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Virology 266, 52–65 (2000)doi:10.1006/viro.1999.0030, available online at http://www.idealibrary.com on

The Distribution and Kinetics of Polyomavirus in Lungs of Intranasally Infected Newborn Mice

K. Gottlieb and L. P. Villarreal1

Department of Molecular Biology and Biochemistry, University of California at Irvine, Irvine, California 92697

Received September 30, 1999; accepted October 4, 1999

The primary cell types that sustain polyomavirus (Py) replication following intranasal infection as well as the nature of thehost cellular response to Py were unknown. As this is an essential and specific site for virus entry, it seems likely that viralgene function must be adapted to these mucosal tissues. Using immunohistochemistry and in situ hybridization, wedetermined the cell types in the lung that support Py gene expression and replication following intranasal inoculation ofnewborn mice within 24 h of birth. Lungs were collected daily from days 1 to 10 postinfection for Py DNA and early T antigenanalysis and for histological examination by H&E staining, using methods that preserve the delicate newborn lungarchitecture. Viral DNA was present in increasing quantities from 2 to 6 dpi in a subset of the Clara cells lining the inner lumenof the bronchi and bronchioles, while T antigen expression was present in a majority of the cells in the bronchi and bronchiolelumen. A distinct and transient pattern of hyperplasia was observed among the cells expressing T antigen and was presentfrom 3 through 6 dpi. Py DNA-containing cells exfoliated into the bronchiole lumen and alveolar ducts, but Py T antigen wasnot detected in these cells. Py DNA was first detected at 2 dpi, increased through 6 dpi, and abruptly declined through 9 dpiat which time there was no sign of viral DNA in the lungs by in situ hybridization. An unusual infiltration of neutrophils beganbefore the presence of exfoliated cells or Py replication and continued for 2–3 days and was followed by a lymphocyticinfiltration at 8–10 dpi lasting 2–3 days. Neither the hyperplasia nor the neutrophil infiltration occurred following infection withthe MOP1033 MT-Ag or RB1 LT-Ag mutants of Py. In addition, both the neutrophil infiltration and the transient hyperplasia arein stark contrast to the heavy macrophage infiltration that follows infection of lungs with mouse adenovirus. Thus it appearsthat Py elicits a distinct host response pattern not seen with other DNA viral infections. © 2000 Academic Press

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INTRODUCTION

Despite decades of polyomavirus (Py) research, theprimary cell types that support mouse polyomavirus rep-lication following natural infection remain unknown. Poly-omavirus was discovered independently by Gross andStewart while attempting to transmit AKR mouse leuke-mia to newborn mice (Stewart, 1955; Stewart et al., 1957).Subsequently, Rowe described inhalation as a portal ofentry for polyomavirus when he discovered that new-borns could be infected intranasally (i.n.) (Rowe et al.,1958). Rowe and colleagues later demonstrated that theintranasal route is very ineffective for inducing tumors,which may explain why wild mice infections or i.n. infec-tion of outbred strains do not normally result in tumorsfrom polyomavirus (Rowe, 1961), although laboratory i.n.-induced tumors are possible in some mouse strains(Dubensky et al., 1991). However, like many other viruses,

olyomavirus must normally infect and travel through theungs prior to reaching its site of persistent infection, theidney. Thus the mucosal respiratory tissue presents the

irst crucial biological and adaptive hurdle for the poly-mavirus proteins (small, middle, and large T antigens),et the functional role of Py proteins in lungs remainsnexplored.

1 To whom correspondence and reprint requests should be ad-ressed. E-mail: [email protected].

0042-6822/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

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Earlier investigations by our lab using a dot-blot sys-em for DNA detection demonstrated that an intranasalnoculation of PyA2 in newborn mice less than 24 h ofge reached peak infection at 6 days postinfection (dpi)nd cleared the virus by 10–12 days (Dubensky andillarreal, 1984). Further investigations using wholeouse hybridization supported these findings in that

iral DNA was initially confined to the respiratory tract,ncreasing from days 2 to 7 postinfection and tapering offy day 9 (unpublished data). These studies, however,either demonstrated a histopathological evaluation ofy replication in the lungs, identified the cell types in-olved, nor evaluated the expression of viral proteins.

The role of the early Py proteins, small, middle, andarge T antigen, as it pertains to transformation has beenhe major focus of polyomavirus research. However, littlenformation has been described about the evolved rolesf these proteins during the acute or persistent infection.ne seemingly obvious role is that the early viral pro-

eins must activate quiescent cells to initiate their DNAeplication machinery, though we have been unable to

easure Py induction of cellular DNA synthesis in vivoMoreno and Villarreal, 1992). Large T antigen (LT-Ag),ssociated with immortalization, initiates viral replica-

ion, transcription, and immortalization of primary cells byinding pRb (Freund et al., 1994; Pilon et al., 1996).iddle T antigen (MT-Ag), associated with transforma-

ion, is needed to increase viral DNA synthesis in cells

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53POLYOMAVIRUS IN NEWBORN MICE

not growing at time of infection (Freund et al., 1992). Inaddition, the antiapoptotic signal transduction inducedby MT-Ag presents a potential in vivo role for establish-ment of productive viral replication in the lung followingintranasal infection (Larose et al., 1991; Dilworth, 1995;Bergqvist et al., 1997; Kennedy et al., 1997).

