Vol. 29, No. 2INFECTION AND IMMUNITY, Aug. 1980, p. 654-6620019-9567/80/08-0654/09$02.00/0

Outcome of Influenza Infection: Effect of Site of InitialInfection and Heterotypic Immunity

ROBERT A. YETTER, SHOSHANA LEHRER,t REUBEN RAMPHAL, AND PARKER A. SMALL, JR.*

Department ofImmunology and Medical Microbiology, University of Florida, College ofMedicine,Gainesville, Florida 32610

An infection established throughout the total respiratory tract of mice with ahighly lung adapted influenza virus (HON1) led to death from viral pneumonia.The 50% lethal dose (LD5o) was approximately the same as the 50% infectiousdose (ID50). An infection with the same virus initiated in the nasal mucosa spreadto the trachea and lungs over a 3- to 5-day period but was not lethal except atvery high infecting doses. The LDr0 was 30,000 times the ID5o. Mice that hadrecovered from a prior infection with A/PC/73(H3N2) demonstrated enhancedrecovery (heterotypic immunity) when challenged with A/PR/8/34(HON1). Het-erotypically immune mice infected while anesthetized with this potentially lethalvirus stopped shedding virus from the nose, trachea, and lungs by day 7 andrecovered. Heterotypically immune mice, infected awake, stopped shedding virusfrom the nose by day 5, and, in fact, the virus did not spread to the trachea orlungs. Thus, some of the variation in the severity of influenza infections may beexplained by two factors: the site of initial infection and previous infection withheterotypic influenza virus.

From the time of Sydenham's classic descrip-tion of influenza in 1679 (11), it has been realizedthat influenza infection may result in a varietyof disease states. These range from inapparentinfection through a mild upper respiratory infec-tion and tracheobronchitis to a severe, occasion-ally lethal, viral pneumonia. The reasons for thisspectrum of severity are not clear.Adaptation of genetic variation of the virus,

site of initiation of infection, and immune statusofthe host may all modify the course ofinfluenzainfection. Genetic variation of the virus has beenshown to occur during sequential passage fromhost to host and to result in increased lethalityof the virus (2, 7). Although this adaptation mayaccount for the increase in the virulence of avirus during the course of an epidemic, it isunlikely that it is the sole reason for the varia-tion in site and severity of disease, since it doesnot account for the age distribution of influenzamortality nor for the range of severity seen inpatients apparently infected from the samesource. In regard to site of infection, viral pneu-monia is the most commonly studied result ofinfluenza infection in the mouse, but the workof Iida and Bang (10) with nasally adapted virusindicated that an influenza infection could berestricted to the nasal epithelium, thereby re-sembling the upper respiratory infection in hu-

t Present address: Institute of Biological Research, Ness-Ziona, Israel.

mans. The immune status of the host also mod-ifies influenza infections. In addition to protect-ing from reinfection with the homotypic virus(3, 8), recovery from prior infections also affectsthe outcome of infection with heterotypic typeA influenza viruses (17). The purpose of thisstudy was to determine the effect of the site ofinitial infection and heterotypic immunity onthe outcome of influenza infection.

MATERIALS AND METHODSAnimals. Seven-week-old male A/J mice were ob-

tained from Jackson Laboratories, Bar Harbor, Maine,and were housed eight per cage. All animals were keptunder conditions which prevent cage-to-cage infection.Mice received food and water ad libitum.

Viruses. The viruses used, A/PR/8/34(HON1) andA/PC/73(H3N2), were obtained from the ResearchResources Branch, National Institute of Allergy andInfectious Diseases, Bethesda, Md. Both viruses werepassaged twice in mouse lungs. A suspension of thetriturated lungs of A/PC/73(H3N2)-infected mice insterile phosphate-buffered saline (PBS), pH 7.2, wasused as a stock virus culture. The A/PR/8/34(HON1)virus was passed once in 10-day-old embryonatedchicken eggs to provide a large stock of virus in allan-toic fluid.

Infection. Mice were infected intranasally whileawake or anesthetized with pentobarbital sodium (0.06mg/g of body weight). Mice given that amount ofpentobarbital sodium were unresponsive to painfulstimuli. Unanesthetized mice received 3 drops (0.03ml) and anesthetized mice received 6 drops (0.06 ml)of virus suspensions. These suspensions were adjusted

654

FACTORS AFFECTING OUTCOME OF INFLUENZA 655

so that both inocula contained the same amount ofvirus.

