Outcome of Influenza Infection: Effect of Site of Initial

Transcription

1 INFECTION AND IMMUNITY, Aug. 1980, p /80/ /09$02.00/0 Vol. 29, No. 2 Oucome of Influenza Infecion: Effec of Sie of Iniial Infecion and Heeroypic Immuniy ROBERT A. YETTER, SHOSHANA LEHRER, REUBEN RAMPHAL, AND PARKER A. SMALL, JR.* Deparmen of Immunology and Medical Microbiology, Universiy of Florida, College ofmedicine, Gainesville, Florida An infecion esablished hroughou he oal respiraory rac of mice wih a highly lung adaped influenza virus (HON1) led o deah from viral pneumonia. The 50% lehal dose (LD5o) was approximaely he same as he 50% infecious dose (ID50). An infecion wih he same virus iniiaed in he nasal mucosa spread o he rachea and lungs over a 3- o 5-day period bu was no lehal excep a very high infecing doses. The LDr0 was 30,000 imes he ID5o. Mice ha had recovered from a prior infecion wih A/PC/73(H3N2) demonsraed enhanced recovery (heeroypic immuniy) when challenged wih A/PR/8/34(HON1). Heeroypically immune mice infeced while anesheized wih his poenially lehal virus sopped shedding virus from he nose, rachea, and lungs by day 7 and recovered. Heeroypically immune mice, infeced awake, sopped shedding virus from he nose by day 5, and, in fac, he virus did no spread o he rachea or lungs. Thus, some of he variaion in he severiy of influenza infecions may be explained by wo facors: he sie of iniial infecion and previous infecion wih heeroypic influenza virus. From he ime of Sydenham's classic descripion of influenza in 1679 (11), i has been realized ha influenza infecion may resul in a variey of disease saes. These range from inapparen infecion hrough a mild upper respiraory infecion and racheobronchiis o a severe, occasionally lehal, viral pneumonia. The reasons for his specrum of severiy are no clear. Adapaion of geneic variaion of he virus, sie of iniiaion of infecion, and immune saus of he hos may all modify he course ofinfluenza infecion. Geneic variaion of he virus has been shown o occur during sequenial passage from hos o hos and o resul in increased lehaliy of he virus (2, 7). Alhough his adapaion may accoun for he increase in he virulence of a virus during he course of an epidemic, i is unlikely ha i is he sole reason for he variaion in sie and severiy of disease, since i does no accoun for he age disribuion of influenza moraliy nor for he range of severiy seen in paiens apparenly infeced from he same source. In regard o sie of infecion, viral pneumonia is he mos commonly sudied resul of influenza infecion in he mouse, bu he work of Iida and Bang (10) wih nasally adaped virus indicaed ha an influenza infecion could be resriced o he nasal epihelium, hereby resembling he upper respiraory infecion in hu Presen address: Insiue of Biological Research, Ness- Ziona, Israel. mans. The immune saus of he hos also modifies influenza infecions. In addiion o proecing from reinfecion wih he homoypic virus (3, 8), recovery from prior infecions also affecs he oucome of infecion wih heeroypic ype A influenza viruses (17). The purpose of his sudy was o deermine he effec of he sie of iniial infecion and heeroypic immuniy on he oucome of influenza infecion. MATERIALS AND METHODS Animals. Seven-week-old male A/J mice were obained from Jackson Laboraories, Bar Harbor, Maine, and were housed eigh per cage. All animals were kep under condiions which preven cage-o-cage infecion. Mice received food and waer ad libium. Viruses. The viruses used, A/PR/8/34(HON1) and A/PC/73(H3N2), were obained from he Research Resources Branch, Naional Insiue of Allergy and Infecious Diseases, Behesda, Md. Boh viruses were passaged wice in mouse lungs. A suspension of he riuraed lungs of A/PC/73(H3N2)-infeced mice in serile phosphae-buffered saline (PBS), ph 7.2, was used as a sock virus culure. The A/PR/8/34(HON1) virus was passed once in 10-day-old embryonaed chicken eggs o provide a large sock of virus in allanoic fluid. Infecion. Mice were infeced inranasally while awake or anesheized wih penobarbial sodium (0.06 mg/g of body weigh). Mice given ha amoun of penobarbial sodium were unresponsive o painful simuli. Unanesheized mice received 3 drops (0.03 ml) and anesheized mice received 6 drops (0.06 ml) of virus suspensions. These suspensions were adjused 654

