Ascorbic acid (vitamin C) and its effects on parainfluenza type 3 virus infection in cotton-topped marmosets

I¿¡o¡¡¡o¡r A¡¡¡rr¡. Sc¡r¡rc¡ Co¡ryrisht @ 1974 by rhc Anrerian Asociation for Laboratory Animal Scicncc Studies on the microbial flora of mannosets by ourselves and others have identified some of the bacteria and viruses to which these a¡rimals are susceptible (1,2). The susceptibility of the marrnoset to various respiratory viruses of presumed human origin suggested that these animals rnay be used to study nutrition-infectiòn interactions. Pauling (3) has hypothesized rhat elevated blood levels of ascorbic acid in hurnans may be associated with a decreased susceptibility to respiratory illness of viral etiology. This paper reports I ) the results of a study in which cotton-topped marmos ets (S øguinus oe dipus) were used as a biomedical model to study this hypothesis, and 2) the effects of long-term ascorl¡ic acid supplementation on these animals. at thc Primatc LabomVol 2{. No I Part II oI Two Paro Ptintù í¡ USA ascoRBlc acrD (VTTAMIN C) A¡{D ITS EIT'ECIS ON PARAINFLUENZA TYP.E Iu VINUS INFECTION IN COTTON. TOPPED MARMOSETISI.2,S B.L. MURpIry, D.tI. KRUSIIAK, J.E. MAYNARD, AND D.W. BRADLEY SUMMARY o Recent sludier suggesl lhol inc¡easod omosal¡ of o¡co¡òic acid (vilømia C) møl iac¡¡or rasr'¡lonce or r,ller lhø clínical rêrPoare ol mon lo rotpìrcloty vi¡øl inleclìoa¡. fo c¡o¡ninc t¡ir gue¡ion in o nonlrurnon primote, o¡co¡ôic ocld wos odded lo tåo diol of colton-loppcd mor¡no¡cl¡ (Soguinut ocdlpur). Along vilh conlrolc' tåese oni¡nols we¡e inocsloled intronasølly wirh o drclín ol paroinlluenzo lypc lll vi¡us ol mo¡mosel orígia. ln*eo¡od o¡nounl¡ ol vilomic C díd nol p¡avc,n1 virus íaleclion or ptimory inmuaologíc rósPonJe in ll¡e onimols, bu, ít did deloy oasel oÍ tàe díreose, ¡educo clinicol raspontct, ond decreose modolily (57/o vs 36o/o). gmr R Fields and G Emcrv ¡nd Barnùart, ¡nd G Blair for tbeii tech, Duf- 'us Merrnrl¡-s AND METHoDs Marmosets: Cotton-topped nutnnosetr (Saguinus oedíþus) were conditioned for 2 mo prior to study. Visual health signs werc rcorded daily, and body weights and temperatures were recorded at specified intervals. The mean body weigtit of the mannosets rxnd in this study was approximately 400 g. All m¿rrmosets were afebrile prior to virus inoculation. Animals were individually caged to assure that each animal would receive its designated amount of daily vitamin C. Parainfluertza tyþe III airus inoculations: Each of 14 vitamin C (ascorbic acid; L-Ascorbic Acid¡l'o supplemented, and Z nonsupplemented (control) marmosets without detectable serum antibody to parainlluenza type III virus (dilution screening level t:5) received intranasal inoculations. All were inoculated with 0.25 ml of 1000 TCID5¡ of parainfluenza type III 108 days after vitar¡in C supplementation was begun. helimÍnary tests showed that 1000 TCIDõ6 of the virus would infect all marmosets a¡¡d produce sero_ conversion. 