Purple protein. Purple protein is a progesterone-induced, basic glycoprotein isolated from the uterinefluid of pigs (5). Radioiodinated purple protein waskindly supplied by R. M. Roberts, of the University ofFlorida. Radioactivity was measured in a well-typegamma counter.Serum antibody titers. Mice were anesthetized

with pentobarbital sodium and then exsanguinatedfrom the retinal artery by removing the eye. Sera usedfor hemagglutination inhibition assays were first ad-sorbed with kaolin and chick erythrocytes and heatedat 560C for 30 min (4). Hemagglutination and hemag-glutination inhibition titers were performed with amicrotiter kit, using disposable microtiter plates(Cooke Engineering Co., Alexandria, Va.) as describedby Sever (18).

Virus isolation and titration. Virus was isolatedfrom the lungs, trachea, and nasal cavity of the miceaccording to the following method. After exsanguina-tion, the skin was swabbed with ethanol and theanimal was opened ventrally, along the midline fromthe xiphoid process to the point of the chin. Thetrachea and lungs were removed aseptically and placedin a sterile petri dish. The lungs were separated fromthe trachea and triturated in 2 ml of PBS. The tracheawas bisected horizontally, and the upper half wastriturated in 1 ml of PBS. The lower half of the tracheawas saved for microscopic studies. The animal wasthen decapitated, the mandible was removed, and a27-gauge 0.5-inch (ca. 1.3-cm) needle was introducedinto the posterior opening of the nasopharynx. Nasalwashes were performed by flushing 1 ml of PBSthrough the nasopharynx and out the external nares.All samples were separated into two equal portionsand frozen at -80'C until they were assayed.

Virus was detected by injecting 0.1 ml ofthe samplesinto the allantoic cavities of 10-day embryonatedchicken eggs which had previously received 0.1 ml ofantibiotic solution containing 250,000 U of penicillinper ml and 250 mg of streptomycin per ml. The eggswere incubated for 3 days at 360C. The allantoic fluidswere harvested and tested for hemagglutination asdescribed by Allan et al. (1). If the sample was positive,serial 10-fold dilutions were injected into eggs in trip-licate, and the 50% egg infectious dose (EID50) wascalculated by the method of Reed and Muench (16).SEM. Tracheas for scanning electron microscopy

(SEM) were placed in 0.1 M sodium cacodylate con-taining 2.5% glutaraldehyde and 0.1% calcium chloride(pH 7.4) and allowed to fix for at least 24 h at 40C.The tracheas were removed from the fixative andbisected longitudinally. One half was returned to thefixative, and the other was dehydrated in graded con-centrations of acetone (70 to 100%). Specimens werecritical-point dried in a Bomar SPC 900/Ex critical-point drying apparatus (Bomar Corp., Tacoma,Wash.), coated with gold palladium in a Hummer II

shadowing machine (Technics, Alexandria, Va.), andexamined with a Novascan 30 electron microscope(Semco, Ottawa, Canada).

Statistical analysis. Mortality was compared byFisher's exact test (19). Viral titers and the course of

virus shedding were compared by Student's t-test (13)and computer-generated regression analysis. For thepurpose of statistical analysis, logo of undetectableamounts of virus was defined as -1.

RESULTS

Effect of anesthesia and volume of inoc-ulum on fluid flow in the respiratory tract.A preliminary experiment was done to quanti-tate the retention of fluid after its instillationinto the nares of anesthetized and unanesthe-tized mice. Radioiodinated purple protein wasdripped into the noses of anesthetized (0.06 ml)and unanesthetized (0.03 ml) mice. Ten minuteslater the mice were exsanguinated, and theblood, head, trachea, lungs, and rest of the bodywere separated and counted in a gamma counter.The results, expressed as percentage of inputradiation corrected for background, are pre-sented in Table 1. Two points are immediatelyevident. Whereas about one-fifth of the inocu-lum was retained in the lungs of the anesthetizedanimals, virtually none of the inoculum wasfound in the lungs ofthe unanesthetized animals.Second, the total amount of inoculum retainedin the anesthetized animals was three to fourtimes that retained in the unanesthetized ani-mals. These results indicated that by using an-esthesia, it was possible to expose the total res-piratory tract, and when the use of anesthesiawas omitted, it was possible to limit the exposurelargely to the nasal epithelium.

