The Pathogenesis of Respiratory Syncytial Virus Infection in Infant Ferrets Gregory A. Prince, DDS, PhD and David D. Porter, MD
The infant ferret is susceptible to respiratory syncytial virus infection in both the upper and lower respiratorv tracts. In the nose, viral replication is restricted to the surface respiratory epithelium in the nasal passages and turbinates. In the lungs, viral replication is of a lower order of magnitude and is localized in the alveolar cells. The pattern of viral replication in nasal tissues is independent of the age of the animal at infection, whereas the pattern in lung tissues shows a striking age dependence, with viral replication progressively decreasing as a function of age. Ihis age dependence appears to be due to an intrinsic age-related mechanism yet to be defined. W e feel that the infant ferret is an acceptable model for the study of respiratory syncytial virus disease and that the study of age dependence observed in ferrets may allow elucidation of the mechanisms involved in the age dependence seen in humans. (Am J Pathol 82:339-352. 1976)
SINCE ITS IN-ITIAL ISOLATION- in 1956,' respiratory syncytial virus (RSV') has come to be recognized as an extremelv prevalent and important human pathogen. One recent study2 suggests, on the basis of seroepidemiologic data, that RSV' infection is almost universal among humans. Initial infection does not confer lasting immunity3 and repeated infections max occur throughout adult life.4 Of particular interest, however, is the age dependence of disease produced by the virus. Reports from several laboratories5-' indicate that, although all age groups are susceptible to infection, the serious forms of the disease (principally bronchiolitis and interstitial pneumonia) are found most frequently in infants under 6 months of age, and that the disease is progressively milder w-ith increasing age. Finally, a report by Gardner and co-workers8 indicates that RSV is the virus most frequentlv associated with fatal respiratory disease in infants. Despite the importance of RSV" disease, fittle is known of the basic mechanisms involved in its pathogenesis. Furthermore, crucial questions regarding these mechanisms are not amenable to study in the human and will require definition in an appropriate animal model. Approaches to successful prophvlaxis and therapy have also been hampered by the lack of an animal model. For example, current efforts to produce a From the Department of Pathology. University of California. Los Angeles. School of Medicine, Los Angeles. California. Presented in part at the 19735 Annual Meeting of the American Society for Experimental Pathology. Atlantic City. N\es Jersev. April 17. 1973. Accepted for publication October 1. 1973. Address reprint requests to Dr Gregors A Prince. National Insitute of Dental Research. Building 30. Room 121. National Institutes of Health. Bethesda. MD 20014. 339
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temperature-sensitive attenuated live virus vaccine would be greatly aided by a suitable animal model in which to test such a vaccine prior to its wvidespread use in humans. Previous Attempts at Producing an Animal Model
Respiratorx syncytial virus produces a clinical disease in chimpanzees that is characterized bv coughing, sneezing, and mucopurulent nasal discharge; in fact, the virus was isolated in this animal before it was implicated as a human pathogen.' However, the chimpanzee is not a suitable laboratory animal for a study of the pathogenesis of RSV disease, due to problems of cost, availability, and handling. Tw-o laboratories9'0 have attempted to use the ferret as a model for RSV7 infection because of its susceptibility- to influenza virus" and canine distemper virus2 -both viruses that are similar to RSV.13 Using adolescent animals, they found that Xviral replication occurred in nasal tissues but not in the lungs. Other attempts at developing a model using the guinea pig'" and cotton rat15 have also met X ith only limited success. WTe hypothesized that a viral disease which in humans produced pulmonarv disease almost exclusivelv in infants might exhibit a similar age dependence in an experimental animal. For this reason, we turned to infant ferrets, rather than the adolescent animals which had been used in previous studies. W\e found the infant ferret to be an acceptable animal model for the stud- of RSV infection in both nasal and pulmonary tissues and also found a striking age dependence in pulmonarv infection in these animals which resembles that seen in humans. Materials and Methods Virus The Long strain of RS\- was obtained from the American Type Culture Collection. After being gro%-n in HEp-2 cells for six passages, the virus was passed five times through cell cultures of ferret lung fibroblasts. Both cell cultures were washed with Eagle's NMinimal Essential \ledium (E\IEMI) without serum prior to the addition of virus. Approximately 10 s PFU of RS\ were added to a 16-ounce culture bottle of cells, incubated for I hour, and \sashed \-.ith EM1EMI. EM1EM1 supplemented with 2.3'% heat-inactivated (56 C for 30 minutes) chicken serum (HICS) was added, and infected cultures were maintained for 3 davs at :37 C in a .5%- COQ atmosphere. \'irus was harvested by one freeze-thaw cycle, followed by centrifugation at 450g to remove particulate material. The virus suspension \-as disided into aliquots and stored at -80 C. Cell Cultures HEp-2 cells -.ere obtained from NMicrobiological Associates, Inc., Bethesda. N.d. Cells sere grown in Eagle's Mfinimum Essential NMedium, supplemented with 1O0c heat-
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inactivated (,56 C for :30 minutes) fetal bosine serum (HIFBS) and subcultured twice a mveek. Frozen stocks of cells Xvere maintained at -80 C in E\IE\M supplemented w-ith 10%7 HIFBS and 10% gIlycerol. Cells \vere used from third to thirtieth passage. Constant monitoring --as necessar- to ensure that stocks retained consistent susceptibility to syncytium formation bv RS\'. 2 Ferret fibroblast cultures \vere prepared from adult female lungs. Animals \were anesthetized mvith pentobarbital and sacrificed by exsanquination from the heart. Lungs w-ere removed and minced into pieces measuring approximately 1mm. and tissue \vas agitated in trypsin solution for 20 minutes at 37 C. Follow ing centrifugation and decanting of the supernatant. ne\ try psin solution was added to the tissues, and they were agitated for 3 hours, The supernatant was decanted and centrifuged at i-5Og for :30 minutes. The cell pellet mvas \sashed t\-ice in E\IE\1 + 10c HIFBS. resuspended in growth medium, and incil)ated in glass bottles at 37 C. Animals
Pregnant female ferrets V Iuf.stela firo). not vaccinated against distemper -irus s-ere obtained from \larshall Research Animals, North Rose. N. Y. Animals were shipped approximately 3 mveeks prepartum and wvere housed in stainless steel rabbit cages to which mvere attached stainless steel-enclosed nesting boxes. The animals were maintained on a diet of canned dog food, supplemented daily with fresh beef liver. Room lighting was regulated to provide 16 hours of light each dav. The controlled lighting and diet -ere essential in stimulating adequate maternal care and a normal growth rate of the infants. respectively. Litters ranged in size from .3 to 13 infant ferrets. with an average of 8 animals. Mode of Infection Ferrets \-ere infected on the day of birth, or at 3. 7. 14. or 28 days of age. All animals ,-ere infected intranasallv under ether anesthesia %vith 3.6 x 103 plaque-forming units PFL' of virus. The volume of inoculum was adjusted, according to the size of the animals. mvith E\IE\I to ensure that the inoculum reached the lungs at the time of infection. and the volume ranged from 0.02.5 to 0.2 ml. The fate of the virus inoculum in each age group mvas determined by using an equivalent volume of dialyzed India ink. Etherized animals in all age groups demonstrated a uniform distribution of the India ink in all of the lobes of the lung. vith no ink noticeable in the esophagus or stomach. Animals inoculated with India ink mvithout ether anesthesia, however, demonstrated most of the ink within the stomach and esophagus. and only traces within the lungs. Infection of animals wvithout ether anesthesia resulted in occasional peak virus titers in the lungs equivalent to those seen in animals infected under ether anesthesia, suggesting no potentiation of infection by ether. Hom-ever. uniform results could not be obtained in unanesthetized animals, since most animals smvallos-ed rather than aspirated, the inoculum.
