Cross-Protection Among Feline Caliciviruses

Transcription

1 INFECrIoN AND IMMUNrrY, My 1975, p Vol. 11, No. 5 Copyright i 1975 Amerin Soiety for Mirobiology Printed in USA. Cross-Protetion Among Feline Cliiviruses CHARLES POVEY1* AND JERRY INGERSOLL Norden Lbortories, Linoln, Nebrsk Reeived for publition 11 November 1974 Eh of five groups of speifi-pthogen-free nd onventionlly rered ts ws infeted with different strin of feline liivirus. Two of the strins were pthogeni, produing hrteristilly fever, depression, loss of ppetite, bul ulertion, nd osionlly inresed oulr nd nsl seretion. Two of the other strins were mildly pthogeni nd ssoited with fever or bul ulertion or both; the fifth strin ws nonpthogeni. The two pthogeni strins plus three others shown lso to be pthogeni were used 3 months fter the initil infetion to hllenge the ts in rerrnged groupings. Of the 28 onventionl ts hllenged, six (21.4%) showed t lest febrile response, lthough none of the 30 speifi-pthogen-free ts showed ny linil signs. After hllenge, virus ws reovered from throt swbs of 37 of the 58 ts (63.8%) inluding the six whih showed symptoms, but the durtion of the exretion of virus ws signifintly less thn tht seen with the initil infetion. The homologous nd heterotypi ntibody responses orrelted well with the linil protetion, or lk of it, seen on hllenge. The results provide further evidene for signifint ross-reltionships between feline liiviruses. Feline liiviruses (FCV) (feline piornviruses) hve been frequently isolted in mny prts of the world; isoltions hve been mde prtiulrly from the respirtory trt of ts with or without linil signs of inflmmtion of tht trt. Preliminry serologil ttempts to lssify the viruses suggested tht numerous distint serotypes existed (2, 3, 5). Subsequent ross-neutrliztion studies (14) of suh isoltes led to the suggestion tht there were in ft onsiderble ntigeni reltionships in the group nd tht the isoltes tested might be regrded s members of single serotype. It ws further postulted (14) tht this reltionship my reflet in ross-protetion in tsinfeted with FCV. To test this postulte, five groups of speifipthogen-free (SPF) nd onventionlly rered ts were eh infeted under refully ontrolled isoltion onditions with n FCV. The linil nd immunologil responses of these ts were monitored, nd then t 3 months the nimls were hllenged with either the homologous infeting virus or heterologous strin, nd gin their responses were determined. MATERIALS AND METHODS Viruses. The following FCV isoltes were used t the pssge level given in prenthesis: M-8 (5th) nd N-3 (6th) were lol isoltes obtined respetively I Present ddress: Deprtment of Clinil Studies, Ontrio Veterinry College, Guelph, Ontrio, Cnd. 877 from kitten with 6-month history of respirtory illness nd from t with ulertive stomtitis. FCV- 255 (14th) ws obtined from D. E. Khn, Ohio Stte University (9). F-17 (2nd fter reeipt), 17-FRV (25th), FC (12th), nd CFI (19th) were reeived from Dorothy Holmes, Cornell University, from stoks prepred for the Ntionl Cner Institute. FCV 5936 ws isolted by R. A. Crndell, University of Illinois, from t with respirtory distress, sneezing, nd orl lesions, nd ws used t its 3rd pssge level. All pssges hd been mde in primry, seondry, or estblished feline tissue ulture. Cell ulture. An estblished line of feline ells (NLFK) ws used for virus propgtion, ssy, nd neutrlizing ntibody studies. Tissue ulture medium routinely used ws: Hnks blned slt solution (10 times onentrted), 10%; ltlbumin supplement, 10%; Egle vitmin solution, 10%; Egle mino ids, 10%; 2.8% NHCO, solution, 5%; peniillin, 50,000 IU; streptomyin, 50 mg/liter; glutmine, 293 mg/liter; nd fetl lf serum, 5% for growth, nd 2% for mintenne, dded just prior to