2 VOL. 29, 1980 so ha boh inocula conained he same amoun of virus. Purple proein. Purple proein is a progeseroneinduced, basic glycoproein isolaed from he uerine fluid of pigs (5). Radioiodinaed purple proein was kindly supplied by R. M. Robers, of he Universiy of Florida. Radioaciviy was measured in a well-ype gamma couner. Serum anibody iers. Mice were anesheized wih penobarbial sodium and hen exsanguinaed from he reinal arery by removing he eye. Sera used for hemaggluinaion inhibiion assays were firs adsorbed wih kaolin and chick eryhrocyes and heaed a 560C for 30 min (4). Hemaggluinaion and hemaggluinaion inhibiion iers were performed wih a microier ki, using disposable microier plaes (Cooke Engineering Co., Alexandria, Va.) as described by Sever (18). Virus isolaion and iraion. Virus was isolaed from he lungs, rachea, and nasal caviy of he mice according o he following mehod. Afer exsanguinaion, he skin was swabbed wih ehanol and he animal was opened venrally, along he midline from he xiphoid process o he poin of he chin. The rachea and lungs were removed asepically and placed in a serile peri dish. The lungs were separaed from he rachea and riuraed in 2 ml of PBS. The rachea was biseced horizonally, and he upper half was riuraed in 1 ml of PBS. The lower half of he rachea was saved for microscopic sudies. The animal was hen decapiaed, he mandible was removed, and a 27-gauge 0.5-inch (ca. 1.3-cm) needle was inroduced ino he poserior opening of he nasopharynx. Nasal washes were performed by flushing 1 ml of PBS hrough he nasopharynx and ou he exernal nares. All samples were separaed ino wo equal porions and frozen a -80'C unil hey were assayed. Virus was deeced by injecing 0.1 ml of he samples ino he allanoic caviies of 10-day embryonaed chicken eggs which had previously received 0.1 ml of anibioic soluion conaining 250,000 U of penicillin per ml and 250 mg of srepomycin per ml. The eggs were incubaed for 3 days a 360C. The allanoic fluids were harvesed and esed for hemaggluinaion as described by Allan e al. (1). If he sample was posiive, serial 10-fold diluions were injeced ino eggs in riplicae, and he 50% egg infecious dose (EID50) was calculaed by he mehod of Reed and Muench (16). SEM. Tracheas for scanning elecron microscopy (SEM) were placed in 0.1 M sodium cacodylae conaining 2.5% gluaraldehyde and 0.1% calcium chloride (ph 7.4) and allowed o fix for a leas 24 h a 40C. The racheas were removed from he fixaive and biseced longiudinally. One half was reurned o he fixaive, and he oher was dehydraed in graded concenraions of aceone (70 o 100%). Specimens were criical-poin dried in a Bomar SPC 900/Ex criicalpoin drying apparaus (Bomar Corp., Tacoma, Wash.), coaed wih gold palladium in a Hummer II shadowing machine (Technics, Alexandria, Va.), and examined wih a Novascan 30 elecron microscope (Semco, Oawa, Canada). Saisical analysis. Moraliy was compared by Fisher's exac es (19). Viral iers and he course of FACTORS AFFECTING OUTCOME OF INFLUENZA 655 virus shedding were compared by Suden's -es (13) and compuer-generaed regression analysis. For he purpose of saisical analysis, logo of undeecable amouns of virus was defined as -1. RESULTS Effec of aneshesia and volume of inoculum on fluid flow in he respiraory rac. A preliminary experimen was done o quaniae he reenion of fluid afer is insillaion ino he nares of anesheized and unanesheized mice. Radioiodinaed purple proein was dripped ino he noses of anesheized (0.06 ml) and unanesheized (0.03 ml) mice. Ten minues laer he mice were exsanguinaed, and he blood, head, rachea, lungs, and