Methykelkulose Overlay A 2 % meth\-lcellulose suspension wlas prepared and autoclaved according to the method of Schulze."6 After cooling to 4:3 C. the suspension was combined with an equal volume of Mxodified Eagle's \ledium (two times) (Grand Island Biological Company. Grand Island, N.Y.). The mixture "vas supplemented w-ith :3%cHICS, 50 gg,,ml gentamicin (Schering Corporation, Port Reading, N.J.), and 2.0 m\1 glutamine (M\icrobiological Associates). Bottles of the overlav medium were stored at -3.5 C until used. Virus Assay
At internals follow-ing infection. infant animals \ere sacrificed by decapitation. Serum %-as stored at -3.3 C for antibody analysis. Lung and nasal tissues were homogenized for virus quantitation. The chest organs were removed from the animal, and the heart.
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thvmus, and trachea were separated from the lungs prior to homogenization. The nasal tissue used included the nasal passage and the nasal turbinates. Fresh tissues were weighed and diluted with an appropriate volume of Hanks' balanced salt solution (dilution ranged from 1:10 to 1:50, depending upon the amount of tissue available). The tissue was homogenized at high speed for 1 minute in an omnimixer (Sorvall, Du Pont Company, Newton, Conn). Following centrifugation at 450g for 20 minutes, 0.2 ml of supernatant was added to monolayers of HEp-2 cells in 60-mm culture dishes. The cell cultures were agitated every 13 minutes for 1 hour to insure uniform distribution of virus over the cell sheet and to prevent drying of the cells. After 1 hour, a 1% methylcellulose overlay was added to the unwashed cultures, which were then maintained at 37 C in a %c CO2 atmosphere for 3 days. After 3 davs' incubation, the cultures were scored for macroscopically visible svncvtia. Virus titers were expressed in terms of plaque-forming units (PFU) per gram of fresh tissue. Imimnofkxwescee Reaents Ferret IgG and rabbit antibody to this protein were prepared by techniques used for the corresponding mink protein."' Antiferret IgM was prepared by adding 10 ml of normal ferret serum heated at 56 C for 30 minutes to 20 ml of rabbit antihuman IgM, which was prepared by inoculating rabbits with human IgM purified by preparative electrophoresis, Sephadex filtration, and diethylaminoethylceliulose chromatography, and absorbed with an insolubilized human IgG-bovine serum albumin (BSA) copolymer. " The reaction mixture contained 0.01 M ethylmedlaine tetiraawetic acid (EDTA), and was incubated 30 minutes at 37 C and overnight at 4 C. The resulting precipitate was washed six times with phosphate-buffered saline (PBS) and divided into three aliquots. Each aliquot was used for inoculation of 4 rabbits at .3-week intervals using complete Freund's adjuvant. The resulting antiserum was absorbed with an insolubilized ferret IgG-BSA copolymer to render it mu-chain specific. Rabbit antiserum to ferret IgA was prepared in a similar fashion, except that rabbit antimink IgA, prepared by the method of Coe and Hadlow " and rendered alpha-chain specific by absorption with a mink IgG-BSA copolymer was
used. Rabbit antiserum to mink gamma chain was prepared by absorbing antiserum to whole mink IgG 17 with a copolymer of mink light chains and BSA. Mink light chains were prepared from purified IgG by reduction, alkylation, and Sephadex filtration using the method of Fleischman et al.20 as modified by Coe.(Z The rabbit antiserums were checked for specificity bv immunoelectrophoresis and double diffusion in agar. Rabbit antiserum to ferret IgG was shown to react with ferret IgG, IgA, and IgM, while rabbit anti-ferret IgM, anti-ferret IgA and anti-mink gamma chain were shown to contain antibody only to ferret mu, alpha, and gamma chains respectivelv. The antiserums were fractionated and labeled with fluorescein isothiocyanate (FITC) as previously done in this laboratory.2 After liver powder absorption, the labeled antiserum was used at a concentration of 0.5 mg/ml in indirect staining procedures. As a further check on the specificity of the reagents, advantage was taken of the extensive cross reactivity with the corresponding mink serum proteins. Mink IgG, IgA, or IgM isolated from the serum of chronically infected mink" was used as the first reagent in an indirect immunofluorescence test for Aleutian disease viral antibody4 at a concentration of 50 jg or 10 mg/ml, and the class-specific antiserums above were used as the second reagent. Each class-specific reagent was shown to be reactive with homologous immunoglobulin at the lower concentration and was unreactive with the other classes of immunoglobulin at the higher concentration. Sam Ab As The presence of neutralizing serum antibody to RSV was tested using a 50%5' plaquereduction neutralization test.25
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Serum samples containing a measurable amount of neutralizinig aitibod! '%ere further studied to determine the class of the antibodv b- indirect immunofluorescenlce. RS\ was gro\-n on glass coverslip cultures of HEp-2 cells, X hich w-ere fixed in acetonie and stored at 4 C until used. Serum samples, diluted 1:10, were tested for RS\' antibody by indirect immunofluorescence using the class-specific labeled reagents described aboxwe The staining techniques, controls emploved, and microscope used have becn described.22 Histoogy Nasal and pulmonary tissues wvere fixed in Bouin's solution for 24 hours, repeatedly vashed in 80%W ethanol, and embedded in paraffin. Lungs were inflated by intratracheal injection of Bouin's solution at the time of fixation in order to preserve the pulmonary architecture in an expanded state. Immunofuoescence M'icoscop Nasal tissue was removed from the animal, immediately immersed in liquid nitrogen. and stored at -80 C. Cryostat sections were cut at 8 p. In order to preserve lung tissue in an expanded state, intratracheal injection of OCT compound (Ames Company. Eckhart. Ind.), diluted 1: 2 with PBS was used immediately prior to freezing.' The indirect immunofluorescence technique was utilized to localize viral antigen in ferret tissues. Unlabeled ferret antiserum to RS%', obtained 28 days following infection of a 7-day-old animal was found to give the brightest indirect staining. This serum had a .50% plaquereduction neutralizing titer of 1:14. It was diluted 1:10 with PBS and applied to the tissues, and was followed by the application of FITC-labeled rabbit antisera to ferret IgG.
Results Cinical Finngs
After infection, no clinical changes were observed in the animals of any age group, and no nasal discharge was seen. The general physical activity of the animals appeared unchanged throughout the course of infection. No animals died from RSV infection. Virus Quantitation
The results of virus quantitation studies of infected ferrets are shown in Text-figure 1. In animals infected on the day of birth, virus was recovered from the lungs beginning on the day after infection, reached a peak titer in each group of approximately 3 X 105 PFU, g, and remained iiM the lungs for a total of 2 days in each litter. Virus reached a higher titer in the nose, about 106 PFU,/g, and remained in nasal tissue for 8 to 9 davs. B\the tenth dav after infection, no virus was recovered from either nose or lung tissue. Animals were monitored for 4 weeks following infection and no secondarv occurrence of virus was observed after the tenth dav. Animals infected at 3 days of age showed a different pattern of infection in the lungs. The peak virus titer in the lungs w-as diminished to ab)ut I(P PFU/g, and virus was recovered from the lungs for a total of 3 days.