use. Cts. Thirty-six SPF ts of 4 to 9 months of ge were imported in individul filtered-ir ontiners from the Lbortory Animls Centre, Crshlton, Englnd, nd from Hill Grove, Englnd, nd were immeditely housed in pirs in gnotobioti-type, flexible-film isoltors. Thirty onventionlly rered ts were housed together in len room (Biolen) from shortly fter wening until use t 6 to 8 months, when they lso were housed in pirs in the isoltors. The isoltors were distributed between five seprte rooms. Isoltor onstrution ws modifition of tht previously published (15). Inoultion nd hllenge. The mjority of ts

2 878 were infeted by diret intrnsl instilltion, using fine dropper, of 0.2 ml of fluids ontining totl of 105. to men tissue ulture infetive doses (TCID50) of virus. The viruses used for primry infetion were M-8, F-17, 255, N-3, nd 17-FRV. Eight ts were infeted by n erosol of virus fluids ontining 106 TCID5gml for 1 min by no. 40 devilbiss nebulizer tthed to smll-niml ether msk pled over the fe of the t. No essentil differene ould be deteted in the response to these two methods of infetion nd thus, for subsequent hllenge 12 weeks fter primry infetion, diret intrnsl instilltion ws used. The viruses used for hllenge were N , FC, CFI, nd 5936 t the sme dose level s for primry infetion. The pthogeniity of the hllenge viruses, FC, CFI, nd 5936, ws heked by infetion of two onventionl nd two SPF ts for eh virus. The groupings of ts for infetion nd hllenge re shown in Tbles 1 nd 7. Before nd fter inoultion nd hllenge ll ts were observed linilly, tempertures were reorded dily, regulr orophryngel swbs were olleted, nd t suitble intervls blood nd nsl seretion smples were tken. The ltter smples were olleted fter synhronous sedtion with ketmine hydrohloride (Ketset, Bristol Veterinry Produts, New York) (7.5 mg/lb of body weight [1 lb equls g]) nd seretory stimultion with pilorpine hydrohloride (0.5 mg/lb of body weight). After olletion tropine sulfte ws given intrmusulrly t 1.0 mg/lb of body weight. Nsl seretions were filtered through disposble filter unit (Miltex, Millipore Corp., Bedford, Mss.) nd s with ser, het intivted t 56 C for 30 min. TABLE 1. Clinil nd virologil response of groups of ts to exposure to vrious feline liiviruses No. from Men No. with whih durtion Group linil virw virus virus ws response reovered reovery (dys) Six onventionl ts infeted with M Six SPF ts infeted with M Six onventionl ts infeted with N Six SPF ts infeted with N Six onventionl ts infeted with F Six SPF ts infeted with F Six onventionl ts infeted with Six SPF ts infeted with Six onventionl ts infeted with FRV Six SPF ts infeted with FRV POVEY AND INGERSOLL Neutrliztion test. The miro-neutrliztion test previously desribed (14) ws used. Briefly, ser or nsl seretion smples were diluted in phosphte-buffered sline using 0.05-ml mirodiluters in 96-well disposble pltes (Mirotest II, Flon Plstis, Oxnrd, Clif.). Virus prediluted to titer of TCID,0/ml ws then dded to 0.05 ml per well nd the mixtures ws gitted on Miromixer (Cooke Engineering). After inubtion t 37 C in 5% CO2 for 1 h, pproximtely 104 NLFK ells ws dded to eh well nd the pltes were reinubted in CO2- enrihed tmosphere for 3 to 4 dys until ontrols showed omplete ytopthi effet. Results were reorded s presene or bsene of visible ytopthi effet in eh well, nd 50% end points were lulted by the method of Reed nd Muenh (16). RESULTS INFECT. IMMUN. Clinil nd virologil results of infetion re shown in Tble 1. Cts infeted with FCV-M-8. The onventionl ts used were found to hve pre-inoultion ntibody titers to FCV-M-8 of between 1:4 nd 1:256. There ws no linil response to infetion, but virus ws reovered from five of the six ts. The six SPF ts in this group lso hd neutrlizing