In addition to their role in altering host cell growthcontrol, it now seems clear that essentially all mamma-lian DNA viruses express early proteins that must con-tend with and alter both the innate and the adaptive hostimmune system. Perhaps the most common DNA virus-directed alteration is to the TNFa response of infectedhost cells, which we have previously observed in Py-infected kidneys (Atencio et al., 1995). Polyomavirus earlygenes may also alter other host responses. Kidneyspersistently infected with polyoma are generally free ofinflammatory response, which suggests a virally sup-pressed host reaction. A few reports have establishedthat Py-infected cells are susceptible to cytotoxic T lym-phocytes, yet a strong response against the virus israrely seen (Sellins and Cohen, 1989; Ramqvist et al.,1989; Reinholdsson-Ljunggren et al., 1993; Lukacher andWilson, 1998). Middle and small T antigens in combina-tion protect against TNFa-induced apoptosis in infected

ells, yet the signal transduction effects of middle T thatrive transformation increase susceptibility to TNFa-in-uced apoptosis (Bergqvist et al., 1997). LT-Ag inhibitsellular responses to interferons, though no report of

nterferon induction by the presence of Py has beeneported (Weihua et al., 1998). That Py middle-T mutantso not replicate in primary cells, but do in establishedell lines, also suggests that the full role of viral earlyenes in host cycle control might be obscured in cellulture systems. It is therefore important to evaluate the

nteraction of Py genes with the more complex tissue andmmunological environment of the newborn mouse lung.

The respiratory route of infection represents the mostommonly used portal of entry for mammalian virusesnd must be crucial for natural Py infection. Studies of

ung infection with other mouse viruses, including Sen-ai virus, pneumonia virus of the mouse, and mouse-dapted strains of influenza, have identified the epithelialells as tropic targets which reach peak titer at 6–7 dpi,

ollowed by immune-mediated clearance (10–14 dpi),hich includes leukocyte infiltration and exfoliation of

nfected cells (Robinson et al., 1968; Wyde and Cate,978; Wyde et al., 1978; Parker and Richter, 1982; Bendert al., 1995; Wijburg et al., 1997). However, because Py

may need cellular differentiation to effectively replicate invivo, this could predict that only those lung cells under-going differentiation would be likely targets of Py infec-tion (Villarreal, 1991; Atencio and Villarreal, 1994). Claracells, which are unusually abundant in mouse relative tohuman bronchi and bronchioles, are the most active indifferentiation, proliferation, and protein synthesis andare also believed to be the progenitor cells for bothnonciliated and ciliated epithelium, the latter recognized

by their characteristic apical projection into the lumenand basal nucleus. (Evans et al., 1978; Reznik-Schullerand Reznik, 1979; Plopper et al., 1980; Kauffman, 1980;Pack et al., 1981). In mice, Clara cells are found in themain, lobar, segmental, and subsegmental bronchi, bron-chioles, and terminal bronchioles, while ciliated cells arefound in the trachea, the upper bronchi, and occasionallythe lower bronchi and bronchioles (Reznik-Schuller andReznik, 1979). The other differentiating cell types of thelung include basal cells, which must be exposed follow-ing desquamation of inner layers of cells to be initiallyinfected, and type II pneumocytes, which are in the lowerregions of the respiratory system. Thus Clara cells wouldseem to be a likely primary target for Py viral replication(Pack et al., 1980, 1981; Villarreal, 1991; Atencio andVillarreal, 1994).

The anatomy of the mouse lung also contains a fewother features distinct from human lungs. Mice lack theability to cough as they don’t have nerve endings in thetrachea and main bronchus, although they still have alimited “expiration reflex” (Pack et al., 1984). In addition,

ice do not have lymphoepithelial tissues in their naso-harynx, a suspected site of initial replication for JCV inumans (Monaco et al., 1998), thus murine Py may notncounter this tissue barrier before entering the lung.

The major aim of this study was to determine the cellropism of Py in the lung, along with the kinetics of theppearance and dissemination of Py DNA by in situybridization, the expression of viral proteins by immu-ohistochemistry, the histopathological changes, andellular infiltration due to the presence of Py. We identify

he Clara cells as the initial site of Py transcription, earlyene expression, and replication, which initiates at 24 hi and peaked at 6 dpi. The virus-replicating cells exfo-

iated into the cavity of the bronchiolar lumen beginningt 3 dpi and were cleared by 10 dpi. However, other

nfected cells became transiently hyperplastic and ex-ressed T-Ag without completing virus replication. Thereas a strong innate immune response consisting of

nfiltrating neutrophils (PMNs) very early in infection,orresponding to the period of exfoliation and a later

ymphocyte infiltration concurrent with an adaptive im-une response.