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PRINCE AND PORTER AGE AT INFECTION: -
10-
o4
-
LLI 104L 0O -: -
_
X
> 10-
TLXT- F-I(._ HE 1-\Virus
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,'
105
*
A
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0
-
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\
:
lo0
-10 IO c,
t eD8AYS
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:
quantitation of tissues from infant ferrets infected swith RS\ Tissues w-ere homogenized in phosphate-buffered saline, and cell-free supernatants were applied to HEp-2 cell monolavers. Cultures ssere scored after :3 davs on the basis of macroscopically visible syncytia. Virus titers are expressed in terms of plaque-forming units per gram of fresh tissue The age of the animals at the time of infection is indic-ated at the upper right corner -irus titers in nasal tissues. solid litnes svirus titers in lungs. brokeni lines)
c^
I0IG 2
4
6
8
10142128 2
4
6
8
10 14 28
DAYS AFTER INFECT I1ON
Although the viral replication pattern in the lung tissue was changed from the earlier animals, the pattern in nasal tissues was essentially the same as that seen in animals infected on the day of birth, both in terms of peak virus titer and duration of viral replication. In animals infected at 7 days of age the peak virus titer in the lungs was further diminished to 10O PFU/g, and virus was recovered from the lungs for only 2 days. The viral replication pattern in the nose was essentially the same as that seen in the younger animals.
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In animals infected at 14 days of age, the peak virus titer in the lungs was further diminished to only 103 PFU/g, and virus was recovered from the lungs for onlv 1 day. Again, the pattern of nasal infection remained essentially the same as that seen in the younger animals with respect to peak virus titer and duration of infection. Finally, in animals infected at 28 days of age, no virus was recovered from the lungs at any time after infection, although the pattern of viral replication in the nose remained essentially the same as that seen in the younger animals. Virus growth in the nose was independent of the age of the animal. In all age groups the peak virus titer in the nose was approximately the same, being about 106 PFU/g. Infectious virus was present in the nose for 6 to 9 days, and in all cases was cleared by the tenth day following infection. Nasal tissues taken at 14, 21, and 28 days after infection failed to vield anv infectious virus. Virus growth in the lungs, however, showed a striking age dependence. The highest peak virus titer was seen in the youngest animals. The peak virus titer progressively decreased in older animals until, in animals infected at 28 days of age, no infectious virus was recovered from the lungs at any time following infection. This was in spite of the fact that the virus inoculum reached the lungs of all age groups of animals at the time of infection. Furthermore, the progressive decrease in the length of time during which virus could be recovered from the lungs paralleled the decrease in peak virus titer. Antldy Response
The humoral antibody response following infection was weak or absent, regardless of the age of the animal at infection, as shown in Table 1. Neutralizing antibodv to RSV was detected in only 10 of 54 animals and was weak in those 10. Table 1-Neutralizing Antibody Titers in Sera of Infant Ferrets Infected With RSV at 0, 3, 7, 14, and 28 Days of Age as Determined by the 50% Plaque Reduction Neutralization Test Reciprocal neutralizing antibody titers 0 days Days after infection 0 days 7 days 3 days 14 days 28 days 1 0,0 0,0 0,0 0,0 0,0 7 0,0 0,39 0,10 0,0 0,0 14 0,15 0,175 0,55 0,30 0,0 21 0,0 0,40 0,0 0,0 0,0 28 0,0 0,10 0 0,14 0 56 0,0,30 84 0,0,0 Each figure represents 1 animal.
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Immunoglobulin class studies of those sera containing neutralizing antibody to RSV are summarized in Table 2. Of the three major serum antibody classes, only IgG was found, using immunofluorescence techniques. to contain RSV antibodv. Localization of Viral Antigen
Sections of nasal passage, nasal turbinate, trachea, and lung were examined, using indirect immunofluorescence staining, to determine the localization of viral antigen. Abundant viral antigen was detected in the surface epithelium of the nasal turbinates (Figure 1). A similar pattern of staining wvas seen in the epithelium of the nasal passages. In both tissues, viral antigen was restricted to the surface epithelial cells. Viral antigen wvas much less abundant in pulmonary tissues (Figure 2). Small foci of staining were seen scattered throughout the lung sections, in contrast to the solid sheet of fluorescent cells seen in both the nasal passages and nasal turbinates. In the lungs, viral antigen was seen almost exclusively in the cells of the alveolar walls. The identitv of the cell type in the lungs which contains viral antigen is uncertain. Onlv two bronchial epithelial cells of the 30 bronchi examined contained viral antigen. No staining was seen in tracheal cells. Hiolo
Histologic examination of nasal tissue demonstrated a mild, focal desquamative rhinitis with occasional polvmorphonuclear leukocytic exudation (Figures :3 and 4). No unequivocal lesions attributable to RSV were seen in pulmonary tissues, but foci of atelectasis were frequent. By contrast, no histopathologic changes were seen in control animals inoculated wvith tissue culture medium. These histologic findings are consistent with the picture seen in immunofluorescence studies, where viral antigens wvere concentrated in nasal tissues but diffuse in the lung. Table 2-Indirect Immunofluorescent Staining of Sera From RSV-lnfected Ferrets to Determine Class Specificity of Antiviral Antibody
Reciprocal neutralization titer 175 40 39 30 30 14 0 (control)
0
=
Antiferret a 0 0 0 0 0 0 0
Antimink
Antiferret
Antiferret
t
A
IgG
0
0 0 0 0 0 0 0
+t
+
0 ++
± ++
0
no immunofluorescence staining, ++++ = brightest staining.