Effect of site of initial infection on sur-vival. The outcome of infection initiated in thetotal respiratory tract or nasal epithelium with

TABLE 1. Inoculum retention 10 min afterinstillation into anesthetized or unanesthetized

mice'Inoculum retention with inoculum

Retention site given while:Asleep Awake

Head 9.2 ± 5.0 3.8 ± 1.6Trachea 0.3 ± 0.6 0.1 ± 0.1Lung 21.8 ± 14.1 O.lb ± 0.1Body 5.9 ± 2.8 6.6 ± 3.5Blood 0.2 ± 0.2 0.2 ± 0.2

a'Two groups of five mice were given a radioiodi-nated protein solution intranasally, one group whileanesthetized and the other without anesthesia. Tenminutes after administration of the solution, the micewere sacrificed and dissected, and organs were assayedfor retained radioactivity. The results given are per-centage of input radiation, less background, ± stan-dard deviation.

bGroup of four; one mouse had 10% retained in thelungs and was excluded because 10% was more than 4standard deviations from the mean.

VOL. 29, 1980

656 YETTER ET AL.

1.6 x 10:' or 6.4 x 104 EID5o of A/PR/8/34(HON1) is presented in Table 2. Mice infectedthroughout the respiratory tract died, whereasthose given an initial nasal infection survivedwith a single exception. Most of these deathswere between days 4 and 7, the exception beingthe single mouse with an initial nasal infectionwho died about day 12. In the seven cases wherenecropsies could be done, death was due to viralpneumonia.To rule out the possibility that mice survived

the initial nasal infection only because such aninfection required much more virus to establishthan did a total respiratory tract infection, the50% infectious dose (ID50) and 50% lethal dose(LD50) for each mode of infection were estab-lished (Table 3). The results indicated that inthe case of total respiratory tract infection, 1

LD50 was approximately the same as 1 IDr,.However, for an initial nasal infection, 1 LD50was approximately 30,000 IDso. It was also foundthat the ID50 for an initial nasal infection re-quired only 60 times as much virus as did theID5o for total respiratory tract initiation.Effect of site of initial infection on course

of virus shedding. Groups of anesthetized andunanesthetized mice were infected with 1.6 x 103EID5o of A/PR/8/34(HON1). Virus sheddingfrom the nose, trachea, and lungs, as well as theoutcome of infection, was evaluated (Fig. 1).Again, mice given an initial infection throughoutthe respiratory tract, i.e., anesthetized mice,died, but those infected while awake survived.The nasal virus titers were not significantly dif-ferent between the two groups on days 1, 3, and5. This was not the case with titers in the lowerrespiratory tract. On day 1, the virus titers in thetrachea and lungs of animals infected initially inthe nasal epithelium were not significantly dif-

TABLE 2. Effect of site of initial infection withinfluenza on survival'

No. of

Dos) Site of initial infection (dead P value

total)

6.4 x 104 Total respiratory 8/8 0.00008Nasal 0/8

1.6 X 103 Total respiratory 7/8 0.005Nasal 1/8

a Four groups of eight mice were infected with A/PR/8/34(HON1) influenza virus. Two groups received6.4 x 104 EID50, and two groups received 1.6 x 10'EID,)o of the virus. One of the groups at each virusdose was infected while awake (nasal initiation), andthe other was infected while anesthetized (total res-

piratory tract initiation). P values were determined byFisher's exact test.

TABLE 3. Effect of site of initial infection on LDr,and ID50o

Infection initiation site LDI, ID1)

Total respiratory 1o-2.4 10-"Nasal 10" ' 10" '

" IDr, and LDr, for both an initial nasal and totalrespiratory tract infection with A/PR/8/34(HON1)influenza virus were determined by infecting groups ofmice (three to eight each) with serial 10-fold dilutionsof stock virus. Mice in the IDrd group were sacrificedon day 1, and the nasal or lung tissues were assayedfor the presence of virus. Mice in the LDro groups wereobserved for 14 days. The results are reported in termsof EID!4 units.