RESULTS

linical disease

As it is often proposed that acute viral infection mustamage host cells during lytic replication, it might bexpected that primary lung infection with high-titer Pyould result in visible symptoms of disease. We moni-

ored newborn mice after high-titer Py intranasal inocu-ation for any signs of disease. Among the four sets ofitters infected (approximately 28 mice), there were noigns of disease or behavioral changes in the newbornice over the week following intranasal (i.n.) inoculation,

lthough some mice weighed less than their control

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54 GOTTLIEB AND VILLARREAL

counterparts. In addition, mothers showed no signs ofdisease after cleaning the newborns of the remaininginoculate, a consequence of this experimental intranasaltechnique.

Gross pathology and histopathology

Using the perfusion methods described under Materi-als and Methods, the lungs of i.n.-infected newborn micewere examined histologically by hematoxylin and eosin(H&E) staining of both frozen and paraffin lung sections.The infected lung tissue appeared to be relatively normalcompared to control mice in terms of overall structureexcept for the appearance of a large number of infiltrat-ing neutrophils in both the perialveolar space (spillagefrom the capillaries lining the alveoli) and the peribron-chiolar space (direct infiltration between the epithelialcells and into the lumen) (Figs. 1A and 1B). Although afew macrophages were observed in the parenchyma,there was neither an increase in the number of mono-cytes or macrophages in uninfected or infected areasnor a significant infiltration. As neutrophils may conceiv-ably have resulted from a bacterial contamination, gramstaining was performed on slides containing neutrophilsbut was negative for bacteria (data not shown). Further-more, mock-infected controls containing the same me-dium and disrupted 3T6 cell supernate intranasally inoc-ulated produced no noticeable inflammatory response(Fig. 1D). Therefore, the neutrophil response is particularto Py infection. Early after the inoculation (2 dpi), the firstindication of viral infection was noted by the appearanceof infiltrating neutrophils, even though in situ hybridiza-ion did not yet show the presence of viral DNA (seeelow, Fig. 2A). At 3 dpi, the appearance of distinct,arkly nucleated cells in the sawtooth portion of the

nner lining of the bronchioles was noted by H&E stain-ng and continued through the course of the infection ashown in Fig. 1C at 5 dpi (left arrow). There was alsoome transient accumulation of cellular debris in the

erminal bronchioles, alveolar ducts, and alveoli. As bothrozen and paraffin, inflated and collapsed (not perfused/ot flushed) sections showed cellular debris in the alve-li (established to be Py DNA positive, see below), thisebris was not a result of the lung perfusion during

ixation. Collapsed tissue, which was not perfused,howed a high level of debris within the lumen of theronchioles and was regarded as a mix of infiltratingells and cells lysed by virus infection. However, therocess of perfusion without a constant pressure systemould likely wash the less stable debris out of these

avities. Yet even under these circumstances of consid-rable viral-induced cellular alterations, no overt histo-

ogical signs of cellular necrosis or apoptosis wereresent. In addition, there were no delays in the growthf the alveoli which normally closely follows birth. Inome (but not all) instances, patches of heavy perialveo-

ar neutrophils appeared during later time points (Fig.

2H). Although there was some mouse-to-mouse varia-tion, a lymphocytic response began around 8–10 dpi andcontinued for 2–3 days. The lymphocytes surrounded thebronchioles and blood vessels in heavy patches (Figs.1E, arrow, and 1F, high magnification).

Hyperplasia in the bronchioles was apparent in thelungs from 3 dpi, becoming more prominent by 6 dpi (Fig.1C, right arrow) and dissipating by 8 dpi (Fig. 1E). Thehyperplasia was seen as multiple undulating layers ofcells extending out of the bronchioles, including seg-mental and subsegmental regions. The cells involved inthe hyperplasia were designated epithelial cells with themajority being Clara cells based on location and mor-phology. The appearance of the hyperplasia was furthermarked by some infiltrating leukocytes. The hyperplasiadissipated during the heavy cellular sloughing seen at5–6 dpi (see inset, Fig. 2D). All of the mice were eutha-nized by CO2 asphyxiation, which can cause small hem-orrhages in the lungs. However, these hemorrhageswere eliminated by infusion with fixative. Therefore thehyperplasia was not an artifact of asphyxiation or han-dling, nor was it present during mock infection.

Kinetics and cellular pattern of Py infection by in situhybridization

To localize Py DNA in specific cells, lung tissue wasexamined by in situ hybridization on formalin-fixed sec-tions as described under Materials and Methods. Over-all, with some mouse-to-mouse variation, the virus ap-peared in almost all the lobes of the lung, though earlytime points showed virus in only one or two lobes.Newborn mice infected intranasally within 24 h of birthshowed only slight signs of Py replication as early as 2dpi (Fig. 2A); however, at 1 dpi lungs were clear ofdetectable Py DNA (data not shown). The virus DNAincreased considerably from 3 through 6 dpi (the peak),with the majority of nuclear viral DNA found in the mostapical cells lining the inner portion of the bronchioles(Figs. 2B–2D). The cells positive for Py DNA were on thesawtooth portion of the bronchioles, the same locationas Clara cells (arrows, Figs. 2B and 2D). Only a fraction(less than 20%) of those cells lining the inner surface ofthe bronchioles showed Py DNA on any given day. How-ever, a substantial amount of material highly positive forPy DNA was also found in exfoliated cells filling thelumen of the bronchioles and the perialveolar spaces(see inset, Fig. 2D). The highly positive cells in the peri-bronchiolar and perialveolar spaces corresponded toinfiltrating neutrophils based on histopathological in-spection and appeared to mainly (95%) consist of theinfiltrating cells, as seen by H&E staining (arrows, Figs.2G and 2H). Py DNA in these locations appeared ex-tranuclear, diffusely present throughout the cell, andseemed to include the cytoplasmic regions, although theintense staining and small size of neutrophils preventeda precise histological analysis. Due to the rapid infusion