0 ++
0
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Natural Infection of Ferret
The possibility of natural transmission of RSV infection in ferrets was studied by examining the serum of mothers at various times after the infection of their litters. Of 7 mothers examined from 12 to 30 days after their litters were infected, 1 contained antibody to RSV, indicating that the virus may be transmitted by means other than direct intranasal inoculation. Discussion Respiratory syncytial virus infection in the nose of infant ferrets may be characterized as an acute epithelial infection often resulting in desquamation of the epithelial cells of the nose and occasionally resulting in an inflammatory exudate composed predominantly of polymorphonuclear leukocytic cells. The histologic picture seen in the infection in ferrets is consistent with the clinical picture of mild rhinitis seen in the human infection. The time course of nasal infection in the two species is similar.4, `7The immunofluorescence findings in the ferret-the restriction of viral antigen to the epithelial cells-are in agreement with the observation of Aheme " that the human nasal infection is restricted to the epithelial cells. In comparison with the picture seen in the nose, RSV infection in the lungs of ferrets is milder in terms of peak virus titer, duration of infection, abundance of viral antigen, and histopathologic change. Since little is known of the amounts or duration of RSV in the lungs in asymptomatic human infections, it is difficult to correlate these findings with the human counterpart. The absence of pathologic changes in the lungs of infected ferrets appears consistent with the lack of pulmonary involvement seen in most cases of RSV infection in humans. Whereas RSV infection appears to be almost universal among humans,2 only a small percentage of infected infants develop pulmonary disease. The factors involved in susceptibility to pulmonary RSV disease in humans remain to be defined. The fact that pulmonarv infection in untreated ferrets does not produce disease may allow for experimental manipulation of the model in an effort to define those factors. The age dependence of RSV infection in the human has eluded explanation.7 Two major hypotheses have been advanced to explain the increased occurrence of serious pulmonary disease in infancy. Some investigators 29.30 have postulated a connection between the prevalence of matemal antibody in the infant and the severity of lower respiratory disease from RSV infection. Since only IgG passes the human placenta, it
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w as postulated that an imbalance between IgG and IgA in infants allowed serious disease to take place. That is, the presence of large quantities of maternal anti-RSV IgG in the infant, in the absence of immune IgA, somehow resulted in the more severe form of the disease seen in infancy. It was noted that at the time when maternal antibody in the human infant disappeared, there was a lessening of the severity of RSV disease. Further clinical evidence to support this hypothesis came as a result of field trials of formalin-killed vaccines.3 32 Such vaccines elicited a strong serum IgG response in the vaccinees in the absence of measurable IgA response. Although the vaccinees were of an older age group than would normally contract severe pulmonary disease from RSV, upon natural infection subsequent to vaccination these infants contracted a disease much more severe than that seen in unvaccinated controls. A unitary hypothesis 2 proposed a single mechanism-antibody imbalance-in both the young infant and the vaccinee. A second theory postulated that serious pulmonary disease from RSV may be due to an allergic reaction based upon prior exposure to the virus.5 Subsequent epidemiologic studies,7 however, have indicated that severe pulmonary disease occurs at an age at which the infant would be most unlikely to have had prior exposure to the virus. The major difficulty in explaining the age dependence seen in the human infant has been the high prevalence of matemally derived RSV antibody in newborn infants. In the infant ferret, we demonstrated an age dependence of virus replication in the lung which seems to mimic that seen in the human, although at an accelerated pace. The severe clinical disease of early infancy in humans correlated well with the high virus titers seen in the infant ferrets. The acceleration of the age dependence seen in the ferret, where bv 28 days of age the animals are no longer susceptible to pulmonarv infection, is not surprising in the light of the difference in life expectancv between these two species; whereas, the human has a life expectancv of approximately 70 years, the ferret has an average life expectancv of 4 to 5 vears. The application of either of the above mentioned hypotheses regarding age dependence (maternal antibody or prior exposure to the virus) fails to explain the age dependence seen in the ferret model. None of the animals in any age group nor the mothers had detectable antibody to RSV at the time of infection, nor was there any exposure to the virus prior to that time. Furthermore, the age dependence is apparently unrelated to the animal's own serum antibody response, as indicated by the fact that, although a striking difference in viral replication in the lungs was noted in
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animals of different ages, there was no significant difference in the serum antibody response of animals of the different age groups. On the basis of our observations in the ferret, we are led to the conclusion that the age dependence seen in the pulmonary component of RSV infection is a function of "aging" or maturation. That is, there is an intrinsic mechanism (or mechanisms), evidently immature in younger animals, -hich accounts for their susceptibilitv to a pulmonary infection with RSV. As the animal ages, this mechanism matures in such a way as to prevent pulmonary infection. Whatever this mechanism is, it appears to operate only in the lungs and not in the nose. This is in agreement with w-hat is seen in the human infection, where pulmonary involvement is limited almost exclusively to voung infants, whereas upper respiratory infection or reinfection by RS\ mav occur at any time through childhood or adult life.34 References 1. 2.
:3. 4.
.3. 6. 7.
8. 9.
10.
11. 12. 13.
Morris JA. Blount RE Jr. Savage RE: Recovery of cytopathogenic agent from chimpanzees with coryza. Proc Soc Exp Biol Ned 92:544-549, 1956 Kim H\. Arrobio JO, Brandt CD, Jeffries BC, Pvles G, Reid JL, Chanock RM. Parrott RH: Epidemiology of respiratonr s%nc-tial virus infection in Washington. D.C. I. Importance of the virus in different respiratory tract disease syndromes and temporal distribution of infection. Am J Epidemiol 98:216-225, 1973 Beem N: Repeated infections with respiratory syncytial virus. J Immunol 98:1113-1122. 1967 Kravetz HM. Knight \V, Chanock RNM, Morris JA, Johnson KM, Rifkind D, Utz JP: Respiratory sy ncytial virus. III. Production of illness and clinical observations in adult volunteers. JAMA 176:657-63, 1961 Gardner PS. MicQuillin J, Court SDM: Speculation on pathogenesis in death from respiratory syncy-tial virus infection. Br Med J 1:327-330, 1970 Jacobs. J\V. Peacock DB, Coiner BD, Caul EO, Clarke SKR: Respiratory syncytial and other viruses associated w-ith respiratory disease in infants. Lancet 1:871-876. 1971 Parrott RH. Kim H\V, Arrobio JO, Hodes DS, Murphy BR, Brandt CD, Camargo E. Chanock RM: Epidemiology of respiratory s ncytial virus infection in WN'ashington. D.C. II. Infection and disease with respect to age, immunologic status, race and sex. Am J Epidemiol 98:289-3300, 1973 Gardner PS, McGuckin R, Beale AJ, Femandes R: Interferon and respiratory svncvtial virus. Lancet 1:374-375, 1970 Coates. H\-. Chanock RM! Experimental infection with respiratory syncytial virus in several species of animals. Am J Hyg 76::302-312, 1962 Pinto CA. Haff RF, Stewart RC: Pathogenesis of and recovery from respiratory svnc\-tial and influenza infections in ferrets. Arch Gesamte \'irusforsch 26:223-237. 1969 Francis T. Stuart-Harris CH: Studies on the nasal histology of epidemic influenza virus infection in the ferret. I. The development and repair of the nasal lesion. J Exp Med 68:789-802. 1938 Dalling T: Experiences with distemper immunisation. \et Rec 10:225-2:34. 19:30 Mlelnick JL: Taxonomy of viruses. Prog Med Virol 19:353-358. 1973