res of he body were separaed and couned in a gamma couner. The resuls, expressed as percenage of inpu radiaion correced for background, are presened in Table 1. Two poins are immediaely eviden. Whereas abou one-fifh of he inoculum was reained in he lungs of he anesheized animals, virually none of he inoculum was found in he lungs of he unanesheized animals. Second, he oal amoun of inoculum reained in he anesheized animals was hree o four imes ha reained in he unanesheized animals. These resuls indicaed ha by using aneshesia, i was possible o expose he oal respiraory rac, and when he use of aneshesia was omied, i was possible o limi he exposure largely o he nasal epihelium. Effec of sie of iniial infecion on survival. The oucome of infecion iniiaed in he oal respiraory rac or nasal epihelium wih TABLE 1. Inoculum reenion 10 min afer insillaion ino anesheized or unanesheized mice' Inoculum reenion wih inoculum Reenion sie given while: Asleep Awake Head 9.2 ± ± 1.6 Trachea 0.3 ± ± 0.1 Lung 21.8 ± 14.1 O.lb ± 0.1 Body 5.9 ± ± 3.5 Blood 0.2 ± ± 0.2 a'two groups of five mice were given a radioiodinaed proein soluion inranasally, one group while anesheized and he oher wihou aneshesia. Ten minues afer adminisraion of he soluion, he mice were sacrificed and disseced, and organs were assayed for reained radioaciviy. The resuls given are percenage of inpu radiaion, less background, ± sandard deviaion. bgroup of four; one mouse had 10% reained in he lungs and was excluded because 10% was more han 4 sandard deviaions from he mean.

3 656 YETTER ET AL. 1.6 x 10:' or 6.4 x 104 EID5o of A/PR/8/ 34(HON1) is presened in Table 2. Mice infeced hroughou he respiraory rac died, whereas hose given an iniial nasal infecion survived wih a single excepion. Mos of hese deahs were beween days 4 and 7, he excepion being he single mouse wih an iniial nasal infecion who died abou day 12. In he seven cases where necropsies could be done, deah was due o viral pneumonia. To rule ou he possibiliy ha mice survived he iniial nasal infecion only because such an infecion required much more virus o esablish han did a oal respiraory rac infecion, he 50% infecious dose (ID50) and 50% lehal dose (LD50) for each mode of infecion were esablished (Table 3). The resuls indicaed ha in he case of oal respiraory rac infecion, 1 LD50 was approximaely he same as 1 IDr,. However, for an iniial nasal infecion, 1 LD50 was approximaely 30,000 IDso. I was also found ha he ID50 for an iniial nasal infecion required only 60 imes as much virus as did he ID5o for oal respiraory rac iniiaion. Effec of sie of iniial infecion on course of virus shedding. Groups of anesheized and unanesheized mice were infeced wih 1.6 x 103 EID5o of A/PR/8/34(HON1). Virus shedding from he nose, rachea, and lungs, as well as he oucome of infecion, was evaluaed (Fig. 1). Again, mice given an iniial infecion hroughou he respiraory rac, i.e., anesheized mice, died, bu hose infeced while awake survived. The nasal virus iers were no significanly differen beween he wo groups on days 1, 3, and 5. This was no he case wih iers in he lower respiraory rac. On day 1, he virus iers in he rachea and lungs of animals infeced iniially in he nasal epihelium were no significanly dif- TABLE 2. Effec of sie of iniial infecion wih influenza on survival' No. of Dos) Sie of iniial infecion (dead P value oal) 6.4 x 104 Toal respiraory 8/ Nasal 0/8 1.6 X 103 Toal respiraory 7/ Nasal 1/8 a Four groups of eigh mice were infeced wih A/ PR/8/34(HON1) influenza virus. Two groups received 6.4 x 104 EID50, and wo groups received 1.6 x 10' EID,)o of he virus. One of he groups a each virus dose was infeced while awake (nasal iniiaion), and he oher was infeced while anesheized (oal respiraory rac iniiaion). P values were deermined by Fisher's exac es. INFECT. IMMUN. TABLE 3. Effec of sie of iniial infecion on LDr, and ID50o Infecion iniiaion sie LDI, ID1) Toal respiraory Nasal 1o " ' 10-" 10" ' " IDr, and LDr, for boh an iniial nasal and oal respiraory rac infecion wih A/PR/8/34(HON1) influenza virus were deermined by infecing groups of mice (hree o eigh each) wih serial 10-fold diluions of sock virus. Mice in he IDrd group were sacrificed on day 1, and he nasal or lung issues were assayed for he presence of virus. Mice in he LDro groups were observed for 14 days. The resuls are repored in erms of EID!4 unis. feren from zero. However, hese iers were significanly (P < 0.01) lower han he iers in racheas and lungs of mice given an iniial oal respiraory rac infecion. By day 3 he racheal iers were comparable for boh infecion modes, bu i was no ill day 5 ha he lung iers of he nasal iniiaion group rose o he levels of hose animals given a oal respiraory rac infecion. Effec of sie of iniial infecion on racheal desquamaion. Figure 2 shows he course of racheal desquamaion afer infecion iniiaed in he nasal or oal respiraory rac. A comparison of he day 3 micrographs shows ha whereas he racheal epihelium of mice ha received a oal respiraory rac infecion was compleely desquamaed, he rachea in he nasally iniiaed infecion was normal. In fac, he rachea was no compleely desquamaed in his case unil day 5, confirming he ime delay necessary for spread of he virus from he nose o he lower respiraory rac. Heeroypic immuniy in mice wih a oal respiraory rac infecion. A proocol for heeroypic immuniy experimens is shown in Figure 3. Mice were made heeroypically immune by infecing heir oal respiraory racs wih 103 EID.% of he nonlehal A/PC/73(H3N2) 30 days before he challenge infecion. These heeroypically immune mice were hen anesheized and challenged wih 0.06 ml of virus suspension conaining 1.6 x 103 EID50 of A/PR/ 8/34(HON1). Mice were sacrificed on days 1, 3, 5, 7, and 9, and virus iers were deermined (Fig. 4). The virgin mice daa depiced in Fig. 4 are he same as hose shown in Fig. 1. None of he virgin mice survived pas day 5. Virus iers in he nose or rachea were no significanly differen beween virgin and heeroypically immune mice on day 1. However, whereas he iers in he virgin mice remained high unil deah, he virus iers in he heeroypically immune mice decreased. The iers were significanly differen

4 VOL. 29, LOGlO 4- EID5O 3- LOGO 4 EID I 6 5. LOGO4 EID I 0 NOSE TRACHEA LUNG i'"s'' \\~~~~~ FIG. 1. Comparison of virus iers shed from virgin mice given an iniial oal respiraory rac (0) or iniial nasal (0) infecion wih A/PR/8/34(HONI). Means and sandard errors are given. If no bar is shown, he sandard error is conained wihin he poin. Five mice were used for each poin in he oal respiraory rac infecion, and seven mice were used for all poins excep day 2 (four mice) in he iniial nasal infecion. The daa for iniial nasal infecion are a combinaion of he resuls of wo experimens. There was no difference beween he resuls of he wo experimens. Log1o of undeecable levels of virus is represened as -1. FACTORS AFFECTING OUTCOME OF INFLUENZA 657 from hose of virgin mice, in he nose by day 3 and in he rachea or lungs by day 5 (P < 0.01). Virus was undeecable in he heeroypically immune mice by day 9. Schulman and Kilbourne's (17) finding ha heeroypic immuniy did no exend o ype B influenza was confirmed (daa no shown). Sudies of racheal desquamaion by SEM corroboraed he viral sudies (Fig. 5). A corresponding imes, he heeroypically immune mice always had less racheal desquamaion han did virgin mice and never suffered oal desquamaion. The day 7 micrographs indicae advanced regeneraion in he heeroypically immune racheas. This could have been due eiher o more rapid regeneraion or o less severe desquamaion of he racheal epihelium. Effec of heeroypic immuniy in iniial nasal infecion. For his sudy, unanesheized heeroypically immune mice were infeced wih 0.03 ml conaining 1.6 X 101 EIDrxo of A/PR/8/ 34(HON1). The daa from he nasal infecion of virgin mice previously shown in Fig. 1 are repeaed in Fig. 