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of formalin, however, many of the loosely connected cellswere likely to have been displaced from the lumen of theupper airway. Though this was not considered a source

FIG. 1. Hematoxylin and eosin (H&E) staining of paraffin lung sectiorrow) and perialveolar infiltration (left arrow) of neutrophils; also notagnification 2003). (B) Neutrophilic infiltration shown in A at 4003

morphology of neutrophils (arrow). (C) 5 dpi—intense hyperplasia amonwithin the lumen of the bronchiole (original magnification 2003). (D) 5-(original magnification 2003). (E) 8 dpi—bronchiolar epithelial layebronchiole (arrow) (original magnification 2003). (F) Lymphocytic infiacentrically located nucleus and narrow rim of cytoplasm morphologyn 10% formalin for 24 h. Intranasal mock infections were performed u

of extensive aberration, sections which were not infused(collapsed) or only partially infused showed higher num-bers of exfoliated cells with Py DNA in the lumen of the

intranasally infected newborn mice. (A) 3 dpi—peribronchiolar (righty nucleated cells in the sawtooth portion of the bronchioles (originalal magnification; note the granular cytoplasm and lobulated nucleilara cells (arrow) and the beginning of exfoliation of a number of cellsmock-infected control demonstrates lack of infiltration or hyperplasia

ars normal while the lymphocytic response appears heavy aroundshown in E at 4003 original magnification; notice the single large,

hocytes. H&E staining was performed on 6-mm paraffin sections fixedme medium and disrupted 3T6 cell supernate as the Py inoculum.

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FIG. 2. In situ hybridization (ISH) for PyA2 of paraffin lung sections from intranasally infected newborn mice. (A) 2 dpi—low, undetectable levelsf viral DNA with surrounding neutrophil infiltration (arrow) already present. (B) 3 dpi—only the very apical cells (arrow) in the bronchiolar lumen areositive for Py (original magnification 4003). (C) 5 dpi—accumulation of Clara cells in a hyperplastic state and only apical cells and exfoliating cellsositive for Py. (D) 6 dpi—the peak of infection with a majority of the surface positive for Py (arrow), inset shows the sloughing of a large number


bronchioles. The thicker sections (10 mm) made fromfrozen tissue also indicated the higher volume of exfoli-ated Py-positive cells (data not shown). After 7 dpi theviral DNA began to dissipate and most of the virus wasfound only in exfoliated cells (Fig. E). In a few instances,heavy patches of perialveolar neutrophils appeared pos-itive for Py DNA, but were often contained to a singlelobe (arrow, Fig. 2H). From 8 to 10 dpi the majority of thevirus was eliminated and did not appear by in situ hy-bridization, except as a few scattered cells and less than5% of those cells lining the inner surface of the bronchi-oles (Figs. 2E and 2F). After 10 dpi, the presence of PyDNA was below the lower limit of detection by in situhybridization.

Immunohistochemistry

The presence of viral early proteins and late proteinswas determined using immunohistochemistry as de-scribed under Materials and Methods. In frozen sections,a significant amount of T-Ag was found in multiple layersof cells lining the inner walls of the bronchioles as earlyas 2 dpi (Fig. 3A). During the time period of 2 to 6 dpi,most epithelial cells lining the inner wall of the bronchi-oles were positive for T-Ag (Figs. 3A–3D). Due to the highnumbers and location of T-Ag-positive cells, both ciliatedand nonciliated (Clara) cells appeared to be infected.The basal pattern of T-Ag expression appeared in con-trast to those cells positive for Py DNA (noted above,Figs. 2B–2D) in that DNA-positive cells were limited tomainly Clara cells only at the apical surfaces of thesaw-toothed portions of the bronchiolar lumen. The T-Agsignal dissipated rapidly over 7–8 dpi, remained at lessthan 5% of the bronchiolar epithelial cells just prior tolymphocytic infiltration, and cleared completely around12 dpi with some mouse variation (Figs. 3E and 3F). Thepositive cells closely corresponded to those areas ofhyperplasia in the bronchiolar lumen noted above (Fig.1C). However, there was no detection of T-Ag expressionin the infiltrating neutrophils, which showed an intensepositive signal for Py DNA. In addition, T-Ag was notfound in the exfoliated cells or the debris positive for PyDNA. Thus, the architecture and pattern of T-Ag expres-sion appeared to identify a significant number of Claracells and other epithelial cells that were positive for T-Ag,but did not show Py DNA by in situ hybridization. How-ever, T-Ag expression closely followed the progression ofhyperplasia and dissipated as the exfoliation increased.In contrast, VP-1 expression corresponded closely withthose cells positive for Py DNA, including the apical cells

of infected cells at 6 dpi. (E) 8 dpi—sloughing of infected cells contininfiltration begins (arrow). (F) 10 dpi—only a small fraction of cells remaiin perialveolar regions (see Fig. 1 for neutrophil location/morphology).distribution of Py DNA-positive cells. ISH was performed on 6-mm pamagnification unless noted otherwise.

in the bronchioles, the cellular debris, and the infiltratingneutrophils (Figs. 3G and 3H).

