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Am J Physiol Lung Cell Mol Physiol 291: L426-L435, 2006. First published March 23, 2006; doi:10.1152/ajplung.00483.2005
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Differential resistance/susceptibility patterns to pneumovirus infection among inbred mouse strains

Dao Bui Tran Anh, Pedro Faisca, and Daniel J.-M. Desmecht

Department of Pathology, University of Liège, Liège, Belgium

Submitted 14 November 2005 ; accepted in final form 10 March 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Respiratory syncytial virus (RSV) is a prominent cause of airway morbidity in children under 1 yr of age. It is assumed that host factors influence the severity of the disease presentation and thus the need for hospitalization. As a first step toward the identification of the underlying genes involved, this study was undertaken to establish whether inbred mouse strains differ in susceptibility to pneumonia virus of mice (PVM), the murine counterpart of RSV, which has been shown to accurately mimic the RSV disease of children. With this purpose in mind, double-chamber plethysmography and carbon monoxide uptake data were collected daily for 7 days after inoculation of PVM in six inbred strains of mice. In parallel, histological examinations and lung viral titration were carried out from day 5 to day 7 after inoculation. Pulmonary structure/function values reflected the success of viral replication in the lungs and revealed a pattern of continuous variation, with resistant, intermediate, and susceptible strains. The results suggest that SJL (resistant) and 129/Sv (susceptible) strains should be used in crossing experiments aimed at identifying genes controlling pneumovirus replication by the positional cloning approach. Similarly, crossing experiments using BALB/c or C57BL/6 (resistant) and DBA/2 or 129/Sv (susceptible) will allow the identification of the genes involved in the control of pulmonary inflammation during pneumovirus infection.

pneumovirinae; respiratory syncytial virus; mouse; genetic resistance

RESPIRATORY SYNCYTIAL VIRUS (RSV) is known to infect ~85% of children by age 18 mo, and virtually all children by age 3 yr (48, 50). About 3% of each year's birth cohort is admitted for RSV respiratory tract infection every winter in Europe, Australasia, and North America (42, 48, 50). Consequently, the need for hospitalization may serve as a marker for the most severe cases of RSV infection. Traditional risk factors for severe disease include premature birth, chronic lung disease, and immunodeficiency, but the great majority of infants admitted to hospitals were previously healthy (42). Therefore, it has been suggested that the differential response to infection with RSV may be related to individual host factors. Because susceptibility to infection is usually a complex genetic trait, assignment of the severity of an infection to one or another gene is difficult. One way of doing this is through population-based association studies that look for increased frequency of particular alleles in severely affected compared with mildly affected infants in the population. This approach requires prior identification of a candidate gene in which the allelic polymorphism can theoretically alter the response to infection. This strategy led recently to the discovery that specific haplotypes at the IL4 (10), IL8 (24), IL10 (51), CCR5 (28), IGHG2 (3), SP-A (23, 33), and SP-D (31) gene loci are associated with the severity of RSV infection in infants. It is clear, however, that obtaining robust replication of population-based association study findings has proven very difficult for a series of reasons that have been reviewed extensively (13, 26, 52).

Alternatively, complex genetic traits can be dissected in genetically well-defined inbred strains of mice in which single gene effects may have naturally segregated. These genes can be mapped in large informative crosses and can be identified by positional cloning. Recently, RSV itself was used to infect a series of inbred mouse strains to identify the genetic factors influencing RSV susceptibility (45). However, RSV infection of mice does not result in any measurable degree of morbidity, evokes a mild mononuclear cell infiltration instead of a profuse granulocytic bronchiolitis, does not result in eosinophil recruitment, never progresses to acute respiratory distress syndrome (ARDS), and generates viral titers systematically lower than that inoculated (9, 11, 21, 32). Together, these characteristics suggest that using the RSV murine model to mimic the human disease is rather counterintuitive.

In this study, the putative segregation of genes conferring innate resistance to pneumoviruses among inbred mouse strains was evaluated in a model using the natural rodent pneumovirus pathogen, which is also the closest phylogenic relative of RSV (11), pneumonia virus of mice (PVM). The crucial advantages of this PVM-associated model include the following: 1) clinical picture—morbidity—consistently mimicking that observed in infants with RSV-associated bronchiolitis; 2) dramatic granulocytic and eosinophilic infiltrations that parallel the pathological changes observed in humans; 3) clear evidence of widespread viral replication in lung tissue, with incremental recoveries that, at peak, are in excess of 108 plaque-forming units (PFU)/g in response to as few as 30 PFU in the inoculum; and 4) clear progression to ARDS, as reported for ~3% of infants with RSV bronchiolitis (9, 25, 32).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Design. Twenty mice from each of six different inbred strains (BALB/c, DBA/2, 129/Sv, SJL, C3H/HeN, and C57BL/6) were inoculated with PVM. These strains were chosen deliberately because they originated from different lineages, as deduced from genealogical and phylogenetic data (2). Respiratory pattern/function values were measured daily in 10 mice from each strain, starting 1 day before inoculation (bi) and going on up to 7 days postinoculation (pi). At selected time intervals, a subset of each strain-specific cohort was euthanized for histological examination and lung viral titer quantitation.