ferent from zero. However, these titers weresignificantly (P < 0.01) lower than the titers intracheas and lungs of mice given an initial totalrespiratory tract infection. By day 3 the trachealtiters were comparable for both infection modes,but it was not till day 5 that the lung titers ofthe nasal initiation group rose to the levels ofthose animals given a total respiratory tractinfection.Effect of site of initial infection on tra-

cheal desquamation. Figure 2 shows thecourse of tracheal desquamation after infectioninitiated in the nasal or total respiratory tract.A comparison of the day 3 micrographs showsthat whereas the tracheal epithelium of micethat received a total respiratory tract infectionwas completely desquamated, the trachea in thenasally initiated infection was normal. In fact,the trachea was not completely desquamated inthis case until day 5, confirming the time delaynecessary for spread of the virus from the noseto the lower respiratory tract.Heterotypic immunity in mice with a to-

tal respiratory tract infection. A protocol forheterotypic immunity experiments is shown inFigure 3. Mice were made heterotypically im-mune by infecting their total respiratory tractswith 103 EID.% of the nonlethal A/PC/73(H3N2)30 days before the challenge infection. Theseheterotypically immune mice were then anes-thetized and challenged with 0.06 ml of virussuspension containing 1.6 x 103 EID50 of A/PR/8/34(HON1). Mice were sacrificed on days 1, 3,5, 7, and 9, and virus titers were determined (Fig.4). The virgin mice data depicted in Fig. 4 arethe same as those shown in Fig. 1. None of thevirgin mice survived past day 5. Virus titers inthe nose or trachea were not significantly differ-ent between virgin and heterotypically immunemice on day 1. However, whereas the titers inthe virgin mice remained high until death, thevirus titers in the heterotypically immune micedecreased. The titers were significantly different

INFECT. IMMUN.

FACTORS AFFECTING OUTCOME OF INFLUENZA 657

5

LOGlO 4-

EID5O 3-

LOGO 4

EID50 3

2

0.

- I

6

5.

LOGO4EID50 3

2

0

NOSE

t

DAY

TRACHEA

1 23 5 7 9

DAY

LUNG

i'"s'' \\~~~~~

2 3 5DAY

7 9

FIG. 1. Comparison of virus titers shed from virginmice given an initial total respiratory tract (0) or

initial nasal (0) infection with A/PR/8/34(HONI).Means and standard errors are given. If no bar isshown, the standard error is contained within thepoint. Five mice were used for each point in the totalrespiratory tract infection, and seven mice were usedfor all points except day 2 (four mice) in the initialnasal infection. The data for initial nasal infectionare a combination of the results of two experiments.There was no difference between the results of thetwo experiments. Log1o of undetectable levels of virusis represented as -1.

from those of virgin mice, in the nose by day 3and in the trachea or lungs by day 5 (P < 0.01).Virus was undetectable in the heterotypicallyimmune mice by day 9. Schulman and Kil-bourne's (17) finding that heterotypic immunitydid not extend to type B influenza was confirmed(data not shown).Studies of tracheal desquamation by SEM

corroborated the viral studies (Fig. 5). At corre-sponding times, the heterotypically immunemice always had less tracheal desquamationthan did virgin mice and never suffered totaldesquamation. The day 7 micrographs indicateadvanced regeneration in the heterotypically im-

mune tracheas. This could have been due eitherto more rapid regeneration or to less severedesquamation of the tracheal epithelium.

Effect of heterotypic immunity in initialnasal infection. For this study, unanesthetizedheterotypically immune mice were infected with0.03 ml containing 1.6 X 101 EIDrxo of A/PR/8/34(HON1). The data from the nasal infection ofvirgin mice previously shown in Fig. 1 are re-peated in Fig. 4. As has been previously shown,the virus spread from the nose to the lowerrespiratory tract of the virgin mice (Fig. 6). Thenasal titers were not significantly different in thetwo groups of mice on days 1 and 2, but by day3 the nasal titer in the virgins was rising whereasthe titer in the heterotypically immune mice wasdropping. By day 5, heterotypically immunemice were not shedding virus from the nose andthe virus had not spread to the lower respiratorytract. The tracheas of heterotypically immunemice given an initial nasal infection never dem-onstrated any signs of desquamation (micro-graphs not shown).Serum antibody response in heterotypic

immunity in mice. The serum hemagglutina-tion inhibition titers of virgin and heterotypi-cally immune mice against A/PR/8/34(HON1)and A/PC/73(H3N2) are summarized in Table4. Contrary to what has been reported by Schul-man and Kilbourne (17), the appearance of se-rum antibody against the challenge virus was noearlier in the heterotypically immune than inthe virgin mice.