Age dependence

The original studies of Rowe and colleagues had in-dicated that only infection of newborn mice resulted inpersistently infected mice that were subsequently able totransmit Py infection to other mice, whereas mice in-fected as adults were unable to transmit Py infections orestablish persistent infections (Rowe et al., 1958). If lunginfections were important for this age restriction, it mightbe expected that lungs become nonsusceptible to Pyinfection soon after birth. To examine this possibility,mouse litters were intranasally inoculated 48 h or 4 daysafter birth. Mice infected 48 h after birth expressed thesame kinetics of Py DNA and T-Ag expression as thoseinoculated 24 h within birth (Figs. 4A–4C). In contrast,there was little or no detection of Py DNA or T-Ag ex-pression in mice inoculated 4 days after birth (data notshown). Thus it appears that some restriction to primaryPy infection of lungs must develop between 2 and 4 daysafter birth. Adult mice over 6 weeks of age were alsointranasally inoculated and showed no signs of Py DNAor T antigen expression (data not shown). Thus theseresults establish that within 4 days of birth, newbornmice become relatively nonsusceptible to lung infectionwith standard intranasal inoculation of Py.

Passive immunity

The passage of maternal antibodies to offspring hasbeen described for polyoma as the primary source ofimmunity for newborn mice (Stewart et al., 1960). Inaddition, the mothers of infected litters seroconvert rap-idly due to the residue of Py following intranasal inocu-lation. Because it is believed that natural Py transmissionwould likely occur from persistently infected mothers topups, it also seems likely that these mothers wouldtransfer passive immunity to their pups. Thus it was ofinterest to determine if the pattern of primary Py lunginfection and host response was altered or prevented inpassively immune pups. Figures 4E and 4F demonstratean increased inflammatory response, including a less-ened neutrophil response and a 2- to 3-day earlier lym-phocytic infiltration, in newborns following intranasal in-oculation of litters of seroconverted mothers. A strikingdifference and much lower amount of Py DNA and T-Agexpression can be seen at 5 dpi between mice withmaternally acquired immunity (Figs. 4G and 4H) andmice born to naive mothers (Figs. 2 and 3). Thus passive

ile the bronchiolar walls return to a single layer and the lymphocyticive for Py. PMN infiltration: (G) 3 dpi—neutrophils positive for Py (arrow)i—late neutrophil infiltration positive for Py DNA (arrow), note unevenections fixed in 10% formalin for 24 h. All sections at 2003 original

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FIG. 3. T antigen expression from PyA2 intranasally infected newborns. Immunofluorescence (IF) was performed on 10-mm frozen sections,aturated with 19% sucrose in PBS and OCT embedded, with a rat anti-T-Ag antibody against all three T antigens. T-Ag was expressed in ciliated and


immunity appears to limit, but not prevent, primary Pylung replication and induces a much stronger and earliercellular lymphocytic infiltration.

Polyoma mutants

Our observation that the basal layers of Clara cellsbecame transiently hyperplastic following Py infectionand were expressing T-Ag led us to consider if viral earlygenes might be involved in this effect. Because it wasknown that some of the Py early gene domains interactwith important cellular regulators of the cell cycle andbecause it was also known that not all of these functionswere needed to replicate Py virus in cell culture, it was ofinterest to determine if any of these early protein do-mains were important for this hyperplastic reaction or thereplication of Py in primary sites of in vivo infection.PyA3-MOP1033 was examined for replication in newbornmouse lung. This virus expresses a truncated polyomaMT-Ag which fails to bind the plasma membrane and isdefective in transformation, although it grows well inestablished cell lines. MOP1033 was made by producingan opal termination codon at nucleotides 1033–1035 inthe reading frame for MT-Ag; however, as a conse-quence there is a proline-to-leucine change in the aminoacid sequence of LT-Ag as well. The lungs of theMOP1033-infected newborn mice showed no signs ofthe hyperplasia present in the wild-type virus infection orthe infiltrating neutrophils or lymphocytes (Fig. 5A). Inaddition, they had low levels (less than 5%) of the viralDNA and T-Ag expression seen in PyA2-infected lungs(Figs. 5B–5D). The background control, PyA3, exhibitedonly slightly lower levels of Py DNA over PyA2 during the7- to 10-day test period (data not shown). This decreasedactivity was taken into account when comparingMOP1033 to PyA2. Thus it is clear that a functionalMT-Ag is important for Py replication and altered cellularmorphology in the newborn mouse lung.