229 Vírologic examínation: Routine throat swabs and blood specimens were obtained from these animals upon admission and dur_ ing quarantine at monthly intervals. Throat swabs were processed as described earlier (2,4). Viral identification and neutralization tests were made by hemadsorption (HAd) ¿nd hemadsorption-inhibition (HAd_Ii tests using guinea pig red blood cells, as described by Hsiung (5). Marmoset serum antibody titers against parainfluenza type III virus were tested prior to inoculation'rrr¿ "t 30 days post-ínoculation. Vítamín C suþþlementation: Marmosets in ^the supplemented group received orally t00 mg crystalline ascorbiclcid twice a day, the e"quivalenr of 35 g per day for a 70 k! man. This amount was used in order to study the effects of large doses of the vitamin on viral respiratory infection. Ascorbic acid sup_ plementation was found to be most effective when the crystaltine vitamin was mixed directly into banana slices which were given prior to feeding. Vitamin Ç supplemenàtion w¿rs removed from the diet at 35 days post_ inoculation. - - Control and supplemented marmosets were bled every 24 wk throughout the course of 230 Flg t. Wñolc blood c¡corbíc øcíd concaàl¡otîon¡ ln conl¡ol ond sopplemcnled mo¡moætt LABORATORY ANT!/!ÂL SCIENCE 60 -ao -20 the study. Bleedings of the supplemented animals were performed prior to daily administration of vitamin C in order to reduce the possibility of measuring transient coneentrations of ascorbic acid. One ml samples of whole blood were taken from the fàmoral artery into syringes containing 0.1 mt (150 units) of heparin. Whole blood ascorbic acid (WBAA) determinations on tríchloroacetic acid stabilized samples (Q 7) were performed according to the method of Roe and Kuether (8) as modified by Lowry et al (g). Yol 24, No 1, Part II Rrsur,rs 20 ¿to The effects of vitamin C supplementation on concentrations of WBAA are shown in Fig I and Table l. Concentrations of WBAA in the supplemented animals appeared to reach their maximum levels dr¡rin! the first month of supplementation (10) and did not further increase with continued supplementation. Small variations in WBAA -"on."rrtr"- tions, however, were seen in both supple_ mented and control groups throughout the course of supplementation. Vítamin C supplementation was discontinued 35 days post_ inoculation, and concentrations of 'WBAA were monitored in order to test the hypo_ !ooo ¡l O I c¡ õ It ¡D G o (, a ô o c' o l¡¡ o ¡ '2 vriuS ttaocuL -l ---* --ì.-"-..--.--.t arrtl c iEÍOVEO Fnot. otEl H SUFPLEIE¡1¡D etOup Þ{ coxtaoL GROuP vtt¡l.tx c SUPPLTTE¡1A'IO¡ loo -ao ÞnElf{ocuLAttox oAYS PO31-lltocuLAftorl 30 February, 1974 VITAMIN C AND MARMOSETS TABLE I Mean concentrations of whole blood ascorbic ac¡d (wBAAt in control and supplemanted marmoseß vs t¡me 23t Days before {-} and after (+l inoculation WBAA in sq % lX + sDl Percent increase (B-Aþ x loo -61 +14 4l 43 8 32 8 14 il 74 -34 7 +42 +56 25,92+ 324 N=8 2285+ 424 N=8 2863 + 504 N=8 2700+ 78t N=7 2608+ 622 N=7 2000 + 363 N=6 2O9O !234 N=3 3656+ 1060 N=14 3214 + 557 N=14 3677 + 61 1 N=.f4 3563 + 891 N=14 3340+ 767 N=14 2271 + 343 N=11 2327 !36A N=9 WBAA b¿seline values on day -l0B; Control: 2266 ! 303 (N = l0l: Group to be supplemented: 2570! 