4. As has been previously shown, he virus spread from he nose o he lower respiraory rac of he virgin mice (Fig. 6). The nasal iers were no significanly differen in he wo groups of mice on days 1 and 2, bu by day 3 he nasal ier in he virgins was rising whereas he ier in he heeroypically immune mice was dropping. By day 5, heeroypically immune mice were no shedding virus from he nose and he virus had no spread o he lower respiraory rac. The racheas of heeroypically immune mice given an iniial nasal infecion never demonsraed any signs of desquamaion (micrographs no shown). Serum anibody response in heeroypic immuniy in mice. The serum hemaggluinaion inhibiion iers of virgin and heeroypically immune mice agains A/PR/8/34(HON1) and A/PC/73(H3N2) are summarized in Table 4. Conrary o wha has been repored by Schulman and Kilbourne (17), he appearance of serum anibody agains he challenge virus was no earlier in he heeroypically immune han in he virgin mice. DISCUSSION This sudy demonsraes ha he sie of iniial infecion and heeroypic immuniy have a major effec on he oucome of influenza infecion. The work of Iida and Bang (10) suggesed ha we could esablish an infecion in he nasal epihelium wihou simulaneously infecing he res of he respiraory rac. This was suppored by he fluid flow experimen in which adminisraion of a radioiodinaed proein soluion showed ha he fluid inoculum reached he lung in he anesheized mouse bu no in hose given he smaller volume of inoculum while awake. This mean ha a virus inroduced in a small volume ino he nosrils of an unanesheized mouse would infec he nasal caviy and no come ino conac wih he res of he respiraory rac a ha ime. The difference in he oucome of he wo ypes of infecions is, quie lierally, ha of life and deah. The amoun of virus required o infec anesheized mice is abou he same as ha required o kill hem; i.e., he LDro -approximaes he ID50. In conras, i akes 30,000 imes he amoun of virus o kill unanes-

5 658 YETTER ET AL. TOTAL RESPIRATORY TRACT INFECT. IMMUN. 3 5 / 9 FIG. 2. Comparison by SEM of racheal desquamaion in mice given an iniial nasal or oal respiraory rac infecion wih A/PR/8/34(HONJ).

6 VOL. 29, 1980 Infec (H3N2) Infec (HON I) i o k Virus serum, racheal SEM, virus from nose, rachea a lung Infec (HONI) # f F V serum, racheal SEM, virus from nose,rochea a lung FIG. 3. Proocol for heeroypic immuniy experimens in mice. heized mice as i does o infec heir noses. The mos obvious conclusion is ha he sie of infecion is crucial in deermining he oucome of he infecion, bu several alernae conclusions mus be ruled ou. Could he difference beween he wo modes of infecion be a funcion of he difference in he volume of he inocula used? An experimen was performed in which unanesheized mice received approximaely 103 EID5o of HONi influenza virus in 0.06 ml and did no develop pneumonia (daa no shown); herefore, he volume does no seem o be criical. A second alernae explanaion is ha he unanesheized mice sneezed ou almos all of he inoculum. This seems highly unlikely since he fluid flow experimens showed ha he unanesheized mice reained abou one-quarer of he inoculum reained by he anesheized animals and required abou 60 imes as much virus o infec i. Incidenally, he 15-fold discrepancy beween hese wo numbers could represen eiher inaccuracies in he ID5o deerminaions or an innae resisance of he nose relaive o he oal respiraory rac, bu in eiher case he 30,000-fold difference beween he LD50 and ID50 in he unanesheized mouse suggess srongly ha he discrepancy beween he anesheized and unanesheized mice canno be due o loss of inoculum due o sneezing. A hird possibiliy is ha he adminisraion of fluid ino he lungs predisposes hem o viral infecion and subsequen lehal pneumonia. An experimen was herefore done wherein mice were anesheized, given 0.06 ml of saline, allowed o wake up, and hen infeced while awake. The oucome of infecion was he same as for conrols no given saline (daa no shown); herefore, fluid in he lungs seems an unlikely explanaion for he observed resuls. We believe ha he survival of mice given an iniial nasal infecion is due o he amoun of ime necessary for he virus o spread hrough he respiraory rac and reach high iers in he lung. The delay was eviden no only in virus FACTORS AFFECTING OUTCOME OF INFLUENZA 659 isolaion bu also in SEM of he rachea. This presumably provided sufficien ime for he immune sysem o respond o he infecion. Alhough here is no sufficien informaion o know wha aspec of he immune sysem is lifesaving, i is ineresing o speculae abou he proecive mechanism. Yap and Ada (22) repored ha he appearance of cyooxic T cells in influenza-infeced mice was followed by deah if he virus iers in he lung were high. However, if sublehal doses of he virus were given, clearance of he virus followed he appearance of cyooxic T cells. Perhaps, in mice given a oal respiraory rac infecion, wih high virus iers in he lung a an early sage and, presumably, considerable alveolar involvemen, cyooxic T 5 LOG1O 4 EID ' -Il 5. LOGO 4 - EID *- -l 6' 5. LOG 1O EID50 3 2' 01 -Il NOSE o - - _- o _ o TRACHEA LUNG I FIG. 4. Comparison of virus shedding from virgin (0) and heeroypically immune (0) mice given a oal respiraory rac infecion wih A/PR/8/ 34(HONI). Means and sandard errors are given. If no bar is shown, he sandard error is conained wihin he poin. Five mice were used for each poin in he virgin group, and hree mice were used for all poins excep day 9 (2 mice) in he convalescengroup. Logio of undeecable levels of virus is represened as -1.

7 660 YETTER ET AL. VIRKIN INFECT. IMMUN. HETEROTYPICALLY IMUNE i FIG. 5. Comparison by SEM of racheal desquamaion in virgin and heeroypically immune mice given a oal respiraory rac infecion wih A/PR/8/34(HONI).

8 VOL. 29, 1980 LOGO 4- EID LOG10 4 EIDSO TRACHE, I I LUNI cells may cause he pahological change bs of viral pneumonia. In mice given an iniial naswal infecus in he ion, he consequen lower levels of viri lungs may resul in less alveolar involvemen and consequenly less cell desrucion by cyo- for oxic T cells which migh hen be respondisible clearance of he virus. The sie of iniial infecion may allso be a major concern in he human disease. II is sill no clear how influenza infecion is ranksmied. Gwalney e al. (9), however, has shoown ha ransmission of rhinovirus infecion in humans is generally by hand-o-hand conac. Should his observaion hold rue for influenza as well, I FACTORS AFFECTING OUTCOME OF INFLUENZA 661 hen many infecions would be iniiaed in he nasal epihelium. This should no be aken o rule ou infecion due o exposure o an aerosol of he virus, which could resul in a oal respiraory rac iniiaion. Thus, i is possible ha infecion may be iniiaed in eiher way in humans. Heeroypic immuniy modified he course of infecions iniiaed eiher in he nose or hrough- 9 ou he respiraory epihelium. Schulman and Kilbourne's observaion ha heeroypic immuniy could be lifesaving in he case of infecions iniiaed hroughou he respiraory rac (17) was confirmed. Furhermore, when he infecions were iniiaed in he nasal epihelium, heeroypic immuniy prevened he spread of he virus from he nose o he res of he respiraory rac. Heeroypic immuniy appears o play a role 9 in recovery bu no in prevenion of influenza E IG infecion. This is suggesed by he fac ha day 1 virus iers from he upper respiraory rac of heeroypically immune and conrol animals were similar, whereas a laer imes he iers from he heeroypically immune animals are 2- lower. Wha migh be he mechanism by which heeroypic immuniy promoes recovery? Possibly heeroypic immuniy is he resul of a 0- I more rapid rise in serum anibody o challenge I T T r virus in he heeroypically immune han in he virgin mice. Alhough reamen wih kaolin FIG. 6. Comparison of virus shedding frcm virgin may remove specific anibodies (13), all sera (0) and heeroypically immune (0) mice afer an were reaed