Similarly, the pRb binding site of polyomavirus LT-Aghas been previously mutated in the PTA background(PTA-RB1). This mutant is unable to immortalize primarycells, but can grow readily in established cell lines. ThePTA parent of this virus was shown to have the samekinetics and distribution as A2 following intranasal inoc-ulation of newborns (data not shown). When mice wereinfected with Py PTA-RB1, these lungs exhibited no PyDNA in two litters inoculated intranasally within 24 h ofbirth and looked similar to the PyA3-MOP1033-infectedlungs (Fig. 5). Thus, although the Rb binding site is notrequired for replication in some cultured mouse cells, itwas clearly required for primary lung infection and hy-perplasia in newborn mice.

nonciliated (Clara cells) epithelial cells of the bronchioles. (A) 2 dpi, (BT-Ag in bronchiole, (F) 9 dpi—note complete lack of T-Ag expression inamount of debris and infiltrating neutrophils as well as bronchiolar epioriginal magnification.

MAV infection

The predominantly neutrophilic infiltration we ob-served following Py lung infection seemed unusual orunexpected for an innate reaction as neutrophils arethought to recruit other immune cells and are not con-sidered highly phagocytic cells. We therefore examinedthe host cellular response to another DNA virus, mouseadenovirus (MAV-1), to determine if this neutrophil reac-tion was unique to Py lung infection. A large plaquevariant of mouse adenovirus was used to infect mice ata titer equal to that of the Py. Following intranasal infec-tion, a strong macrophage infiltration was seen in thelungs within 3 dpi (Fig. 6A). This was a response dis-tinctly different from what we observed with Py and moresimilar to influenza-infected mouse lung, in which a rapidand strong macrophage infiltration was also seen. WithPy, few if any macrophages were seen. With MAV-1, fewif any neutrophils were observed. Thus it seems clearthat the neutrophil response is not a general antiviralresponse and likely reflects some aspect of Py geneactivity and tropism.

DISCUSSION

The major site of Py replication in newborn lungsinfected intranasally within 24 h of birth was found to bea selected subset of the Clara cells lining the inner wallsof the bronchioles. Py DNA-positive cells had a distinc-tive appearance and could be identified without in situhybridization in that they had condensed nuclei and wereelevated from the lining cells such that they appeared tobe in the process of exfoliation. The presence of earlyproteins showed a pattern somewhat different from thatof Py DNA. Although this was also mainly limited to Claracells, these T-Ag-expressing cells were basal to the cellswith Py DNA and were found in the majority of cells liningthe bronchioles (including areas with ciliated and non-ciliated epithelium) and maintained T-Ag expressionfrom 2 through 7 dpi (Fig. 3) with a few cells remainingpositive through 10 dpi. This appears to establish thatmany lung cells, although infected with Py, were notpermissive for replication. Curiously, the Py DNA-positive“exfoliating” cells showed little if any T-Ag, which suggesta strong down regulation of T-Ag levels during viral DNAreplication.

Clara cells represent over 70% of the lung epitheliumand are found throughout the respiratory system of themouse, including nasal cavities, trachea, bronchi, andbronchioles (Matulionis and Parks, 1973; Reznik-Schullerand Reznik, 1979; Pack et al., 1981; Villaschi et al., 1991).In the bronchial epithelium, where Py replication is most

, (C) 4 dpi, (D) 5 dpi, (E) 7 dpi—note low numbers of cells positive forhiole. IF for VP1, one of the three Py capsid proteins, (G) 4 dpi—largecells are positive for VP1, (H) 5 dpi—same as G. All sections at 2003

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FIG. 4. Paraffin lung sections from mice intranasally inoculated 48 h after birth. (A) 5 dpi—H&E staining; note hyperplasia of Clara cells. (B) 5dpi—ISH, only the apical cells show Py DNA in a pattern similar to mice intranasally inoculated within 24 h of birth. (C) 5 dpi—IF for T-Ag. (D) 5-daymock-infected control; note lack of hyperplasia and evenness of epithelial layers. Lung sections from PyA2 intranasally inoculated newborns of

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apparent, they create the saw-tooth appearance (Reznik-Schuller and Reznik, 1979; Plopper et al., 1980). Claracells are roughly cuboidal in shape, project their cyto-plasm into the airway lumen, have an irregularly shapednucleus at the base of the cell, and are abundant in bothendoplasmic reticulum and secretory granules (Plopperet al., 1980; Massaro et al., 1994). Basal cells and type II

neumocytes, both differentiating cell types, are oftennfected by other viruses and would seem to be likelyargets for Py virus replication. However, neither of theseell types was shown to be positive for Py DNA or T-Ag.lthough mice do not have an extensive amount of end-tage cilia in their upper respiratory tract relative toumans, these ciliated cells are sufficiently abundant totill represent the first epithelial cells that Py virus is

ikely to encounter during lung infection in the tracheand upper bronchi (Pack et al., 1980). However, the end-tage ciliated cells in the lower bronchi and bronchioles,

seroconverted mothers (previous litter also intranasally inoculated). (E ainfiltration in perivascular and peribronchiolar space at this early time(H) 5 dpi—IF for T-Ag; still a number of cells expressing T-Ag without