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14. Hambling NiH: Antibody response in guinea-pigs following intranasal inoculation of respiratory svncytial virus. J Pathol Bacteriol 91:623-629, 1966 13. Dreizin RS, Vvshnevetskaia LO, Bagdamian EE, Yankevich OD. Tarasova LB. Klenova AV: [Study of experimental respiratory syncytial virus infection in cotton rats: Virologic and immunofluorescent studies.] Vopr Virusol 16:67-0-676. 1971 16. Schulze IT. Schlesinger RW: Plaque assay of dengue and other group B arthropodborne viruses under methyvl cellulose overlay media. Virology 19:40-48. 196.3 17. Porter DD, Dixon FJ, Larsen AE: Metabolism and function of gamma globulin in Aleutian disease of mink. J Exp Med 121:889-900, 1965 18. Avrameas S, Ternynck T: The cross-linking of proteins with glutaraldehyde and its use for the preparation of immunoadsorbents. Immunochemistry 6:53:-66. 1969 19. Coe JE. Hadlow WVJ: Studies on immunoglobulins of mink: Definition of IgG. IgA. and IgM. J Immunol 108:530-337, 1972 20. Fleischman JB, Pain RH, Porter RR: Reduction of Y-globulins. Arch Biochem Biophys (Suppl) 1:174-180, 1962 21. Coe JE: Studies on immunoglobulins of mink: Definition of two populations of light chains. Immunochemistry 9:147-131, 1972 22. Porter. DD, Porter HG, Deerhake BB: Immunofluorescence assay for antigen and antibody in lactic dehvdrogenase virus infection of mice. J Immunol 102:431-436. 1969 23. Porter DD. Porter HG: Unpublished data 24. Porter DD, Larsen AE, Porter HG: The pathogenesis of Aleutian disease of mink. I. In vivo viral replication and the host antibody response to viral antigen. J Exp Med 130:575-7393, 1969 23. Coates HV. Alling DWX', Chanock RNM: An antigenic analysis of respiratory syncvtial virus isolates by a plaque reduction neutralization test. Am J Epidemiol &3:299-313. 1966
26. Prince GA, Porter DD: Crvostat microtomy of lung tissue in an expanded state. Stain Technol .30:4:3-4, 1973. 27. Beem M, Hamre D: Respiratory svncytial virus. Diagnostic Procedures for Viral and Rickettsial Infections. Edited by EH Lennette, NJ Schmidt. New York, American Public Health Association, 1969, pp 491-503 28. Aherne W, Bird T, Court SDM, Gardner, PS, McQuillin J: Pathological changes in virus infections of the lower respiratory tract in children. J Clin Pathol 23:7-18, 197-0 29. Chanock RM, Kapikian AZ, Mills J, Kim HXN', Parrott RH: Influence of immunological factors in respiratory svncytial virus disease of the lower respiratory tract. Arch Environ Health 21:347-5355, 1970 :30. Kapikian AZ, Mitchell RH, Chanock RNM, Shvedoff RA. Stew-art CE: An epidemiologic study of altered clinical reactivity to respiratory syncvtial (RS) virus infection in children previously vaccinated with an inactivated RS virus vaccine. Arm J Epidemiol 89:405-421, 1969 31. Chin J. Magoffin RL, Shearer LA, Schieble JH, Iennette EH: Field evaluation of a respiratory svncytial virus vaccine and a trivalent parainfluenza virus vaccine in a pediatric population. Am J Epidemiol 89:449-46:3. 1969 :32. Fulginiti VA, Eller JJ, Sieber OF, Joyner JW', Minamitani N., Meiklejohn G: Respiratory virus immunization. I. A field trial of two inactivated respiratory virus vaccines; an aqueous trivalent parainfluenza virus vaccine and an alum-precipitated respiratory svncytial 'irus vaccine. Am J Epidemiol 89:435-448. 1969
Fe= 1-Immunofluorescence photomicrograph of RSV-infected ferret nasal turbinate showing viral antigen in the surface epithelium. Frozen sections were cut on a cryostat at 8 g and stained using indirect immunofluorescence technique. (x 250) Fue 2-Immunofluorescence photomicrograph of RSVinfected ferret lung showing viral antigen in cells of the alveolar walls (x 250).
3
11
4
i-
5 c. V% f
s
atw
jr'"
'AfI;
-
Fgu 3-Nasal turbinate of a ferret inoculated with virus-free tissue culture medium. No histopathologic changes are evident. (H&E, x 110) Fure 4Nasal turbinate of a ferret infected with RSV. Epithelium is clefted and portions of desquamated epithelium are seen in the acute inflammatory exudate. Detachment of epithelium from lamina propria is an artifact of fixation. (H&E, x 110)
The infant ferret is susceptible to respiratory syncytial virus infection in both the upper and lower respiratorv tracts. In the nose, viral replication is restricted to the surface respiratory epithelium in the nasal passages and turbinates. In the lungs, viral replication is of a lower order of magnitude and is localized in the alveolar cells. The pattern of viral replication in nasal tissues is independent of the age of the animal at infection, whereas the pattern in lung tissues shows a striking age dependence, with viral replication progressively decreasing as a function of age. Ihis age dependence appears to be due to an intrinsic age-related mechanism yet to be defined. W e feel that the infant ferret is an acceptable model for the study of respiratory syncytial virus disease and that the study of age dependence observed in ferrets may allow elucidation of the mechanisms involved in the age dependence seen in humans. (Am J Pathol 82:339-352. 1976)
SINCE ITS IN-ITIAL ISOLATION- in 1956,' respiratory syncytial virus (RSV') has come to be recognized as an extremelv prevalent and important human pathogen. One recent study2 suggests, on the basis of seroepidemiologic data, that RSV' infection is almost universal among humans. Initial infection does not confer lasting immunity3 and repeated infections max occur throughout adult life.4 Of particular interest, however, is the age dependence of disease produced by the virus. Reports from several laboratories5-' indicate that, although all age groups are susceptible to infection, the serious forms of the disease (principally bronchiolitis and interstitial pneumonia) are found most frequently in infants under 6 months of age, and that the disease is progressively milder w-ith increasing age. Finally, a report by Gardner and co-workers8 indicates that RSV is the virus most frequentlv associated with fatal respiratory disease in infants. Despite the importance of RSV" disease, fittle is known of the basic mechanisms involved in its pathogenesis. Furthermore, crucial questions regarding these mechanisms are not amenable to study in the human and will require definition in an appropriate animal model. Approaches to successful prophvlaxis and therapy have also been hampered by the lack of an animal model. For example, current efforts to produce a From the Department of Pathology. University of California. Los Angeles. School of Medicine, Los Angeles. California. Presented in part at the 19735 Annual Meeting of the American Society for Experimental Pathology. Atlantic City. N\es Jersev. April 17. 1973. Accepted for publication October 1. 1973. Address reprint requests to Dr Gregors A Prince. National Insitute of Dental Research. Building 30. Room 121. National Institutes of Health. Bethesda. MD 20014. 339
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temperature-sensitive attenuated live virus vaccine would be greatly aided by a suitable animal model in which to test such a vaccine prior to its wvidespread use in humans. Previous Attempts at Producing an Animal Model