tivity ginst M-8 present in their ser prior to infetion, but levels were miniml (<1:4). The group showed no symptoms fter infetion, but gin virus ws reovered from ll six ts infeted. Cts infeted with FCV-N-3. Only one of the onventionl ts showed pre-inoultion homologous ntibody, but ll six showed titers of 1:2 to 1:200 to the heterologous liviruses (M-8, 255, et.). Four of the six onventionl ts inluding the only one with pre-inoultion neutrlizing ntibody titer (1:32) showed febrile response (> 103 F), whih in three ses ws biphsi, the initil rise being t 2 dys nd the seond between dys 4 nd 9 post-inoultion. Svmptoms in these four ts vried from osionl sneezing, through ler oulr nd/or nsl dishrge with tongue ulertion, to frnk onjuntivitis, photophobi with purulent oulr nd nsl dishrge, mrked lingul ulertion, trnsient dirrhe, nd generl depression. The two remining onventionl ts showed neither febrile response nor symptoms. Virus ws reovered from ll six ts in the onventionl group nd three ts were still positive t 37 dys but not 57 dys postinfetion. Of the SPF group, none of whih hd pre-inoultion ntibody, homologous or heterologous, two ts showed trnsient fever. Tongue ulers developed in four ts, nd two of these hd erosions on the hrd plte lso. One t hd vesile on the lip nd nother hd erosion of the externl

3 VOL. 11, 1975 CROSS-PROTECTION AMONG FELINE CALICIVIRUSES nres. Virus ws reovered from four of the SPF ts. Cts infeted with FCV-F-17. All six onventionl ts showed preinoultion homologous neutrlizing ntibody in their ser (1:2 to 1:10) nd some heterologous tivity lso. In this group no definite febrile response ws deteted nd no ts showed ny symptoms, lthough virus ws reovered from ll ts. For the SPF group, two of whih hd miniml levels of homologous neutrlizing tivity pre-inoultion (<1:8), two ts showed trnsient fever, two hd lingul ulers, one hd hrd-plte erosions nd ulertion of the externl nres, nd one hrd-plte erosion only. Virus ws reovered s lte s 14 dys post-inoultion from four of the six ts in the group. Cts infeted with FCV-255. Two of the onventionl ts showed pre-inoultion serum neutrlizing tivity (1:10 nd 1:20). Febrile responses ourred in only two ts nd were not lerly biphsi. Only one t showed ny other signs of infetion, solitry tongue uler present from dys 9 through 11. Virus ws reovered from ll ts exept one between dys 4 nd 22 post-inoultion. By ontrst, the SPF ts, none of whih hd pre-inoultion serum neutrlizing tivity, ll showed some symptoms inluding fever (three ts); lingul ulers prtiulrly of the nterodorsl re (four); hrd-plte ulertion (four); mild oulr nd nsl dishrge (two); onjuntivitis (one); nd dirrhe (one). Virus ws reovered for up to 5 weeks for ll ts. Cts infeted with FCV-FRV. The ser of ll six onventionl ts hd pre-inoultion neutrlizing ntibody ginst FCV-FRV of 1:2 to 1:4. No definite febrile response ourred mong this onventionl group, lthough one t showed lingul uler nd one showed slight nsl dishrge, tongue ulertion, nd erosion of the externl nres. This ltter t filed to reover from ketmine sedtion t 8 weeks post-inoultion. Virus ws reovered from ll ts between dys 2 nd 16 post-inoultion. Ulertion of the externl nres ws feture of infetion in five of the SPF ts inluding the three whih showed preinfetion serum neutrlizing tivity between 1:2 nd 1:3. One of the five ts hd nsl dishrge nd ws sneezing. Two ts showed febrile episode, nd virus reovery ws suessful from five ts. Cts infeted with FCV-FC. A mrked onjuntivitis ourred in one of the pir of onventionl ts. With the SPF ts, one showed lingul uler nd both hd ulertion of the externl nres. 879 Cts infeted with FCV-CFI. Both onventionl kittens showed trnsient pyrexi, depression, nd poor ppetite, with