DISCUSSIONThis study demonstrates that the site of initial

infection and heterotypic immunity have a ma-jor effect on the outcome of influenza infection.The work of Iida and Bang (10) suggested thatwe could establish an infection in the nasal epi-thelium without simultaneously infecting therest of the respiratory tract. This was supportedby the fluid flow experiment in which adminis-tration of a radioiodinated protein solutionshowed that the fluid inoculum reached the lungin the anesthetized mouse but not in those giventhe smaller volume of inoculum while awake.This meant that a virus introduced in a smallvolume into the nostrils of an unanesthetizedmouse would infect the nasal cavity and notcome into contact with the rest of the respiratorytract at that time. The difference in the outcomeof the two types of infections is, quite literally,that of life and death. The amount of virusrequired to infect anesthetized mice is about thesame as that required to kill them; i.e., the LDro-approximates the ID50. In contrast, it takes30,000 times the amount of virus to kill unanes-

VOL. 29, 1980

I

658 YETTER ET AL.

TOTAL RESPIRATORY TRACTINFECT. IMMUN.

DAY 3

DAY 5

t

t

DAY /

DAY 9

FIG. 2. Comparison by SEM of tracheal desquamation in mice given an initial nasal or total respiratorytract infection with A/PR/8/34(HONJ).

VOL. 29, 1980

Infect(H3N2)

i 27

FACTORS AFFECTING OUTCOME OF INFLUENZA 659

Infect(HON I)

1 3 5 7 9-30 0 o k

Virusserum, tracheal SEM,

virus from nose, trachea a lung

Infect(HONI)

# 3 5 7 90 t f Ft V

serum, tracheal SEM,virus from nose,trochea a lung

FIG. 3. Protocol for heterotypic immunity experi-ments in mice.

thetized mice as it does to infect their noses. Themost obvious conclusion is that the site of infec-tion is crucial in determining the outcome of theinfection, but several alternate conclusions mustbe ruled out.Could the difference between the two modes

of infection be a function of the difference in thevolume of the inocula used? An experiment wasperformed in which unanesthetized mice re-ceived approximately 103 EID5o of HONi influ-enza virus in 0.06 ml and did not develop pneu-monia (data not shown); therefore, the volumedoes not seem to be critical. A second alternateexplanation is that the unanesthetized micesneezed out almost all of the inoculum. Thisseems highly unlikely since the fluid flow exper-iments showed that the unanesthetized miceretained about one-quarter of the inoculum re-tained by the anesthetized animals and requiredabout 60 times as much virus to infect it. Inci-dentally, the 15-fold discrepancy between thesetwo numbers could represent either inaccuraciesin the ID5o determinations or an innate resist-ance of the nose relative to the total respiratorytract, but in either case the 30,000-fold differencebetween the LD50 and ID50 in the unanesthetizedmouse suggests strongly that the discrepancybetween the anesthetized and unanesthetizedmice cannot be due to loss of inoculum due tosneezing. A third possibility is that the admin-istration of fluid into the lungs predisposes themto viral infection and subsequent lethal pneu-monia. An experiment was therefore donewherein mice were anesthetized, given 0.06 mlof saline, allowed to wake up, and then infectedwhile awake. The outcome of infection was thesame as for controls not given saline (data notshown); therefore, fluid in the lungs seems anunlikely explanation for the observed results.We believe that the survival of mice given an

initial nasal infection is due to the amount oftime necessary for the virus to spread throughthe respiratory tract and reach high titers in thelung. The delay was evident not only in virus

isolation but also in SEM of the trachea. Thispresumably provided sufficient time for the im-mune system to respond to the infection. Al-though there is not sufficient information toknow what aspect of the immune system is life-saving, it is interesting to speculate about theprotective mechanism. Yap and Ada (22) re-ported that the appearance of cytotoxic T cellsin influenza-infected mice was followed by deathif the virus titers in the lung were high. However,if sublethal doses of the virus were given, clear-ance of the virus followed the appearance ofcytotoxic T cells. Perhaps, in mice given a totalrespiratory tract infection, with high virus titersin the lung at an early stage and, presumably,considerable alveolar involvement, cytotoxic T

5

LOG1O 4

EID503

2

0'

-Il

5.

LOGO 4 -

EID50 3

2

-0*

-l

6'

5.