Respiratorx syncytial virus produces a clinical disease in chimpanzees that is characterized bv coughing, sneezing, and mucopurulent nasal discharge; in fact, the virus was isolated in this animal before it was implicated as a human pathogen.' However, the chimpanzee is not a suitable laboratory animal for a study of the pathogenesis of RSV disease, due to problems of cost, availability, and handling. Tw-o laboratories9'0 have attempted to use the ferret as a model for RSV7 infection because of its susceptibility- to influenza virus" and canine distemper virus2 -both viruses that are similar to RSV.13 Using adolescent animals, they found that Xviral replication occurred in nasal tissues but not in the lungs. Other attempts at developing a model using the guinea pig'" and cotton rat15 have also met X ith only limited success. WTe hypothesized that a viral disease which in humans produced pulmonarv disease almost exclusivelv in infants might exhibit a similar age dependence in an experimental animal. For this reason, we turned to infant ferrets, rather than the adolescent animals which had been used in previous studies. W\e found the infant ferret to be an acceptable animal model for the stud- of RSV infection in both nasal and pulmonary tissues and also found a striking age dependence in pulmonarv infection in these animals which resembles that seen in humans. Materials and Methods Virus The Long strain of RS\- was obtained from the American Type Culture Collection. After being gro%-n in HEp-2 cells for six passages, the virus was passed five times through cell cultures of ferret lung fibroblasts. Both cell cultures were washed with Eagle's NMinimal Essential \ledium (E\IEMI) without serum prior to the addition of virus. Approximately 10 s PFU of RS\ were added to a 16-ounce culture bottle of cells, incubated for I hour, and \sashed \-.ith EM1EMI. EM1EM1 supplemented with 2.3'% heat-inactivated (56 C for 30 minutes) chicken serum (HICS) was added, and infected cultures were maintained for 3 davs at :37 C in a .5%- COQ atmosphere. \'irus was harvested by one freeze-thaw cycle, followed by centrifugation at 450g to remove particulate material. The virus suspension \-as disided into aliquots and stored at -80 C. Cell Cultures HEp-2 cells -.ere obtained from NMicrobiological Associates, Inc., Bethesda. N.d. Cells sere grown in Eagle's Mfinimum Essential NMedium, supplemented with 1O0c heat-
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inactivated (,56 C for :30 minutes) fetal bosine serum (HIFBS) and subcultured twice a mveek. Frozen stocks of cells Xvere maintained at -80 C in E\IE\M supplemented w-ith 10%7 HIFBS and 10% gIlycerol. Cells \vere used from third to thirtieth passage. Constant monitoring --as necessar- to ensure that stocks retained consistent susceptibility to syncytium formation bv RS\'. 2 Ferret fibroblast cultures \vere prepared from adult female lungs. Animals \were anesthetized mvith pentobarbital and sacrificed by exsanquination from the heart. Lungs w-ere removed and minced into pieces measuring approximately 1mm. and tissue \vas agitated in trypsin solution for 20 minutes at 37 C. Follow ing centrifugation and decanting of the supernatant. ne\ try psin solution was added to the tissues, and they were agitated for 3 hours, The supernatant was decanted and centrifuged at i-5Og for :30 minutes. The cell pellet mvas \sashed t\-ice in E\IE\1 + 10c HIFBS. resuspended in growth medium, and incil)ated in glass bottles at 37 C. Animals
Pregnant female ferrets V Iuf.stela firo). not vaccinated against distemper -irus s-ere obtained from \larshall Research Animals, North Rose. N. Y. Animals were shipped approximately 3 mveeks prepartum and wvere housed in stainless steel rabbit cages to which mvere attached stainless steel-enclosed nesting boxes. The animals were maintained on a diet of canned dog food, supplemented daily with fresh beef liver. Room lighting was regulated to provide 16 hours of light each dav. The controlled lighting and diet -ere essential in stimulating adequate maternal care and a normal growth rate of the infants. respectively. Litters ranged in size from .3 to 13 infant ferrets. with an average of 8 animals. Mode of Infection Ferrets \-ere infected on the day of birth, or at 3. 7. 14. or 28 days of age. All animals ,-ere infected intranasallv under ether anesthesia %vith 3.6 x 103 plaque-forming units PFL' of virus. The volume of inoculum was adjusted, according to the size of the animals. mvith E\IE\I to ensure that the inoculum reached the lungs at the time of infection. and the volume ranged from 0.02.5 to 0.2 ml. The fate of the virus inoculum in each age group mvas determined by using an equivalent volume of dialyzed India ink. Etherized animals in all age groups demonstrated a uniform distribution of the India ink in all of the lobes of the lung. vith no ink noticeable in the esophagus or stomach. Animals inoculated with India ink mvithout ether anesthesia, however, demonstrated most of the ink within the stomach and esophagus. and only traces within the lungs. Infection of animals wvithout ether anesthesia resulted in occasional peak virus titers in the lungs equivalent to those seen in animals infected under ether anesthesia, suggesting no potentiation of infection by ether. Hom-ever. uniform results could not be obtained in unanesthetized animals, since most animals smvallos-ed rather than aspirated, the inoculum.
Methykelkulose Overlay A 2 % meth\-lcellulose suspension wlas prepared and autoclaved according to the method of Schulze."6 After cooling to 4:3 C. the suspension was combined with an equal volume of Mxodified Eagle's \ledium (two times) (Grand Island Biological Company. Grand Island, N.Y.). The mixture "vas supplemented w-ith :3%cHICS, 50 gg,,ml gentamicin (Schering Corporation, Port Reading, N.J.), and 2.0 m\1 glutamine (M\icrobiological Associates). Bottles of the overlav medium were stored at -3.5 C until used. Virus Assay
At internals follow-ing infection. infant animals \ere sacrificed by decapitation. Serum %-as stored at -3.3 C for antibody analysis. Lung and nasal tissues were homogenized for virus quantitation. The chest organs were removed from the animal, and the heart.
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thvmus, and trachea were separated from the lungs prior to homogenization. The nasal tissue used included the nasal passage and the nasal turbinates. Fresh tissues were weighed and diluted with an appropriate volume of Hanks' balanced salt solution (dilution ranged from 1:10 to 1:50, depending upon the amount of tissue available). The tissue was homogenized at high speed for 1 minute in an omnimixer (Sorvall, Du Pont Company, Newton, Conn). Following centrifugation at 450g for 20 minutes, 0.2 ml of supernatant was added to monolayers of HEp-2 cells in 60-mm culture dishes. The cell cultures were agitated every 13 minutes for 1 hour to insure uniform distribution of virus over the cell sheet and to prevent drying of the cells. After 1 hour, a 1% methylcellulose overlay was added to the unwashed cultures, which were then maintained at 37 C in a %c CO2 atmosphere for 3 days. After 3 davs' incubation, the cultures were scored for macroscopically visible svncvtia. Virus titers were expressed in terms of plaque-forming units (PFU) per gram of fresh tissue. Imimnofkxwescee Reaents Ferret IgG and rabbit antibody to this protein were prepared by techniques used for the corresponding mink protein."' Antiferret IgM was prepared by adding 10 ml of normal ferret serum heated at 56 C for 30 minutes to 20 ml of rabbit antihuman IgM, which was prepared by inoculating rabbits with human IgM purified by preparative electrophoresis, Sephadex filtration, and diethylaminoethylceliulose chromatography, and absorbed with an insolubilized human IgG-bovine serum albumin (BSA) copolymer. " The reaction mixture contained 0.01 M ethylmedlaine tetiraawetic acid (EDTA), and was incubated 30 minutes at 37 C and overnight at 4 C. The resulting precipitate was washed six times with phosphate-buffered saline (PBS) and divided into three aliquots. Each aliquot was used for inoculation of 4 rabbits at .3-week intervals using complete Freund's adjuvant. The resulting antiserum was absorbed with an insolubilized ferret IgG-BSA copolymer to render it mu-chain specific. Rabbit antiserum to ferret IgA was prepared in a similar fashion, except that rabbit antimink IgA, prepared by the method of Coe and Hadlow " and rendered alpha-chain specific by absorption with a mink IgG-BSA copolymer was