4gO(N = lal thesis that animals removed from vitamin C supplementation would become scorbutic under otherwise normal dietary conditions (10). Parainfluenza type III virus was isolated from throat swabs taken from all of the animals. The virus was isolated as early as 3 days post-inoculation and as long as 14 days post-inoculation. No differences in virus isolation rates at 3, 7, 10, and, 14 days postinoculation were observed between supplemented and non-supplemented animals. No statistically significant differences in serum antibody response (geometric means) were detected betrveen the supplemented and nonsupplemented groups. HAd-I tirers (30 days post-inoculation) in the supplemented group ranged from l: 16 to t:1024, and in the control group they ranged from 1:16 to l:128. Marmosets were observed for morbidity signs daily. Signs of illness rvere nasal discharge, ocular discharge, transient fever up to l05oF (ca 4lcÇ¡, and lertrargy. All nonsupl/lerr.er¡ted ¿tr.d i c,l. l* ru¡,[/lerr¡r:r¡ted, rrrarrr.c:Ë¡s e-*.ibirad :izr.i '..,itt ir, 3 ða',i; trr;,,rever. rle o*,et 7 sup[,¡er¡er¡ted rr¡arrr¡oiets did not exhibit signs until 7 days pnst-inoculatiorr. \fr,rrtaliry \r'as otrscrrcd in 5 of 14 (36%) of the supplemented animals and 4 of 7 (57/o) of the control animals. The mean duration of illness in supplemented survivors was 9 days compared with 20 da¡æ in the control group. D¡scussroN Marmosets supplemented with vitamin C were slìown to be as susceptible to primary infection with parainfluenza virus as control marmosets not receiving the vitamin supplementation. However, there appeared to be some therapeutic value of vitamin C since supplemented animals had fewer mean days of illness and experienced less mortality postinoculation than did non-supplemented marmosets. Two recent publications by Wilson et øl (ll) and Anderson et al (12) have also reported a correlation between vitamin C utilization and the duration of "cold symptorrravtlrtgy" ir¡ hr¡rnar¡s. It sl¡rrulrl be runterJ tt.a,t aftr:r vitzrrtitt () ','tut Íeßú¿vtd Írort II*. s(:urvy r.¡r suL;,r¡orrr¡al concer¡tratior¡s of WITAA r^,.crc fr,r¡r,d. 'I1¡is ir¡dicates that vitarni¡¡ C :232 LABORÁ,TOR,Y ANTMAL SCTENCE supplementation does not involve the risk of post-supplementation scurvy. Large doses of the vitamin did not pre_ vent virus infection, suggesting that the me_ chanism of ascorbic acid does not involve direct interaction with the virus. The results of our study with marmosets indicate that vitamin C exerts its effects on the host, possibly by maintaining cellular and tissue integrity. Further investigations should be continued using this biomedical model to answer current questions on the effects of ascorbic acid on respiratory disease. RET'ERENCES 4. Murphy BL, Jvfaynard JE, Krushak DlH, et aI: gccurrence of a carrier state for Herþewínr ta-ntdrínus in ma¡mosets. AþþI Ulioúìol Ztz 50-52, lgTl 5. Hsiung GD, Henderson JR: Dídgnostìc Virolog2. .New flaven, yale ûniversiti iress, tSOl, p 124 6. Bradley. DW, Emery G, Maynard fE: Vitamin u-tn ptasma: A compatative study-of the vita_ min stabilized with tii.t to.oàc.t¡"'".iã o, _.t"- plgsphoric_ acid and_the effeiæ-oi-itãog* "t ;70: q,-To c,..4o c, and ?¡ã c;;îe-stabi. ¡¡zeo vrramin. clin chìm Acta 44: 47_52, lg7, 7. lr:d1? l_y,, lr"yn..ra ¡r, Èmery -c:' c-#rpari_ son of ascorbic acid concentratíons in r,rihote blood obtained trv.^venipu-n.tuiu -ãi üy fing.i ^ prick._C_tín Chcm'rtz éoõ:rizq iõzá' ", ' 8. Roe.JH,.-Klerher CA: Determ¡nition of "r_ corbic acid in whoteblood and "ri"ã-ìfr."ugh l, 4-dinitrophenylhydrazin" ¿"riu"i¡n.- oi'i.rr"- droascorbic'aa.í. í B;o, ch;;-l;;; ä'rrïii: l94E 9. !g"rl. 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