onjuntivitis nd erosions on the hrd plte in one. Appetite ws similrly depressed in the SPF ts, nd one hd solitry lingul uler. Cts infeted with FCV Trnsient pyrexi ourred in both onventionl kittens, with onjuntivitis, nsl dishrge, nd sneezing in one. On utopsy, t 7 dys postinfetion, foi of interstitil pneumoni were present in the seond kitten. The two SPF kittens showed fever, norexi, nd tongue nd externl nres ulers, with nsl dishrge in one kitten. Serologil response to primry infetion. (i) Serum. Tbles 2 through 6 show the development of neutrlizing ntibody in eh group, both ginst the originl infeting virus nd ginst some of the other FCV under study, tht is, the homologous nd heterologous response. The homologous response is shown grphilly in Fig. 1 nd the mens between groups for the heterologous responses re sho,.-n in Fig. 2. (ii) Nsl seretion. Mny smples proved ytotoxi t low dilutions, nd nsl seretion neutrlizing tivity ws in generl of low order (< 1:16). Mny of the onventionl ts showed low-level homologous nd heterologous virus neutrliztion in pre-exposure smples, but prt from two SPF ts in the M-8-infeted group (titers of 1:4 nd 1:7), none of the SPF ts showed suh preinfetion tivity. After infetion, the mjority of the onventionl ts showed neutrlizing bility of nsl seretions but, prt from ts in the M-8-infeted group, where titers rehed s high s 1:200 t 4 weeks post-inoultion, titers were between 1:2 nd 1:16. For the SPF nimls, the pek titers rehed were 1:20, but mny nimls filed to show detetble tivity. Clinil response to hllenge t 12 weeks post-inoultion. This is summrized in tble 7. There ws no linil response to the hllenge infetions in ny t originlly infeted with either M-8 or N-3 viruses. Of the ts infeted initilly with F-17 virus, two of the onventionl group, both reeiving FC s the hllenge virus, showed, respetively, trnsient fever with solitry lingul vesile nd lingul uler without fever. Three ts, ll in the onventionl group, whih hd been infeted with 255 virus gve linil response to hllenge. Of these, one t, hllenged with virus 5936, showed fever nd erosion of the externl nres, nd the other two ts, both hllenged, s they hd been initilly infeted, with 255 virus, showed fever, norexi, nsl dishrge,

4 880 POVEY AND INGERSOLL INFECT. IMMUN. TABLE 2. Geometri men serum neutrlizing ntibody titers (-log2) for groups of ts pre- nd to feline liivirus M-8 Virus Weeks M-8 N FRV F < 1.0 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0 < NT 5.3 NT NT NT NT NT C, Conventionl ts; S, SPF ts; NT, not tested. TABLE 3. Geometri men serum neutrlizing ntibody titers (-log2) for groups of ts pre- nd to feline liivirus N-3 Virus Weeks M-8 N FRV F < <1.0 <1.0 < < < NT 6.3 NT NT NT NT NT C, Conventionl ts; S, SPF ts; NT, not tested. TABLE 4. Geometri men serum neutrlizing ntibody titers (-log2) for groups of ts pre- nd to feline liivirus F-17 Virus Weeks M-8 N FRV F <1.0 <1.0 < < < < < < NT NT NT C, Conventionl ts; S, SPF ts; NT, not tested. TABLE 5. Geometri men serum neutrlizing ntibody titers (-log2) for groups of ts pre- nd to feline liivirus 255 Virus Weeks M-8 M N-3 N 2255 FRV F F < < < < < < NT 5.0 NT NT NT NT NT C, Conventionl ts; S, SPF ts; NT, not tested.