Mice, virus, and inoculation. The experiments were conducted with specific pathogen-free 14-wk-old female mice obtained from Charles River Laboratories. The mice were made familiar with the experimental environment by placing them into the plethysmograph for 15 min a day, starting 3 days bi. Housing, inoculation, data collection, and euthanasia procedures complied with National Institutes of Health guidelines, and the experimental protocol was approved by the Bioethics Committee of the University of Liège. PVM strain J3666 (generously supplied by A. Easton, University of Warwick, Coventry, UK) was first passed in 10-wk-old BALB/c mice and then grown once onto BS-C-1 cells to produce the stock solution. The stock solution was then diluted to 10–5 in MEM, divided into aliquots, and stored at –80°C to serve as inoculum. Randomly selected aliquots yielded highly reproducible titers on BS-C-1 cells, amounting to ~2 x 105 PFU/ml. The inoculation procedure consisted of slowly instilling 50 µl of the viral suspension (i.e., ~103 PFU) into the nostrils of the anesthetized mouse maintained in a vertical position (35 mg/kg pentobarbital sodium ip).

Examination of respiratory function and virus yields. Respiratory pattern/function (RPF) values were measured in 10 mice per strain with the two-chambered, whole body plethysmograph devised by Buxco (model no. PLY-3351), with practical procedures, quality controls, raw data processing, and respiratory flow curve analysis that were validated previously in our laboratory (20). A series of parameters was measured directly on the basis of the thoracoabdominal flow curve: duration of inspiration (TI), duration of expiration (TE), tidal volume (TV), time needed to exhale the first 30% of the TV (TE30%), and time needed to exhale the last 5% of the TV (TE5%). Furthermore, the delay observed between nasal and thoracoabdominal flows (dT) was measured and used to calculate the specific resistance of the airways: sRaw = [(TI + TE)/(2 x {pi}) x (Patm – 47) x 1.36 x 2 x {pi} x dT/(TI + TE)], where Patm is atmospheric pressure (20). Finally, on the basis of the parameters measured above, the respiratory rate [RR = 60/(TI + TE)], the minute ventilation (MV = RR x TV), and the duty cycle [%TI = TI/(TI + TE)] were calculated. CO uptake values were measured in the same mice with the CO uptake monitor devised by Columbus Instruments, with the operational flowchart and quality controls recommended by the manufacturer.

At selected time intervals (5, 6, and 7 days pi), some mice (5, 5, and 10 respectively) were overdosed with pentobarbital sodium and exsanguinated by cutting the renal artery. The right lung was weighed, homogenized in 10% PBS, and clarified (3000 g for 10 min), and the supernatant was used for virus titration by plaque assays onto BS-C-1 cells. Once quasi-confluent monolayers grown in 24-well plaques were obtained, the wells were filled with 200 µl of successive 10-fold dilutions of the lung homogenates. After the viral suspensions were left to adsorb for 3 h at 31°C, the wells were washed with PBS and the cell monolayers were covered with 1 ml of 0.6% agarose (wt/vol) in MEM into which FBS (2% wt/vol) had been incorporated. After incubation at 31°C for 12 days, the agar overlay was removed and the remaining cells were stained with crystal violet. The plaques were counted, and the viral titer per unit of right lung weight was calculated. Preliminary studies had shown that lung viral titers measured on day 4 pi were far lower than those retrieved on day 5 pi, whatever the strain inoculated. Distribution of PVM antigens was sought by fluorescence microscopy, after dewaxed sections were sequentially incubated with anti-PVM antiserum and FITC-conjugated anti-rabbit IgG antibody (Molecular Probes).

Assessment of lung lesion severity. At the same time, the left lung was inflated with 4% formalin under a pressure of ~3 kPa for 24 h and then processed along with pieces of nasal turbinates/trachea as for routine histopathological examination. The lung slides were examined independently by two individuals who were never aware of the strain considered. Lesions were graded from "N" (normal) to "+++." Scattered single inflammatory cells in the alveolar spaces were given a value of "+," dispersed clusters of inflammatory cells were given a value of "++," and dispersed confluent areas of inflammatory cells and various amounts of cell debris were given a value of "+++." Interstitium cell infiltration was scored "+" if only small foci of mononuclear cell infiltration were observed; a score of "++" signified that those foci were multifocal and moderate in extension; and finally a score of "+++" was given when the interstitium was infiltrated by mononuclear cells in a diffuse manner. Lymphoid accumulations were scored "+" if they were focal and caused no thickening of the submucosa, "++" if they caused mild thickening, and "+++" if they were extensive and complete and resulted in substantial thickening. Between-examiner reproducibility of eosinophil infiltration scoring was good for a Boolean reporting (presence or absence).