LOG1OEID50 3

2'

01

-Il

NOSE

t

3 5 7 9DAY

TRACHEA

t

3 5 7 9DAY

LUNG

I 3 5 7 9

DAY

FIG. 4. Comparison of virus shedding from virgin(0) and heterotypically immune (0) mice given atotal respiratory tract infection with A/PR/8/34(HONI). Means and standard errors are given. Ifno bar is shown, the standard error is containedwithin the point. Five mice were used for each pointin the virgin group, and three mice were used for allpoints except day 9 (2 mice) in the convalescentgroup.Logio of undetectable levels of virus is represented as-1.

o - - _- o _ _ _ o

660 YETTER ET AL.

HETEROTYPICALLY ItMUNE

DAY 1

DAY 3

DAY 5

DAY 7 i

FIG. 5. Comparison by SEM of tracheal desquamation in virgin and heterotypically immune mice given a

total respiratory tract infection with A/PR/8/34(HONI).

VIRKIN

t

INFECT. IMMUN.

FACTORS AFFECTING OUTCOME OF INFLUENZA 661

LOGO 4-

EID50 3-

2-

0-

5-

LOG104

EIDSO 3-

2-

0-

I

2 3 5 7DAY

TRACHE,

I2 3 5

DAY7

LUNI

I T T r2 3 5 7

DAY

FIG. 6. Comparison of virus shedding frc(0) and heterotypically immune (0) miceinitial nasal infection with A/PR/8/3Means and standard errors are given. If i

shown, the standard error is contained wpoint. Seven mice were used for each point e.2, when four mice were used, and day 9, wimice were used for each point. The dataare a combination of the results of two exp

There was no difference between the resulttwo experiments. Loglo of undetectable levelis represented as -1.

cells may cause the pathological changepneumonia. In mice given an initial nastion, the consequent lower levels of virilungs may result in less alveolar invoand consequently less cell destructiontoxic T cells which might then be respondclearance of the virus.The site of initial infection may al

major concern in the human disease. I

not clear how influenza infection is tranGwaltney et al. (9), however, has shotransmission of rhinovirus infection inis generally by hand-to-hand contact.this observation hold true for influenza

E then many infections would be initiated in thenasal epithelium. This should not be taken torule out infection due to exposure to an aerosolof the virus, which could result in a total respi-ratory tract initiation. Thus, it is possible thatinfection may be initiated in either way in hu-mans.

Heterotypic immunity modified the course ofinfections initiated either in the nose or through-

9 out the respiratory epithelium. Schulman andKilbourne's observation that heterotypic im-

munity could be lifesaving in the case of infec-tions initiated throughout the respiratory tract(17) was confirmed. Furthermore, when the in-fections were initiated in the nasal epithelium,heterotypic immunity prevented the spread ofthe virus from the nose to the rest of the respi-ratory tract.

Heterotypic immunity appears to play a role9 in recovery but not in prevention of influenza

IG infection. This is suggested by the fact that day1 virus titers from the upper respiratory tract ofheterotypically immune and control animalswere similar, whereas at later times the titersfrom the heterotypically immune animals arelower. What might be the mechanism by whichheterotypic immunity promotes recovery? Pos-sibly heterotypic immunity is the result of amore rapid rise in serum antibody to challengevirus in the heterotypically immune than in thevirgin mice. Although treatment with kaolin

m virgin may remove specific antibodies (13), all sera

after an were treated in the same manner. That being34(HONI). the case, the observation that such a rise inno bar is serum antibody did not occur indicates that)ithin the serum antibody is unlikely to be the mechanismexcept day of heterotypic immunity. This hypothesis is sup-hen threePresentedweriments. TABLE 4. Serum hemagglutination inhibition titerss of these after a nasal infection with HONI influenza virus"Is of virus

bs of viralWal infec-us in thelvementby cyto-isible for

lso be a

It is stillksmitted.own thathumansShouldas well,

Assay Titer on day:Group Virus

1 3 5 7 9

Virgin H3N2 <8 <8 <8 <8 <8HONI <8 <8 <8 <8" 48

Heterotypi- H3N2 64 79 28 111 194cally im-mune

HONI <8 <8 <8 <8 21'

"Sera of two groups of mice, virgin and heterotyp-ically immune, were tested for hemagglutination in-hibition activity against A/PC/73(H3N2) and A/PR/8/34(HON1) after the mice were given an initial nasalinfection. The heterotypically immune mice had beenpreviously infected with A/PC/73(H3N2). The sera offour mice were tested on each of 5 days.