5 VOL. 11, 1975 CROSS-PROTECTION AMONG FELINE CALICIVIRUSES 881 TABLE 6. Geometri men serum neutrlizing ntibody titers (-log2) for groups of ts pre- nd to feline liivirus FRV Virus Weeks M-8 N FRV F-17 I s s s l s I s < 1.0 < 1.0 < 1.0 < 1.0 < < 1.0 < 1.0 < < NT NT NT NT C, Conventionl ts; S, SPF ts; NT, not tested. A. A. 1,.0, >% I- 1- I V* 2 r %A I I II "I B. /j? FRV 42S5 11 ~ C14 9 >1 2 m 2F ta nd lingul nd hrd-plte ulertion between dys 5 nd 12 post-hllenge. In the group originlly infeted with FRV, single onventionl t showed trnsient fever fter hllenge with N-3 virus. Virus reovery fter hllenge. Virus reovery from throt swbs ws suessful not only from eh of the six ts tht showed linil response to hllenge, but lso from further 31 ts mong those tht hd shown ny symptoms fter hllenge infetion. The men dur week following exposure B. <Ii I 6 2 i & 8 i0 i2 week following exposure II._ z >1 I week following exposure FIG. 1. Geometri men homologous serum neutrlizing ntibody titers in groups of ts (A, onventionl; B, SPF) exposed to feline liiviruses week following exposure FIG. 2. Heterologous response fter exposure to feline liiviruses. Geometri men serum neutrlizing ntibody titers to eh virus of ll ts (A, onventionl; B, SPF) exept those originlly infeted with tht virus. tion of exretion ws 3.1 dys for the onventionl ts, exluding those showing symptoms fter hllenge, nd the men for the SPF ts ws 0.9 dys. By ontrst, the men durtion of exretion for those six onventionl ts tht showed symptoms ws 14.3 dys. Serologil response to hllenge. Figures 3 nd 4 show the serologil responses in terms of neutrlizing ntibody in serum to the originl

6 882 POVEY AND INGERSOLL INFECT. IMMUN. TABLE 7. Grouping nd response of ts to hllenge with vrious feline liiviruses No. No. Men Originl Chl- No. nd showing from durtion infeting lenge type of linil whih virus virus virus ts response virus to hl- ws re- reovery reveys lenge overed (dys) M-8 CFI 1 Con CFI 2 SPF Con N-3 2 Con FC 2 SPF SPF N-3 CFI 2 Con Con N-3 4 SPF FC 2 SPF F17 CF1 2 Con SPF N-3 2 Con FC 2 Con FC 2 SPF SPF CFI 2 SPF Con SPF N-3 2 SPF FC 2 Con FRV CFI 2 SPF SPF N-3 1 Con FC 2 Con Con SPF Con-n (Convrntionllv rered: + two ts _vvl died._'a,- from inidentl uses, prior to hllenge. infeting virus nd to the hllenge virus in the 8 weeks fter hllenge infetion. Those ts in the groups originlly infeted with F-17 nd 255 viruses tht suumbed to hllenge showed mrkedly lower men serum neutrlizing ntibody to the originl infeting virus t the time of hllenge thn did resistnt ts in their groups. This differene is not pprent for the single t in the FRV group tht showed trnsient pyrexi on hllenge. However, when the ntibody levels to the hllenge virus re exmined the differenes re onsistent. Thus, the t tht suumbed to hllenge with FC virus hd n FC ntibody titer of 1.7 (-log2) ompred with the men for the FC-hllenged onventionl nimls of 5.5. The t suseptible to 5936 hllenge hd titer to 5936 t hllenge of 1.0 (men for group, 2.8); the ts suseptible to 255 hllenge hd titers to 255 t hllenge of 2.3 nd 1.3, respetively (men for the rest of 255-inoulted group, 6.3; men for the rest of 255-hllenged group, 5.7); nd the t suseptible to N-3 hllenge hd titer to N-3 t hllenge of 3.3 s opposed to men for the group of 7.3. Antibody to viruses other thn those used in the originl or hllenge infetion ws lso mesured (results not shown) nd prlleled the piture seen with those ntibodies in terms of boosting where titers were initilly low but little hnge where titers were higher. DISCUSSION Reent in vitro studies (14) suggesting ross-tivity between 46 isoltes of FCV hve now been extended to the in vivo sitution for totl of eight isoltes. The serologil rossobtined, nd more signifintly retion the A. C-li F 2 78 :e zm U.,CD 0 V) (, B. l 9 'E -< ,',. ~-_ F-17,.FRVi 9 j-'f RVi ii F-17i; O week following hllenge 11O A- N-3 < 6-4; 2 21 = v~~~~em-m8 *. ~~~~~FRV F I week following hllenge FIG. 3. Geometri men serum neutrlizing ntibody ginst originl infeting virus in (A) onventionl nd (B) SPF ts fter hllenge with the sme (*) or heterologous feline liivirus. Mens for those ts showing linil response to hllenge (F-17ii, FRVii, 255ii) re plotted seprtely from those resisting hllenge (F-17i, FRVi, 255i).