Statistical analysis. Within each strain, consecutive means of RPF values were analyzed by performing the general linear model procedure. Comparisons with day 1 bi were then made with Dunnett's post hoc test. Effect of strain on log10 means of lung virus titers was analyzed by one-way analysis of variance, and between-strain pairwise comparisons were then made with Bonferroni's post hoc test. Comparisons that yielded P values of <0.05 were considered statistically significant. Finally, titer-against-day linear regressions were made within each strain and, whenever significant, yielded a slope value representing the strain-specific lung viral clearance. All statistical analyses were performed with Minitab Release 14 Minitab statistical software.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Clinical illness and survival. All strain- and day-specific values (means ± SD) are presented in Table 1 or Figs. 1–5 or as a data supplement (Supplemental Tables S1–S10).1 None of the 60 inoculated SJL (20), BALB/c (20), or C57BL/6 (20) mice died spontaneously during the experiment. Cumulated mortality rates on days 5, 6, and 7 pi were 0%, 10%, and 10% in DBA/2, 0%, 10%, and 15% in 129/Sv, and 5%, 10%, and 20% in C3H/HEN, respectively. As far as body weight (BW) was concerned, two trends were distinguishable (Fig. 1). The first was a gradual decrease in BW from day 2 (BALB/c) or day 4 (129/Sv, C3H/HeN, DBA/2) that reached a nadir on day 7 with a weight loss of ~15%. In contrast, SJL mice maintained their weight throughout the observation period (Fig. 1).


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  		Table 1. Effect of strain on lung histopathology after pneumonia virus of mice infection

 

Figure 1
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  		Fig. 1. Effect of mouse strain on body weight after infection with pneumonia virus of mice (PVM). Relative values (%) are given, as calculated with respect to preinoculation control values (means ± SD). Within each strain, means significantly different from baseline are indicated (*P < 0.05, **P < 0.01).

 

Figure 5
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  		Fig. 5. Effect of mouse strain on lung viral titers (means ± SD) 5–7 days after infection with PVM. Titers are expressed in log10 plaque-forming units (PFU)/g of lung. Titers significantly different from isotime values in 129/Sv are indicated (*P < 0.05). Titer-against-day linear regressions were made within each strain that, whenever significant (*P < 0.05), yielded a slope value that can be interpreted in terms of lung viral clearance.

 
RPF data. Regarding the respiratory pattern, two specific types of reaction were observed. The first, typical of DBA/2, C3H/HeN, 129/Sv, and BALB/c strains, consisted of a stable MV (Fig. 2A, Supplemental Table S1) up to day 4, followed by a progressive decrease the nadir of which was reached at day 7 and amounted to 51%, 65%, 78%, and 79% of the preinoculation levels in DBA/2, C3H/HeN, 129/Sv, and BALB/c, respectively. The stable MV was achieved via a stable RR-TV (Fig. 2B, Supplemental Tables S2 and S3) combination, whereas the hypoventilation period was always characterized by an increased RR (120%, 130%, 115%, and 115%, respectively) on the one hand and a dramatically collapsed TV on the other (50%, 50%, 65%, and 70%, respectively). The second type of reaction was shared by C57BL/6 and SJL and consisted of a constant MV throughout the experiment, achieved through a constant RR-TV combination up to day 4 but via a faster and shallower breathing thereafter. Between the two strains, the C57BL/6 response was obviously more intense (~150% RR bi combined with ~60% TV bi on day 7) than that of SJL (127% and 83%, respectively).


Figure 2
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  		Fig. 2. Strain-specific trends in selected respiratory pattern/function values after experimental infection with PVM: minute volume (A), respiratory rate (B), duty cycle (C), expiratory balance (D), specific airway resistance (E), and CO uptake (F). Day-specific means within each strain are reported in % as calculated with respect to preinoculation control values (day 1 bi). Solid lines and filled symbols, extreme strains in term of resistance (SJL) and susceptibility (129/Sv); dotted lines and open symbols, intermediate strains as indicated. Absolute means ± SD are available as supplemental data in the online version of this article, along with day-to-day statistical comparisons. TI, duration of inspiration; Ttot, TI + duration of expiration; bi, before inoculation; pi, postinoculation.

 
When the compartmentation of the duration of each breathing cycle into its inspiratory (TI) and expiratory (TE) components was examined (Fig. 2C, Supplemental Table S4), a similar grouping was found, with a constant compartmentation in C57BL/6 and SJL and a dramatic decrease in the other four strains. Once more, the amplitude and timing of the changes paralleled those of the respiratory pattern, with a significant decrease on and after day 5 in DBA/2 (nadir ~50% on day 7) and C3H/HeN (55% on day 7), on and after day 6 in 129/Sv (~75% on day 7), and only on day 7 in BALB/c (85%).