FIG. 5. Lung sections from mice intranasally inoculated with PyA3-Mfound in wild-type Py. (B) 5 dpi—ISH; lack of Py replication as evidenexpression in only a fraction of the bronchiolar epithelial cells. (D) 6 dpperformed on 6-mm paraffin sections of formalin infused and fixed lung

here the majority of Py DNA was present, appeared toe only abortively infected by Py, as they seemed to bemong the cells expressing T-Ag but did not replicate PyNA. This apparent blockage of Py replication in a sub-et of bronchiolar epithelial cells established that Pyarly proteins were unable to induce these cells into aroductive S-like phase and appeared to establish that

he ability of Py early genes to support Py DNA replica-ion was restricted to a specific cell type. Other scatteredells in the alveoli did appear Py DNA positive by in situybridization, but these cells did not express T antigens,

hus they were not considered permissive for Py replica-ion. It seems likely that these cells corresponded tonfiltrating neutrophils and to a lesser extent alveolar

acrophages, which may be scavenging virus or exfoli-ted cellular debris, as this Py DNA appeared cytoplas-ic.Clara cells are an interesting site for Py replication

dpi—H&E staining shows presence of both neutrophil and lymphocyteG) 5 dpi—ISH; lack of Py DNA in cells, though still present in debris.ting DNA. All sections at 2003 original magnification.

3. (A) 5 dpi—H&E staining; lack of hyperplasia or immune infiltrationsundetectable levels of Py DNA. (C) 3 dpi—IF for T-Ag; note very lowe as C; only a few cells expressing T-Ag. ISH and H&E staining were

as performed as for Fig. 3. All sections at 2003 original magnification.

nd F) 5point. (replica

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since they are known to secrete proteins, including theClara cell secretory protein (CCSP), that can downgradethe lung inflammatory response to acute virus infection(Harrod et al., 1998). CCSP has been shown to limit bothadenovirus-initiated macrophage and neutrophil infiltra-tion and cytokine responses, including those of TNFa,IL-1b, IL-3, IL-6, and chemokines MIP-1a, MIP-2, and

CP-1 (Harrod et al., 1998). The predominantly neutro-hilic infiltration following Py infection may suggest thaty early gene expression disturbs Clara cell function andlters the usual host cellular infiltration. In addition, Claraells are actively maturing and differentiating in theouse at birth and support Py replication, whereas other

ronchiolar epithelial cells expressed only Py T-Ag andid not support Py replication (Evans et al., 1978;epulveda and Velasquez, 1982; Villarreal, 1991;arkema et al., 1991; Atencio and Villarreal, 1994; Mas-

aro et al., 1994). This appears to be in agreement withur proposal that Py replication requires ongoing hostell differentiation in vivo.

We have also confirmed that mouse lungs are suscep-tible to Py infection only during the first 2 days after birthunder standard intranasal inoculation conditions per-formed here. This result appears to finally clarify theearly and still unexplained observation that only new-borns are permissive for Py infections which allow sub-sequent viral transmission (Dawe et al., 1959; Rowe,1961). The age dependence of Py replication is notunique to Py virus, as many other viruses, includingherpes simplex virus, coxsackie virus, poxviruses arbo-viruses, and many of the oncogenic viruses, show asimilar unexplained restriction (Allison, 1967; Subrah-manyan, 1968; Hirsch et al., 1970; Zisman et al., 1971;Rager-Zisman and Allison, 1973; Tardieu et al., 1980). Ouranalysis establishes that mice infected 4 days after birthno longer showed evidence of even abortive infection(early region expression) in the lungs nor signs of hyper-plasia or neutrophil infiltration. This suggests that Py wasunable to reach or enter its target cells and would ap-

FIG. 6. Mouse adenovirus intranasal infection. (A) 3 dpi—H&E staininumbers of infiltrating macrophages and lymphocytes surrounding thoriginal magnification. Intranasal mock infections were performed using

pear to preclude the involvement of any innate or adap-tive immune response which requires expression of viralgenes. It is possible that mechanical processes, such asthe development of the mucociliary blanket, or otheranatomical changes, such as decreased aspiration, maybe preventing Py lung infection as these systems arelikely to be immature at birth. Further investigation by usis under way to address this issue.

The two main histological observations we made werethe appearance of transient hyperplasia in bronchiolarClara cells expressing T-Ag and the presence of exfoli-ated cellular debris highly positive for Py DNA that cor-responded to the infiltrating neutrophils. These observa-tions are in notable contrast to mouse lung infection withSendai virus, which shows an extensive necrosis andsubstantial inflammatory response, including macro-phages, neutrophils, and lymphocytes, followed by cel-lular exfoliation, transient hyperplasia, and viral clear-ance by 13 dpi (Robinson et al., 1968; Ward, 1974; Ishidaand Homma, 1978). The hyperplastic abortive Py-infectedClara cells suggest that Py early genes may directlymanipulate host cell division or induce exfoliation inlinkage to the activation of Py DNA replication and lategene expression. These particulars of Py-infected Claracells were shown in Figs. 1–3, which note the distinctionbetween basal Clara cells that express Py early genesand are hyperplastic and those apical cells that expresslate genes, have low T-Ag, replicate Py DNA, and exfo-liate. The inference is that a subset of T-Ag-expressingClara cells will go on to replicate Py DNA (perhaps thoseinduced to produce replication factors), express lategenes, and exfoliate while suppressing the production ofT-Ag, whereas the other T-Ag-expressing Clara cells be-come transiently hyperplastic and then rapidly disap-pear, without progressing to virus production. However,the rapid disappearance of Py replication in lungs seemscurious relative to the delayed replication of Py in sec-ondary organs (such as the kidney). We have no obviousexplanation as to why the first round of lung Py replica-

ng section from a mouse intranasally inoculated with MAV. Note largechiole. (B) 3-day mock-infected lung section. Both sections at 2003me medium and disrupted 3T6 cell supernate as in the MAV inoculum.