7 - >1 z VOL. 11, x- U- B. 9 W >1 8 FIC body (B) S ross prov nee CROSS-PROTECTION AMONG FELINE CALICIVIRUSES,ssitte reonsidertion of the former onept neou nt on from with lss thou viru re r do o file thre4 nti~ se, TI sso fetii supp re: pth t with hroni upper respirtory disese. In 10 other studies (15) M-8 t dosge level some 10-fold higher thn used in this work did show FC 8-,_Nj==-~3 some pthogeniity in terms of mild oulr < 2554CFI dishrge nd lingul ulertion. N-3, 255, FC, CF1, nd 5936 re signifint pthogens, how- *.:.- - ever, nd were vlid hllenge viruses. *.._' - -._ ~5936 The homologous serum neutrlizing ntibody 4. response to initil infetion ws generlly rpid, being well developed t 2 weeks. This ws in 2- greement with the finding of Olsen et l. (11) for FCV-F9. The response ws prtiulrly mrked with the onventionl ts infeted <]o 2 i 6 l'01 i with M-8, where there were signifint preweek following hllenge inoultion titers. It seemed to be seondry 10 response nd presumbly refleted prior exposure to n FCV, probbly M-8 whih ws indeed N-3 originlly isolted from the olony from whih C -*CFI the mjority of the onventionl ts originted. Pre-inoultion ntibody titers ginst the 6-,, 9255 other FCV mong the onventionl ts were of low order nd ould still represent the hetero *5935 typi response indued by n M-8 exposure..' The low-level neutrlizing tivity seen in the SPF ser of severl ts in different groups prior 2- to infetion is probbly nonspeifi sine there hd not been ny linil or virologil evidene <1..,,.. of livirus infetion in the SPF olony from 2 4o 6 8 l0 2 whih these ts were obtined immeditely week following hllenge prior to use. 4. Geometri men serum neutrlizing nti- Olsen et l. (11), working with two liiviginst hllenge virus in (A) onventionl nd ruses, found tht serum neutrlizing ntibody 'PF t groups, before nd fter hllenge. to the heterotypi strin developed, but t only low level, by dy 35 postinfetion, lthough s-protetion hieved between the viruses, heterotypi omplement fixtion inhibiting ide onfirmtion of these erlier results nd ntibody hd ppered by dy 21. Exept for virus N-3, where the heterotypi serum neutrlof the feline liiviruses being heteroge- izing response ws delyed prtiulrly in the 5S group with only low-level, nonsignifi- onventionl group, the heterologous ntibody ntigeni interreltionship. This erlier response in the present work followed very Wept me bout in prt by extrpoltion losely the development of homologous serum the sitution with the humn rhinoviruses, neutrlizing, lthough t generlly lower order whih the feline liiviruses hve been of mgnitude by ftor of between two nd 64. 3ified in the fmily of piornviruses, l- Titers to M-8 gin were generlly higher nd igh in seprte gener. Even with the rhino- this gin presumbly is refletion of prior ses, lthough numerous distint serotypes exposure to this ntigen in the onventionl eognized, heterologous ntibody responses group, together with possibly more effiient ur (7, 6). Other workers, however (17), neutrliztion of M-8 by ntibody thn of the!d to show suh heterologous responses to other viruses. e rhinovirus serotypes in vines, but the Present work hs not eluidted the imporgeniity of the vines ws poor in two tne of nsl or other lol ntibody reltive to s. serum ntibody suh s those found working he vrible, generlly mild symptomtology with humn rhinoviruses (4, 10, 12, 13). The ited with t lest experimentl FCV in- neutrlizing tivity present in nsl seretions on hs been noted previously (8, 15) nd is ws of low order, if detetble t ll, but did ported by this work, where F-17 nd FRV orrelte somewht with the level of serum mildly pthogeni nd M-8 ppers non- neutrlizing ntibody. Titers of 1:20 were obogeni inspite of its originl isoltion from 883 tined in few ts, nd this pek ws generlly