Focusing on expiration, a qualitative examination of the shape of the breathing waves suggested considerable differences among strains at the time of the final phase of expiration (Fig. 3). For this reason, in addition to the duration of expiration (TE; Supplemental Table S5), it was necessary to be able to study the evolution of the initial (TE30%, Supplemental Table S6) and terminal (TE5%; Supplemental Table S7) phases of expiratory emptying, as well as their respective weight in the determination of TE (expiratory balance, TE30%/TE; Fig. 2D and Supplemental Table S8). In short, these times measured on the expiratory wave made it possible to distinguish objectively three "expiratory entities" (Fig. 2D). The first, typical of SJL and C57BL/6, was characterized by a parallel and progressive decrease of TE, TE30%, and TE5%, thus with no recompartmentation of expiration. The second, typical of BALB/c, was constant TE up to day 7, but with accelerated TE30% and decelerated TE5%. The third profile, shared by 129/Sv, C3H/HeN, and DBA/2, consisted of a continuous decrease of TE30% with bell-shaped evolutions of TE and TE5% (first shortening, then lengthening). More specifically, TE decreased from day 3 to day 5 and then progressively lengthened up to day 7, recovering its initial value in 129/Sv and even largely exceeding its initial value in C3H/HeN and DBA/2 (~130% of TE bi). TE5% evolved in roughly the same manner, increasing dramatically on and after day 5. Conversely, no parallelism was ever detected between the evolution of TE30%, which decreased gradually, and the evolution of TE or TE5%. The evolution of the disease therefore included recompartmentation of expiration, light in BALB/c, moderate in 129/Sv, and severe in C3H/HeN and DBA/2, between its initial and terminal components. The expiratory balance parameter provides a good illustration (Fig. 2D and Supplemental Table S8), with total stability throughout the experiment in SJL and C57BL/6 and a decrease of ~10% in BALB/c, ~50% in 129/Sv, and up to 65% in C3H/HeN and DBA/2.


Figure 3
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  		Fig. 3. Effect of mouse strain on thoracoabdominal flow curves after infection with PVM. In each strain, day 1 bi (black) is compared with day 7 pi (gray). Flow trace below 0 represents inhalation and above 0 exhalation. Arrows indicate the lengthening of the final part of expiration.

 
As far as respiratory mechanics was concerned, and in particular sRaw, three categories became apparent (Fig. 2E and Supplemental Table S9). In DBA/2, C3H/HeN, and 129/Sv, sRaw increased on and after day 5, reaching >200% of its initial value. In BALB/c, the trend was the same, reaching significance only on day 7 at ~200% of the initial value. Finally, in C57BL/6 and SJL, there was no detectable change in sRaw.

When CO uptake was examined (Fig. 2F and Supplemental Table S10), between-strain differences were even more marked. It remained unaltered in SJL and was reduced to 83% of its preinoculation level on day 7 in BALB/c. In C57BL/6, the amplitude of the peak decrease was similar to that of BALB/c (17% of CO bi), but the process began earlier (on day 5). Likewise, the CO uptake was maintained no more on and after day 5 in C3H/HeN and DBA/2, but the decrease was steeper, reaching 45% on day 7. Finally, the earliest and most dramatic failure of gas exchanges occurred in 129/Sv, in which a significant decrease was already observed on day 3 and reached 75% on day 7.

Histological data. Nasal turbinates and tracheas displayed very subtle changes, consisting of degeneration of individual epithelial cells and infiltration of some mononucleated inflammatory cells in the lamina propria. At first sight, the morphological aspect of cartilaginous as well as membranous intrapulmonary airways was similar among strains, with very modest and rare regressive epithelial lesions (degeneration, necrosis, exfoliation) and luminal exudates and total absence of epithelial hyperplasia or syncytia formation. Two clearly different histological profiles were identified on the basis of qualitative and quantitative criteria applied to the peribronchial/peribronchiolar cuffs, SJL on the one hand and the five other strains on the other (Table 1). In SJL, there was little difference between the lamina propria of infected animals and the normal morphology of murine airways. Specifically, only a few animals exhibited a moderate infiltration with mononuclear inflammatory cells, and there was no eosinophilic infiltration. Conversely, in the other five strains, most of the animals displayed significant to severe infiltration of the lamina propria with mononucleated inflammatory cells but also with eosinophils. Among the five strains, eosinophils were readily identified in 30% of BALB/c, in 70–80% of C57BL/6, C3H/HeN, and DBA/2, and in virtually all 129/Sv.

Considering the lungs (Fig. 4), SJL also differed from the other five strains in that the alveolar spaces remained empty, with the exception of a few macrophages the density of which was comparable to that observable in a healthy lung (Table 1). In the interstitium, cell density appeared slightly elevated, multifocally, in roughly half of the animals. The histopathological diagnosis most compatible with SJL-specific observations was slight mononuclear multifocal interstitial pneumonitis. The lung histological profile of the other five strains was qualitatively similar, consisting of the presence of alveolar exudates and diffuse interstitial infiltration. The alveolar exudates was a more (score +++) or less (+) concentrated mixture of cell debris, pleiomorphic lymphoid cells, neutrophils, and morphologically altered macrophages (cytoplasmic vacuolation, pyknosis, karyorhexis). Quantitatively speaking, most of the alveolar spaces were optically empty, but often (~50% of individuals in BALB/c) or rather systematically (C57BL/6, C3H/HeN, DBA/2, and 129/Sv), groups of alveoli were observed which contained the exudates described above. In general, these alveolar clusters were distributed in a random fashion and in C3H/HeN and 129/Sv tended to merge, giving the impression of conquering the more peripheral spaces gradually. Overall, the estimated number of alveoli concerned varied between strains, from a few dozen in BALB/c to 15% (C57BL/6, DBA/2, and 129/Sv) or 20% (C3H/HeN) of the lung section area. The lung interstitium, when examined away from the areas of alveolitis, was multifocally (C57BL/6, BALB/c) or diffusely (C3H/HeN, DBA/2, 129/Sv) infiltrated by cells with round nuclei of the monocytoid or lymphoid type. The histopathological diagnoses most compatible with these observations were multifocal mononuclear alveolitis and moderate (C57BL/6, BALB/c) to severe (C3H/HeN, DBA/2, 129/Sv) diffuse mononuclear interstitial pneumonia.


