITERS OF 26 BANGKOK DENGUE MOUSE SERA TO DENGUE 1 (HAWAII) AND DENGUE TH-SMAN ANTIGENS Reciprocl titer vs. dengue 1 (Hwii) ntigen Reciproc 1 4 titer vs. 4 TH-Smn 8 ntigen 16 Reciprocl CF titer Tble 4 CORRELATION TABLE SHOWING CF ANTIBODY TITERS IN 44 BANGKOK DENGUE VIRUS MOUSE IMMUNE SERA TO DENGUE 2 (NEW GUINEA C) AND TH-36. Reciprocl titers vs. dengue 2 (New Guine C) ntigen Reciproc 1 titer vs. TH-36 Reciprocl CF titer
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