in he same manner. Tha being iniial nasal infecion wih A/PR/8/334(HONI). he case, he observaion ha such a rise in Means and sandard errors are given. If ino bar is serum anibody did no occur indicaes ha shown, he sandard error is conained w)ihin he serum anibody is unlikely o be he mechanism poin. Seven mice were used for each poin e. excep day of heeroypic immuniy. This hypohesis is sup- hree 2, when four mice were used, and day 9, wihen mice were used for each poin. The daa Presened are a combinaion of he resuls of wo expwerimens. TABLE 4. Serum hemaggluinaion inhibiion iers There was no difference beween he resuls of hese afer a nasal infecion wih HONI influenza virus" wo experimens. Loglo of undeecable levelis of virus is represened as -1. Assay Tier on day: Group Virus Virgin H3N2 <8 <8 <8 <8 <8 HONI <8 <8 <8 <8" 48 Heeroypi- H3N cally immune HONI <8 <8 <8 <8 21' "Sera of wo groups of mice, virgin and heeroypically immune, were esed for hemaggluinaion inhibiion aciviy agains A/PC/73(H3N2) and A/PR/ 8/34(HON1) afer he mice were given an iniial nasal infecion. The heeroypically immune mice had been previously infeced wih A/PC/73(H3N2). The sera of four mice were esed on each of 5 days. "One mouse of 4 had a ier of 16. Range from 8 (hree mice) o 1,024 (one mouse).

9 662 YETTER ET AL. pored by he finding ha passively adminisered aniserum does no proec or enhance recovery of he upper respiraory rac for influenza (15). Alhough he possibiliy ha his enhanced recovery is he resul of a rapid rise in local secreary anibody has no been ruled ou, i is perhaps more likely ha he mechanism of heeroypic immuniy is relaed o cyooxic T- cell funcion. The increased incidence of influenza pneumonia observed in young children (6) may be due o he absence of heeroypic immuniy. I has been repored ha aduls no only have lower incidence of viral pneumonia bu also shed virus for a shorer ime han do children (21). This decreased ime of shedding resembles he effecs of heeroypic immuniy seen in animals. The drawback o posulaing a role for heeroypic immuniy in he human disease has been he repored shor-erm naure of he phenomenon (12), bu our sudies have indicaed ha heeroypic immuniy is sill funcional a leas 18 monhs afer he firs infecion and consequenly could play a significan role in he modificaion of he disease (23). Thus, he sie of iniial infecion and heeroypic immuniy play major roles in deerminaion of he course and oucome of influenza infecion in mice. In humans, lack of heeroypic immuniy and an iniial nasal infecion may lead o he spread of he virus down he respiraory rac and a severe bu probably nonlehal disease. The combinaion of heeroypic immuniy and iniial nasal infecion migh resul in an infecion limied o he upper respiraory rac and hence a relaively mild disease. Togeher wih he inheren pahogeniciy of he virus and geneic facors of he hos, hese facors may explain he wide range of disease saes ha are seen in influenza in humans. ACKNOWLEDGMENTS The sudy was suppored by Public Healh Service grans AI from he Naional Insiue of Allergy and Infecious Diseases. R.A.Y. was a recipien of Public Healh Service gran AI 0128 from he same Insiue. We graefully acknowledge Rober Cogliano and George Gifford for many helpful discussions. We also acknowledge Harold S. Ginsberg for his suggesion ha he amoun of inoculum reained in awake versus anesheized mice should be quaniaed. LITERATURE CITED 1. Allan, W. H., C. R. Madeley, and A. P. Kendall Sudies wih avian influenza A viruses: cross proecion experimens in chickens. J. Gen. Virol. 12: Andrewes, C. H., P. 0. Laidlaw, and W. Smih The suscepibiliy of mice o he viruses of human and INFECT. IMMUN. swine influenza. Lance ii: Andrewes, C. H., P. O. Laidlaw, and W. Smih Influenza: observaions on he recovery of virus from man and on he anibody conen of human sera. Br. J. Exp. Pahol. 16: Barber, W. H., and P. A. 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