used. Rabbit antiserum to mink gamma chain was prepared by absorbing antiserum to whole mink IgG 17 with a copolymer of mink light chains and BSA. Mink light chains were prepared from purified IgG by reduction, alkylation, and Sephadex filtration using the method of Fleischman et al.20 as modified by Coe.(Z The rabbit antiserums were checked for specificity bv immunoelectrophoresis and double diffusion in agar. Rabbit antiserum to ferret IgG was shown to react with ferret IgG, IgA, and IgM, while rabbit anti-ferret IgM, anti-ferret IgA and anti-mink gamma chain were shown to contain antibody only to ferret mu, alpha, and gamma chains respectivelv. The antiserums were fractionated and labeled with fluorescein isothiocyanate (FITC) as previously done in this laboratory.2 After liver powder absorption, the labeled antiserum was used at a concentration of 0.5 mg/ml in indirect staining procedures. As a further check on the specificity of the reagents, advantage was taken of the extensive cross reactivity with the corresponding mink serum proteins. Mink IgG, IgA, or IgM isolated from the serum of chronically infected mink" was used as the first reagent in an indirect immunofluorescence test for Aleutian disease viral antibody4 at a concentration of 50 jg or 10 mg/ml, and the class-specific antiserums above were used as the second reagent. Each class-specific reagent was shown to be reactive with homologous immunoglobulin at the lower concentration and was unreactive with the other classes of immunoglobulin at the higher concentration. Sam Ab As The presence of neutralizing serum antibody to RSV was tested using a 50%5' plaquereduction neutralization test.25
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Serum samples containing a measurable amount of neutralizinig aitibod! '%ere further studied to determine the class of the antibodv b- indirect immunofluorescenlce. RS\ was gro\-n on glass coverslip cultures of HEp-2 cells, X hich w-ere fixed in acetonie and stored at 4 C until used. Serum samples, diluted 1:10, were tested for RS\' antibody by indirect immunofluorescence using the class-specific labeled reagents described aboxwe The staining techniques, controls emploved, and microscope used have becn described.22 Histoogy Nasal and pulmonary tissues wvere fixed in Bouin's solution for 24 hours, repeatedly vashed in 80%W ethanol, and embedded in paraffin. Lungs were inflated by intratracheal injection of Bouin's solution at the time of fixation in order to preserve the pulmonary architecture in an expanded state. Immunofuoescence M'icoscop Nasal tissue was removed from the animal, immediately immersed in liquid nitrogen. and stored at -80 C. Cryostat sections were cut at 8 p. In order to preserve lung tissue in an expanded state, intratracheal injection of OCT compound (Ames Company. Eckhart. Ind.), diluted 1: 2 with PBS was used immediately prior to freezing.' The indirect immunofluorescence technique was utilized to localize viral antigen in ferret tissues. Unlabeled ferret antiserum to RS%', obtained 28 days following infection of a 7-day-old animal was found to give the brightest indirect staining. This serum had a .50% plaquereduction neutralizing titer of 1:14. It was diluted 1:10 with PBS and applied to the tissues, and was followed by the application of FITC-labeled rabbit antisera to ferret IgG.
Results Cinical Finngs
After infection, no clinical changes were observed in the animals of any age group, and no nasal discharge was seen. The general physical activity of the animals appeared unchanged throughout the course of infection. No animals died from RSV infection. Virus Quantitation
The results of virus quantitation studies of infected ferrets are shown in Text-figure 1. In animals infected on the day of birth, virus was recovered from the lungs beginning on the day after infection, reached a peak titer in each group of approximately 3 X 105 PFU, g, and remained iiM the lungs for a total of 2 days in each litter. Virus reached a higher titer in the nose, about 106 PFU,/g, and remained in nasal tissue for 8 to 9 davs. B\the tenth dav after infection, no virus was recovered from either nose or lung tissue. Animals were monitored for 4 weeks following infection and no secondarv occurrence of virus was observed after the tenth dav. Animals infected at 3 days of age showed a different pattern of infection in the lungs. The peak virus titer in the lungs w-as diminished to ab)ut I(P PFU/g, and virus was recovered from the lungs for a total of 3 days.
344
American Journal of Pathology
PRINCE AND PORTER AGE AT INFECTION: -
10-
o4
-
LLI 104L 0O -: -
_
X
> 10-
TLXT- F-I(._ HE 1-\Virus
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,'
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:
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quantitation of tissues from infant ferrets infected swith RS\ Tissues w-ere homogenized in phosphate-buffered saline, and cell-free supernatants were applied to HEp-2 cell monolavers. Cultures ssere scored after :3 davs on the basis of macroscopically visible syncytia. Virus titers are expressed in terms of plaque-forming units per gram of fresh tissue The age of the animals at the time of infection is indic-ated at the upper right corner -irus titers in nasal tissues. solid litnes svirus titers in lungs. brokeni lines)
c^
I0IG 2
4
6
8
10142128 2
4
6
8
10 14 28
DAYS AFTER INFECT I1ON
Although the viral replication pattern in the lung tissue was changed from the earlier animals, the pattern in nasal tissues was essentially the same as that seen in animals infected on the day of birth, both in terms of peak virus titer and duration of viral replication. In animals infected at 7 days of age the peak virus titer in the lungs was further diminished to 10O PFU/g, and virus was recovered from the lungs for only 2 days. The viral replication pattern in the nose was essentially the same as that seen in the younger animals.
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In animals infected at 14 days of age, the peak virus titer in the lungs was further diminished to only 103 PFU/g, and virus was recovered from the lungs for onlv 1 day. Again, the pattern of nasal infection remained essentially the same as that seen in the younger animals with respect to peak virus titer and duration of infection. Finally, in animals infected at 28 days of age, no virus was recovered from the lungs at any time after infection, although the pattern of viral replication in the nose remained essentially the same as that seen in the younger animals. Virus growth in the nose was independent of the age of the animal. In all age groups the peak virus titer in the nose was approximately the same, being about 106 PFU/g. Infectious virus was present in the nose for 6 to 9 days, and in all cases was cleared by the tenth day following infection. Nasal tissues taken at 14, 21, and 28 days after infection failed to vield anv infectious virus. Virus growth in the lungs, however, showed a striking age dependence. The highest peak virus titer was seen in the youngest animals. The peak virus titer progressively decreased in older animals until, in animals infected at 28 days of age, no infectious virus was recovered from the lungs at any time following infection. This was in spite of the fact that the virus inoculum reached the lungs of all age groups of animals at the time of infection. Furthermore, the progressive decrease in the length of time during which virus could be recovered from the lungs paralleled the decrease in peak virus titer. Antldy Response
The humoral antibody response following infection was weak or absent, regardless of the age of the animal at infection, as shown in Table 1. Neutralizing antibodv to RSV was detected in only 10 of 54 animals and was weak in those 10. Table 1-Neutralizing Antibody Titers in Sera of Infant Ferrets Infected With RSV at 0, 3, 7, 14, and 28 Days of Age as Determined by the 50% Plaque Reduction Neutralization Test Reciprocal neutralizing antibody titers 0 days Days after infection 0 days 7 days 3 days 14 days 28 days 1 0,0 0,0 0,0 0,0 0,0 7 0,0 0,39 0,10 0,0 0,0 14 0,15 0,175 0,55 0,30 0,0 21 0,0 0,40 0,0 0,0 0,0 28 0,0 0,10 0 0,14 0 56 0,0,30 84 0,0,0 Each figure represents 1 animal.