Figure 4
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  		Fig. 4. Effect of mouse strain on the lung histology and distribution of viral antigens 6 days after experimental infection with PVM. Representative lung sections are shown for 129/Sv (A and D), SJL (B and E), and negative control (C and F). Lung sections were stained with hematoxylin and eosin for histological evaluation (A–C), and viral antigens (D–F) were detected by immunofluorescence using specific antibodies. Replacement of antiPVM serum by normal rabbit serum suppressed immunofluorescence positivity, which demonstrates the specificity of the staining (F). Original magnification x20.

 
Virological data. The titers (PFU/g of lung) obtained on days 5, 6, and 7 are presented in Fig. 5. The time required to reach the peak viral titer differed between strains: 5 days in C3H/HeN, DBA/2, and SJL, 6 days in BALB/c and DBA/2, and 7 days or more in C57BL/6. The 129/Sv strain displayed the highest peak viral load (on day 6) and SJL the lowest (on day 5). Only day 5 to day 7 SJL and day 7 C3H/HeN values were statistically different from 129/Sv isotime values (P < 0.05). Among strains in which the peak viral titer was reached on day 5, C3H/HeN and SJL displayed comparable day 5 to day 7 clearances (minus 1.14 vs. 1.09 log unit), whereas that of DBA/2 was not significant. Thus SJL combined the shorter time-to-peak viral titer, the lowest peak viral titer, and, with C3H/HeN, the best lung clearance. Topological distribution of viral antigens within the lungs (immunofluorescence) also generated very different results according to the strain (Fig. 4). The 129/Sv strain was distinguished by the presence of virus-positive bronchial/bronchiolar epithelial cells, type 1 and 2 pneumocytes, and macrophages homogeneously distributed throughout the lungs. The types of virus-positive cells were similar in DBA/2, C3H/HeN, C57BL/6, and BALB/c, but the virus-positive cells occupied an accumulated area of ~60–80% of the sample section area. In SJL, only few and far between virus-positive bronchial cells and subpleural pneumocytes were identified.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
One hundred twenty mice from six inbred strains were nasally inoculated with a standardized suspension containing PVM. The procedure caused a respiratory illness, as demonstrated by the changes in RPF values, respiratory histology, and pulmonary viral titers recorded 5–7 days thereafter. Significant differences appeared between strains, to the extent that a wide phenotypic spectrum was established for survival rate, clinical and tissue responses (bronchial cuffing, alveolar exudates, interstitial infiltration), eosinophil infiltration (present or not), lung viral peak titer (high, medium, low), and clearance (effective or not).

Methodological aspects. The priority was to achieve a level of standardization that would in fine enable the differences in the severity of the disease induced to be explained in terms of genetic background, thus making it possible, at least in theory, to designate a "resistant" and a "susceptible" strain at the end of the experiment. Regarding the pathogen, all mice were infected with an identical number of plaque-forming units from the same stock of a well-characterized viral strain (47) diluted in an identical volume of MEM. Preliminary experiments had duly established that the volume instilled in the nostrils with the selected procedure systematically caused diffuse deposition in the lungs while avoiding sneezing at the time of inoculation (which would have reduced the effective viral load) and the presence of changes in the breathing pattern during the following hours. As far as the hosts were concerned, all the mice were female, aged 14 wk, and seronegative for PVM and the most common murine pathogens. Thus the functional parameters reported were unaffected by changes related to sex and somatic growth (20). The methods chosen (double-chamber plethysmography and CO uptake) had been revealed to be reliable and sensitive techniques to evaluate the severity of respiratory diseases. Before inoculation, they generated reproducible baseline physiological measurements on the same animal subjects. After inoculation, they generated reliable and repeated ventilatory, mechanics, and gas exchange values that were well correlated with BW and histological and virological data. The experimental protocol as a whole generated an infectious model that was neither too benign (which would have reduced sensitivity) nor too severe (which would have gone against specificity and hindered the statistical analysis). The initial decision to restrict virological examination to days 5–7 pi was based on preliminary experiments (data not shown) that had established that peak viral titers were never reached before the fifth day pi, regardless of strain. The lung viral titers were not examined after the seventh day pi because the mortality rates among C3H/HeN, DBA/2, and 129/Sv were such that between-strain statistical comparisons lost significance. The peak titers were recorded on day 5 pi in SJL, C3H/HeN, and DBA/2 and on day 6 pi in BALB/c and 129/Sv and declined after, suggesting that shifts in the time course of viral replication among strains could be interpreted in terms of differences in innate (peak) or adaptive (clearance) immune responses. For C57BL/6J however, it is not certain that the peak was reached on day 7 pi, as a slight increase still occurred. Because the day 6–7 increase was only subtle, the day 7 value was considered as the peak. Each of the methods used to monitor the induced disease generated objective categories, ranging from "favorable" to "unfavorable." The combination of the functional, histological, and virological categories led to a classification of the strains in terms of resistance to the disease induced by PVM: SJL > C57BL/6 > BALB/c > C3H/HeN > DBA/2 > 129/Sv.