8 884 POVEY AND INGERSOLL INFECT. IMMUN. rehed 2 to 4 weeks fter infetion or hllenge nd fell rpidly. The onventionl ts infeted with M-8 virus were gin n exeption, showing titers s high s 1:200, probbly refleting both the prior sensitiztion of this group nd lso the generlly higher ntibody responses to M-8 ntigen. Virl shedding by the mjority of ts posthllenge, in the bsene of ny linil symptoms nd in the presene of ntibody, is not unexpeted, hving been noted previously with FCV hllenge (15) nd with other viruses, for instne humn rhinovirus (4). It is quite pprent, however, tht the durtion of exretion fter hllenge (overll men, 3.1 dys) ws muh shorter thn fter initil infetion (overll men, 11.3 dys). Brtholomew nd Gillespie (1) reported tht ts reovered from infetion with FCV-CFI did not resist hllenge with FCV-FJ, but their riteri for this were linil illness nd/or virus isoltions, so it is not ler whether ny of the ts hllenged showed linil signs. Of the six ts tht showed ny symptoms fter hllenge, only two were signifintly severe. These were ts whih were hllenged, s well s primrily infeted, with FCV 255. With these ts, lthough neither hd preinoultion ntibody to FCV 255, the linil response to the originl infetion hd been mild nd one t did not yield virus fter infetion. The low serologil response orrelted with this, but fter hllenge titers rose to level on pr with the rest of the group. Bsed on the results of the hllenge experiment, it would seem tht serum neutrlizing titer of 4.0 (1:16) or greter indites protetion ginst hllenge with vrious FCV, t lest t the TCID5o hllenge level. Further, serum neutrlizing titer of 2.6 (1:7) or less orreltes with suseptibility to hllenge with heterologous FCV. The protetive titer of neutrlizing tivity in nsl seretion is diffiult to quntify, but ll 20 ts tht hd suh tivity detetble (men level, 2.3 or 1:5) did resist hllenge linilly lthough they still showed trnsient virus shedding (men durtion, 2.6 dys). The likely persistene of the ntibody response is impossible to forest, but there is no tendeny for deline of titers up to 8 weeks post-hllenge. It would hve been desirble to inlude in the experiment some FCV isoltes from other prts of the world besides the United Sttes, but importtion lienses were impossible to obtin t the time. The extensive ross-neutrlizing tivity previously reported (14), however, whih inluded results using isoltes from severl ountries, would indite tht vlid extrpoltion ould be mde of the in vivo ross-protetion obtined with the isoltes urrently tested, to inorporte most, if not ll, feline liiviruses. Work will ontinue to test this hypothesis. ACKNOWLEDGMENTS We would like to thnk W. H. Bekenhuer, Vie-President of Reserh nd Development, Norden Lbortories, for his ontinuous interest in the support of this work, E. V. Dvis for enourgement nd dvie, Gry Mihud for onsientious re of the ts, nd finlly R. H. Johnson for his omments on the drft of this pper. LITERATURE CITED 1. Brtholomew, P. T., nd J. H. Gillespie Feline viruses. 1. Chrteriztion of four isoltes nd their effet on young kittens. Cornell Vet. 58: Bittle, J. L., C. J. York, J. W. Newberne, nd M. Mrtin Serologi reltionship of new feline ytopthogeni viruses. Am. J. Vet. Res. 21: Burki, F Piornviruses of ts. Arh. Gesmte Virusforsh. 15: Busho, R. F., J. C. Perkins, J. L. S. Knopf, A. Z. Kpikin, nd R. M. Chnok Further hrteriztion of the lol respirtory trt ntibody response indued by intrnsl instilltion of intivted rhinovirus 13 vine. J. Immunol. 108: Crndell, R. A A desription of eight feline piornviruses nd n ttempt to lssify them. Pro. So. Exp. Biol. Med. 126: Fleet, W. F., R. G. Dougls, Jr., T. R. Cte, nd R. B. Couh Antibody to rhinovirus in humn ser. II. Heterotypi responses. Pro. So. Exp. Biol. Med. 127: Hmre, D., A. P. Conelly, Jr., nd J. J. Proknow Virologi studies of ute respirtory disese in young dults. 3. Some biologi nd serologi hrteristis of 17 rhinovirus serotypes isolted Ot June J. Lb. Clin. Med. 64: Hoover, E. A., nd D. E. Khn Lesions produed by feline piornviruses of different virulene in pthogen-free ts. Vet. Pthol. 10: Khn, D. E., nd J. H. Gillespie Feline viruses. X. Chrteriztion of newly-isolted piornvirus using interstitil pneumoni nd ulertive stomtitis in the domesti t. Cornell Vet. 60: Knopf, H. L. S., J. C. Perkins, D. M. Bertrn, A. Z. Kpikin, nd R. M. Chnok Anlysis of the neutrlizing tivity in nsl wsh nd serum following intrnsl vintion with intivted type 13 rhinovirus. J. Immunol. 104: Olsen, R. G., D. E. Khn, E. A. Hoover, N. J. Sxe, nd D. S. Yohn Differenes in ute nd onvlesent-phse ntibodies of ts infeted with feline piornviruses. Infet. Immun. 10: Perkins, J. C., D. 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