"One mouse of 4 had a titer of 16.Range from 8 (three mice) to 1,024 (one mouse).

I I

VOL. 29, 1980

662 YETTER ET AL.

ported by the finding that passively adminis-tered antiserum does not protect or enhancerecovery of the upper respiratory tract for influ-enza (15). Although the possibility that this en-hanced recovery is the result of a rapid rise inlocal secretary antibody has not been ruled out,it is perhaps more likely that the mechanism ofheterotypic immunity is related to cytotoxic T-cell function.The increased incidence of influenza pneu-

monia observed in young children (6) may bedue to the absence of heterotypic immunity. Ithas been reported that adults not only havelower incidence of viral pneumonia but also shedvirus for a shorter time than do children (21).This decreased time of shedding resembles theeffects of heterotypic immunity seen in animals.The drawback to postulating a role for hetero-typic immunity in the human disease has beenthe reported short-term nature of the phenom-enon (12), but our studies have indicated thatheterotypic immunity is still functional at least18 months after the first infection and conse-quently could play a significant role in the mod-ification of the disease (23).Thus, the site of initial infection and hetero-

typic immunity play major roles in determina-tion of the course and outcome of influenzainfection in mice. In humans, lack of heterotypicimmunity and an initial nasal infection may leadto the spread of the virus down the respiratorytract and a severe but probably nonlethal dis-ease. The combination of heterotypic immunityand initial nasal infection might result in aninfection limited to the upper respiratory tractand hence a relatively mild disease. Togetherwith the inherent pathogenicity of the virus andgenetic factors of the host, these factors mayexplain the wide range of disease states that areseen in influenza in humans.

ACKNOWLEDGMENTSThe study was supported by Public Health Service grants

AI 07713 from the National Institute of Allergy and InfectiousDiseases. R.A.Y. was a recipient of Public Health Servicegrant AI 0128 from the same Institute.We gratefully acknowledge Robert Cogliano and George

Gifford for many helpful discussions. We also acknowledgeHarold S. Ginsberg for his suggestion that the amount ofinoculum retained in awake versus anesthetized mice shouldbe quantitated.

LITERATURE CITED1. Allan, W. H., C. R. Madeley, and A. P. Kendall. 1971.

Studies with avian influenza A viruses: cross protectionexperiments in chickens. J. Gen. Virol. 12:79-84.

2. Andrewes, C. H., P. 0. Laidlaw, and W. Smith. 1934.The susceptibility of mice to the viruses of human and

swine influenza. Lancet ii:859-862.3. Andrewes, C. H., P. O. Laidlaw, and W. Smith. 1935.

Influenza: observations on the recovery of virus fromman and on the antibody content of human sera. Br. J.Exp. Pathol. 16:566-582.

4. Barber, W. H., and P. A. Small, Jr. 1974. Disseminationof influenza virus between anatomically isolated sites inferrets. Infect. Immun. 9:530-533.

5. Chen, T. T., F. W. Bazer, J. J. Cetorelli, W. Pollard,and R. M. Roberts. 1973. Purification and propertiesof a progesterone-induced basic glycoprotein from theuterine fluid of pigs. J. Biol. Chem. 248:8560-8566.

6. Foy, H. M., M. K. Cooney, R. McMahan, and J. T.Grayston. 1973. Viral and mycoplasmal pneumonia ina prepaid medical care group during an eight-year pe-riod. Am. J. Epidemiol. 97:93-102.

7. Francis, T., Jr. 1934. Transmission of influenza by afilterable virus. Science 80:457-459.

8. Francis, T., Jr., and C. H. Stuart-Harris. 1938. Studieson the nasal histology and epidemic influenza virusinfection in the ferret. III. Histological and serologicalobservations on ferrets receiving repeated inoculationsof epidemic influenza virus. J. Exp. Med. 68:813-830.

9. Gwaltney, J. M., Jr., P. B. Moskalski, and J. 0.Hendley. 1978. Hand-to-hand transmission of rhinovi-rus colds. Ann. Intern. Med. 88:463-467.

10. Iida, T., and F. B. Bang. 1963. Infection of the upperrespiratory tract of mice with influenza A virus. Am. J.Hyg. 77:169-176.

11. Major, R. H. 1932. Classic descriptions of disease, p. 148.Charles C Thomas, Publisher, Springfield, Ill.

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