Functional severity. Specific access to the respiratory system was given by the monitoring of plethysmographic and CO uptake values, especially when considering the descriptive parameters of global breathing, expiration, and gas exchanges. In general, inoculation of PVM resulted in a combination of tachypnea, decreased TV, braking of expiratory emptying, and alteration in CO uptake. On the favorable side, besides a level of tachypnea roughly comparable to that displayed by other strains, SJL was the only strain of which the expiratory pattern, respiratory mechanics (sRaw), and CO uptake remained unchanged throughout the experiment, suggesting that PVM caused a symptomatic respiratory disease that was compensated by accelerating RR without altering the inspiration/expiration compartmentation (Supplemental Table S4). The "favorable" nature of SJL response was further confirmed by the observation that the mice were able to maintain their BW in face of the experimental disease induced. BALB/c and C57BL/6 differed from SJL mice in that they displayed an increased sRaw and developed a level of tachypnea that was not able to maintain the CO uptake.

Increased resistance (sRaw) points to a diminished cross-sectional area of the airways (20, 38). To the best of our knowledge, partitioning of sRaw between the different segments of the mouse respiratory tree is not available yet, which renders reliable interpretation in terms of location of the source of the added mechanical constraint very speculative. However, it was recently shown that nasal obstruction alone can substantially increase sRaw in mice (34, 35). Because turbinate histology did not differ among the six strains and the mouse breathing pattern response typical of nasal irritation or narrowing (slowing down of the beginning of expiration) was never observed (1, 6, 19, 49), it is tempting to conclude that the increased sRaw in C57BL/6, BALB/c, C3H/HeN, DBA/2, and 129/Sv results from tracheobronchial alterations rather than from superior nasal congestion and/or thickening of the mucosa. Furthermore, the fact that inspiratory duration, peak flow, and flow trace were never lengthened, blunted, or reshaped also argues against an increased resistance to airflow in the upper airways as an important facet of the pathological process (19). A decreased CO uptake theoretically points to hypoventilation, V/Q mismatch, blood shunting, and/or altered diffusion of gases from alveoli to capillaries. As far as C57BL/6 is considered, it can be seen that mice displayed a reduced CO uptake despite a normal MV, suggesting that the three other processes play the major role. This hypothesis is substantiated further by the observation that the lung histological changes were significant and extensive in all strains but SJL (see below). Also, BALB/c were distinguished from C57BL/6 in that they were not able to accelerate the final phase of expiration (TE5%), thus leading to a slightly altered expiratory compartmentation (EB), stable TE, and altered %TI. With this specific slightly altered expiratory pattern, BALB/c stands as an intermediate strain between SJL and C57BL/6 on the one hand and the three following strains on the other.

On the unfavorable side, C3H/HeN, DBA/2, and 129/Sv displayed qualitatively similar functional changes, which therefore generate a similar interpretation, but these were far exacerbated, which explains why death ensued in a significant fraction of the mice. In addition, three events were typical of these three strains: 1) lengthening of the terminal phase of expiratory emptying (TE5%), 2) abrupt decrease of ventilation (MV), and 3) dramatic reduction of CO uptake to <60% of baseline. The expiratory flow profiles recorded in these three strains (Fig. 3) closely resemble those known as being typical of deep pulmonary irritation/inflammation (1, 6, 34–36, 40, 49). Together with the fact that, in this study, only those strains with nonshortened/lengthened TE5% were subsequently shown to develop severe alveolar alterations, it is suggested that PVM infection results in deeper and/or more extensive lesions in those strains than in SJL, C57BL/6, and BALB/c. It therefore appears that the abatement of MV in face of a decreased gas diffusion capacity is at least partly attributable to the braking of lung emptying. Moreover, the hypoventilation contributed to the dramatic lessening of CO uptake, the latter unequivocally appearing as the most credible cause of death.

As the CO uptake is never decreased in Sendai virus (SeV)-infected BALB/c, SJL, C57BL/6, C3H/HeN, and DBA/2 mice (P. Faisca, unpublished observations), it can be deduced that PVM-infected mice are more likely suffering from hypoxemia and hypercapnia than SeV-infected mice. In this connection, a striking observation is that, 129/Sv excepted, SeV-infected mice display an intense hyperpnea (18) when PVM-infected mice do not, thus suggesting that compensatory reflexes are not working in the same way. Interestingly, in the SeV model, 129/Sv is the only strain in which 1) diffuse alveolitis and interstitial pneumonia and 2) abatement of CO uptake occur (18). Conversely, in the PVM model presented here, the sole strain in which CO uptake is not affected is also the only strain in which no pulmonary lesions are seen (see below). Thus, as a first approximation, the data are compatible with the hypothesis that a deep pulmonary disease in mice results in a braking of the compensatory ventilatory pattern changes.