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American Journal of Pathology
Immunoglobulin class studies of those sera containing neutralizing antibody to RSV are summarized in Table 2. Of the three major serum antibody classes, only IgG was found, using immunofluorescence techniques. to contain RSV antibodv. Localization of Viral Antigen
Sections of nasal passage, nasal turbinate, trachea, and lung were examined, using indirect immunofluorescence staining, to determine the localization of viral antigen. Abundant viral antigen was detected in the surface epithelium of the nasal turbinates (Figure 1). A similar pattern of staining wvas seen in the epithelium of the nasal passages. In both tissues, viral antigen was restricted to the surface epithelial cells. Viral antigen wvas much less abundant in pulmonary tissues (Figure 2). Small foci of staining were seen scattered throughout the lung sections, in contrast to the solid sheet of fluorescent cells seen in both the nasal passages and nasal turbinates. In the lungs, viral antigen was seen almost exclusively in the cells of the alveolar walls. The identitv of the cell type in the lungs which contains viral antigen is uncertain. Onlv two bronchial epithelial cells of the 30 bronchi examined contained viral antigen. No staining was seen in tracheal cells. Hiolo
Histologic examination of nasal tissue demonstrated a mild, focal desquamative rhinitis with occasional polvmorphonuclear leukocytic exudation (Figures :3 and 4). No unequivocal lesions attributable to RSV were seen in pulmonary tissues, but foci of atelectasis were frequent. By contrast, no histopathologic changes were seen in control animals inoculated wvith tissue culture medium. These histologic findings are consistent with the picture seen in immunofluorescence studies, where viral antigens wvere concentrated in nasal tissues but diffuse in the lung. Table 2-Indirect Immunofluorescent Staining of Sera From RSV-lnfected Ferrets to Determine Class Specificity of Antiviral Antibody
Reciprocal neutralization titer 175 40 39 30 30 14 0 (control)
0
=
Antiferret a 0 0 0 0 0 0 0
Antimink
Antiferret
Antiferret
t
A
IgG
0
0 0 0 0 0 0 0
+t
+
0 ++
± ++
0
no immunofluorescence staining, ++++ = brightest staining.
0 ++
0
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Natural Infection of Ferret
The possibility of natural transmission of RSV infection in ferrets was studied by examining the serum of mothers at various times after the infection of their litters. Of 7 mothers examined from 12 to 30 days after their litters were infected, 1 contained antibody to RSV, indicating that the virus may be transmitted by means other than direct intranasal inoculation. Discussion Respiratory syncytial virus infection in the nose of infant ferrets may be characterized as an acute epithelial infection often resulting in desquamation of the epithelial cells of the nose and occasionally resulting in an inflammatory exudate composed predominantly of polymorphonuclear leukocytic cells. The histologic picture seen in the infection in ferrets is consistent with the clinical picture of mild rhinitis seen in the human infection. The time course of nasal infection in the two species is similar.4, `7The immunofluorescence findings in the ferret-the restriction of viral antigen to the epithelial cells-are in agreement with the observation of Aheme " that the human nasal infection is restricted to the epithelial cells. In comparison with the picture seen in the nose, RSV infection in the lungs of ferrets is milder in terms of peak virus titer, duration of infection, abundance of viral antigen, and histopathologic change. Since little is known of the amounts or duration of RSV in the lungs in asymptomatic human infections, it is difficult to correlate these findings with the human counterpart. The absence of pathologic changes in the lungs of infected ferrets appears consistent with the lack of pulmonary involvement seen in most cases of RSV infection in humans. Whereas RSV infection appears to be almost universal among humans,2 only a small percentage of infected infants develop pulmonary disease. The factors involved in susceptibility to pulmonary RSV disease in humans remain to be defined. The fact that pulmonarv infection in untreated ferrets does not produce disease may allow for experimental manipulation of the model in an effort to define those factors. The age dependence of RSV infection in the human has eluded explanation.7 Two major hypotheses have been advanced to explain the increased occurrence of serious pulmonary disease in infancy. Some investigators 29.30 have postulated a connection between the prevalence of matemal antibody in the infant and the severity of lower respiratory disease from RSV infection. Since only IgG passes the human placenta, it
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w as postulated that an imbalance between IgG and IgA in infants allowed serious disease to take place. That is, the presence of large quantities of maternal anti-RSV IgG in the infant, in the absence of immune IgA, somehow resulted in the more severe form of the disease seen in infancy. It was noted that at the time when maternal antibody in the human infant disappeared, there was a lessening of the severity of RSV disease. Further clinical evidence to support this hypothesis came as a result of field trials of formalin-killed vaccines.3 32 Such vaccines elicited a strong serum IgG response in the vaccinees in the absence of measurable IgA response. Although the vaccinees were of an older age group than would normally contract severe pulmonary disease from RSV, upon natural infection subsequent to vaccination these infants contracted a disease much more severe than that seen in unvaccinated controls. A unitary hypothesis 2 proposed a single mechanism-antibody imbalance-in both the young infant and the vaccinee. A second theory postulated that serious pulmonary disease from RSV may be due to an allergic reaction based upon prior exposure to the virus.5 Subsequent epidemiologic studies,7 however, have indicated that severe pulmonary disease occurs at an age at which the infant would be most unlikely to have had prior exposure to the virus. The major difficulty in explaining the age dependence seen in the human infant has been the high prevalence of matemally derived RSV antibody in newborn infants. In the infant ferret, we demonstrated an age dependence of virus replication in the lung which seems to mimic that seen in the human, although at an accelerated pace. The severe clinical disease of early infancy in humans correlated well with the high virus titers seen in the infant ferrets. The acceleration of the age dependence seen in the ferret, where bv 28 days of age the animals are no longer susceptible to pulmonarv infection, is not surprising in the light of the difference in life expectancv between these two species; whereas, the human has a life expectancv of approximately 70 years, the ferret has an average life expectancv of 4 to 5 vears. The application of either of the above mentioned hypotheses regarding age dependence (maternal antibody or prior exposure to the virus) fails to explain the age dependence seen in the ferret model. None of the animals in any age group nor the mothers had detectable antibody to RSV at the time of infection, nor was there any exposure to the virus prior to that time. Furthermore, the age dependence is apparently unrelated to the animal's own serum antibody response, as indicated by the fact that, although a striking difference in viral replication in the lungs was noted in
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animals of different ages, there was no significant difference in the serum antibody response of animals of the different age groups. On the basis of our observations in the ferret, we are led to the conclusion that the age dependence seen in the pulmonary component of RSV infection is a function of "aging" or maturation. That is, there is an intrinsic mechanism (or mechanisms), evidently immature in younger animals, -hich accounts for their susceptibilitv to a pulmonary infection with RSV. As the animal ages, this mechanism matures in such a way as to prevent pulmonary infection. Whatever this mechanism is, it appears to operate only in the lungs and not in the nose. This is in agreement with w-hat is seen in the human infection, where pulmonary involvement is limited almost exclusively to voung infants, whereas upper respiratory infection or reinfection by RS\ mav occur at any time through childhood or adult life.34 References 1. 2.
:3. 4.
.3. 6. 7.
8. 9.
10.
11. 12. 13.
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Fe= 1-Immunofluorescence photomicrograph of RSV-infected ferret nasal turbinate showing viral antigen in the surface epithelium. Frozen sections were cut on a cryostat at 8 g and stained using indirect immunofluorescence technique. (x 250) Fue 2-Immunofluorescence photomicrograph of RSVinfected ferret lung showing viral antigen in cells of the alveolar walls (x 250).
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Fgu 3-Nasal turbinate of a ferret inoculated with virus-free tissue culture medium. No histopathologic changes are evident. (H&E, x 110) Fure 4Nasal turbinate of a ferret infected with RSV. Epithelium is clefted and portions of desquamated epithelium are seen in the acute inflammatory exudate. Detachment of epithelium from lamina propria is an artifact of fixation. (H&E, x 110)