Morphological severity. Although nonspecific, the monitoring of live weight enabled three main categories to be determined: favorable for SJL, which did not lose weight, fairly unfavorable for C57BL/6J and BALB/c (–10 and –13%), and catastrophic for C3H/HeN, DBA/2, and 129/Sv, the BW loss of which exceeded 15% in 7 days.

In general, PVM infection caused epithelial regressive (deciliation, degeneration, necrosis, and exfoliation) lesions and inflammatory granulocytic/mononuclear infiltrations, with very little epithelial hyperplasia. Whereas no obvious between-strain difference was recorded in turbinates and tracheae, lung lesions were clearly strain specific, confirming that the differences recorded in the RPF values are not attributable to the upper airways. In SJL, epithelial regressive lesions were extremely rare, luminal exudates were absent, and inflammatory infiltrations were very scarce, suggesting that viral replication is tightly controlled. In particular, eosinophils were not detected. Among the other five strains, epithelial regressive lesions were not prominent either, but there were widespread luminal granulocytic exudates, thick and dense eosinophilic/mononuclear peribronch(iol)ic cuffing, and severe interstitial infiltrations. Overall, the severity of interstitial mononuclear infiltration and the estimated fraction of alveoli and airway lumina filled with exudates correlated with survival and amplitude of functional changes, with 129/Sv and C3H/HeN showing the most dramatic changes. Conversely, the intensity of peribronchial lymphoid and eosinophilic recruitment did not correlate with survival or severity of functional changes. For example, C57BL/6 and BALB/c exhibited the most intense lymphoid cuffing and displayed an eosinophilic infiltration quantitatively comparable to that of C3H/HeN and 129/Sv while, on the other hand, showing only moderate BW loss and breathing pattern changes. Taking these five strains, a categorization based on the thickness of the peribronchial cuffs (C57BL/6 > BALB/c > DBA/2 > C3H/HeN > 129/Sv) correlates with clinical/functional severity, yielding a caricatural "the thicker the cuff the more resistant the mouse." However, SJL is by far the most resistant strain but peribronchial cuffs were never seen, thus invalidating the hypothesis according to which thick peribronchial cuffing protects against viral disease. With respect to the type of inflammatory cells involved, previous reports of PVM infection in mice have repeatedly emphasized the typical eosinophilic nature of the inflammatory response (14). Although this was confirmed in C57BL/6B, BALB/c, C3H/HeN, DBA/2, and 129/Sv, it is striking to note that eosinophils were absent from SJL. Moreover, PVM infection of knockout mice in which the macrophage inflammatory protein (MIP)-1{alpha}-chemokine receptor (CCR)1 axis is disrupted results in an attenuated neutrophilic/eosinophilic infiltration but exacerbated viral replication and mortality, suggesting that an intact acute neutrophilic/eosinophilic inflammation is protective (14). Here, the SJL response to PVM rather suggests that strong innate resistance occurs without significant neutrophilic/eosinophilic recruitment. This may be due to an SJL-specific early repression of PVM amplification at the site of entry or to a different kind of acute/early inflammatory response to the virus.

Virological severity. The results show that 1) the strain affects the maximum viral titer, 2) the strain affects the decrease of viral titer from day 5 to day 7, 3) there is no absolute correlation between the peak titer and the day 5 to day 7 decrease in viral titers, and 4) there is no clear correlation between lung titers and disease severity/survival (Fig. 5). The lung viral titers gathered here (104-108 PFU/g of lung weight) closely resemble those previously reported (12, 14–17). If SJL is excluded, which makes sense because this strain was never tested before, the range of peak titers exactly reproduces that reported in the literature (106-108). Similarly, the time-to-peak titer was always superior to 5 days, which also confirms the data available from the literature. With respect to the lung clearance of virions, two groups clearly emerged: SJL and C3H/HeN on the one hand, with a significant day 5 to day 7 pi abatement, and the other four strains, with rather a plateauing of viral titers. It is noteworthy that the severity of the induced disease can be roughly similar despite differences in peak titers or differences in lung clearance.

Resistance/susceptibility patterns to PVM in mouse. This study has made it possible to characterize several phenotypes relating to the reaction of murine hosts to PVM. The results suggests that resistance is associated either with the host capacity to repress the initial multiplication of the virus and to eliminate it or with a better tolerance to a nonrestricted viral growth/persistence within the lungs. SJL exhibit a symptomatic disease that is easily compensated by ventilatory adaptation, with no impact on the lung gas exchange capacity, generating slight mononucleated infiltration of the interstitium and peribronchic/vascular walls, in which viral replication is repressed and the virus swiftly eliminated. A striking aspect of PVM infection in SJL is the fact that neutrophilic exudates were scarce and eosinophilic infiltration was either absent or already extinct, which contrasts with available data. A possible explanation could be that the type and/or extent of the inflammatory response depends on the initial level of viral replication, a strong initial repression of PVM thus eliciting few or no early response. Alternatively, a silencing of the typical early inflammatory events may have occurred because of the SJL-specific genetic background. Whatever the cause, the picture of PVM infection in SJL shows that absence of granulocytic inflammation is not automatically associated with enhanced recovery of infectious virions and increased mortality, as previously demonstrated in MIP-1{alpha}/CCR1-knockout mice (14). Comparing the course of the disease in SJL with that in the other five strains studied here and with all the PVM infection data available, it is unambiguously concluded that PVM infection in SJL exhibits unique features related to the control of viral growth and elimination. It is hypothesized that these differences are genetically determined. C57BL/6 and BALB/c showed a symptomatic resistant-type respiratory disease with insufficient ventilatory compensation, as suggested by the alteration of the pulmonary gas exchange capacity. In those strains, PVM inoculation led to widespread granulocytic exudates, eosinophilic cuffing of bronchi/bronchioli, and severe interstitial infiltrations, with a plateauing of viral titers after day 5 pi. Finally, in C3H/HeN, DBA/2, and 129/Sv, a qualitatively similar picture was observed, but with far more intense lung inflammatory infiltrations associated with far more severe dysfunctions, which resulted in expiratory disturbances, collapse of the gas exchange capacity, and death. Among these five strains, C3H/HeN attracts attention because it associates the best "virological resistance" (in terms of peak titers and clearance) with the most dramatic global dysfunction (collapse of CO uptake) and BW loss and, logically, the highest mortality within the time window chosen. As the lung structure/function of C3H/HeN displays important strain-specific features, i.e., larger pulmonary volumes, higher compliance, and larger alveoli (27, 39, 41, 43, 46), it is anticipated that the total gas exchange area is probably lower, which could explain why the disease was more severe in this strain than in 129/Sv or DBA/2. In terms of resistance/susceptibility patterns to PVM, the study thus allows the delineation of two specific phenotypes, one related to the control of the viral amplification (SJL vs. C57BL/6, BALB/c, DBA/2, and 129/Sv) and the second related to the extent of the alveolar inflammatory response (C57BL/6 and BALB/c vs. DBA/2 and 129/Sv). These observations, made on strains that have been inbred for a long time, suggest plainly that genetic background strongly influences the apparent virulence of PVM.

Candidate resistance genes. A series of genetic loci have already been identified allelic variation of which underlies patterns of susceptibility/resistance to viral infection in mice, i.e., Mx1 for influenza viruses (44), Mx2 for vesicular stomatitis virus and hantaviruses (29, 30), Rmp for mousepox virus (8), Ly49h for cytomegalovirus (7), Bgp1 for mouse hepatitis virus (37), and Flv for flaviviruses (22). These loci are unlikely to play a role here. Indeed, the six strains used had been proven to carry the susceptible alleles at Mx1 and Mx2 loci; BALB/c and DBA/2 share a disease-susceptible phenotype against mousepox virus, whereas they significantly differ in resistance here; BALB/c carries the susceptible allele at the Ly49h locus and C57BL/6J the resistant allele, whereas they share a similar degree of resistance to PVM; and none of the strains used carries the resistant allele at the Flv locus. Therefore, only the Bgp1 locus could, at least theoretically, play a role in explaining the results gathered here because the SJL strain combines a resistant phenotype against PVM and mouse hepatitis virus, the latter being associated with a specific allele at this locus. However, the contrast between the pneumotropism of PVM on the one hand and the biliary tract-specific expression of Bgp1 on the other rather pleads against the hypothesis that Bgp1 could be involved. Thus it is anticipated that resistance to PVM is not underlined by antimicrobial resistance loci identified so far. Consistent with the protection conferred by manipulating the mouse MIP-1{alpha}-CCR1 axis (4, 5), it is anticipated that gene products underlying or acting on this axis should control the severity of pulmonary inflammation in response to PVM infection. Identifying differential expression of this chemokine and its receptor is therefore a work that should be done first in the hunt for candidate genes. The present study suggests that SJL and 129/Sv strains should be used in crossing experiments aimed at identifying the genes involved in resistance to PVM replication by the positional cloning approach. The genetic control of PVM-induced inflammation could in turn be approached by crossing BALB/c or C57BL/6 with DBA/2 or 129/Sv. Alternatively, experiments directly comparing the successive steps of the PVM biological cycle between SJL- and 129/Sv-derived primary lung epithelial cells in vitro could help in elucidating the mechanism opposed by SJL to viral replication. As a first step, such studies should consider the possibility that SJL mice might have fewer or lower-affinity receptors for PVM, which may or may not be relevant to RSV disease per se.


   ACKNOWLEDGMENTS
 
We are grateful for the scientific expertise provided by Dominique Cassart, Etienne Baise, Michaël Leroy, Thierry Flandre, and Jean-Adélite Habyarimana. We also thank Michaël Sarlet and Grégory Pire for excellent technical skills and enthusiasm.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. J.-M. Desmecht, Sart Tilman FMV-B43, Univ. of Liège, B-4000 Liège, Belgium (e-mail: daniel.desmecht{at}ulg.ac.be)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The online version of this article contains supplemental data. Back


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