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tion (peak at 4 dpi) is not followed by a second round oflytic infection since replication in secondary sites (kid-ney) will not have peaked until 8–12 dpi, the time afterwhich lung infections are cleared of Py DNA. It seemslikely that a rapid and local host response (or limited hostcell differentiation) is somehow limiting continuedrounds Py lung replication. Alternatively, the hyperplasiaand subsequent exfoliation of a high number of cellsaround 5–6 dpi may be directly responsible for the elim-ination of a majority of the virus-permissive cells from thebronchioles, acting as a highly specialized transient re-sponse which prevents subsequent rounds of virus in-fection.

The primarily neutrophilic response to Py was unex-pected in that this rapid (2 dpi) chemotactic neutrophilresponse was not followed by an expected macrophageresponse as typically seen in adult mouse viral infectionsor as we observed with MAV-1-infected lungs. With Py,few macrophages were seen to follow the neutrophilresponse and a lymphocytic response did not begin untilaround 8 dpi. The delayed onset of a macrophage orlymphocytic response could be due to the inability ofimmature macrophages to present antigen in newbornmice (Lu et al., 1979). However, such a delay would seemto give Py an opportunity to spread to secondary sites(such as the kidney) and establish persistence beforethe adaptive immune system controls virus spread. How-ever, passively immune pups did show a more rapid andstronger lymphocytic response, suggesting that passiveimmunity can bypass this putative macrophage immatu-rity.

The inability of the PTA-RB1 mutant to replicate innewborn lungs clearly establishes the need for LT-Ag RBinteraction in this tissue and distinguishes lung fromestablished cell culture for the replication of this virus. Itis not unexpected that whole tissues and cell culturewould respond differently to virus infection, such as bythe induction of innate immune reactions or other cellu-lar signaling. The failure of PTA-RB1 to replicate might beexplained by a failure of this mutant T-Ag to inactivate anantiviral response, such as apoptosis, as reported inLT-Ag transgenic lung cells (Lebel et al., 1995). However,

reliminary experiments by us have failed to observe wtr mutant Py-induced apoptosis in vivo (unpublishedbservation). Alternatively, Rb binding may be needed toediate cell-cycle induction by LT-Ag in vivo, even

hough newborn lungs are an actively differentiating tis-ue. A continued viral genetic analysis should clarify this

ssue. Py MT-Ag also appears to have distinct require-ents in vivo compared to in culture. The lack of repli-

ation or even T-Ag expression in the PyA3-MOP1033iddle T-Ag mutant demonstrated the need for the func-

ions of MT-Ag in establishing even the initial phase of Pynfection. Possibly, MT-Ag is needed to prevent prema-ure cell death following Py infection. As MT-Ag appearso affect many aspects of the host apoptotic responsend cellular signal transduction, it seems likely that an

xpanded analysis of polyomavirus middle T and small Tutants, including mutants in the PP2A binding region

nd the tyrosine and serine phosphorylation sites, in theung would further elucidate the degree of Py control ofntiviral cellular responses in vivo (Dilworth, 1995;

Bergqvist et al., 1997; Kennedy et al., 1997; Mullane et al.,1998).

MATERIALS AND METHODS

Mice and virus

Balb/c mice were obtained from The Jackson Labora-tory (Bar Harbor, ME) and bred in the University of Cali-fornia Irvine Animal Care Facilities. Mice were main-tained and bred according to NIH guidelines. Litters of 6to 10 newborn mice less than 24 h of age were inocu-lated intranasally with 15 ml containing 7.5 3 106 to 1.5 3

07 PFU of wild-type polyomavirus, strain A2 (PyA2).ild-type PyA2 was propagated in mouse 3T6 cells as

reviously described (Eckhart, 1969). Mutant virusyPTA-RB1 (a gift from Robert Freund, University of Mary-

and School of Medicine, Baltimore, MD) was used at3 1010 PFU/mouse. Mutant virus PyA3-MOP1033 (a gift

rom Walter Eckhart) was used at 1.2 3 107 PFU/mouse.ouse adenovirus (a gift from Katherine Spindler, Uni-

ersity of Georgia, Athens) was propagated in mouseT6 cells and used at 2 3 106 PFU/mouse.

Mothers of previously infected litters used in thesexperiments were bred again with uninfected males. The

esulting litters were also intranasally inoculated as de-cribed above.

ung histopathology

As newborn mouse lungs are highly prone to collapse,t was necessary to develop perfusion methods thatreserved the architecture and histology of these deli-ate tissues. Starting with day 2 postinfection, mice wereuthanized each day until the entire litter was used. Miceere carefully dissected and the peritoneal organs were

emoved to allow access to the pleural cavity. The skinbove the rib cage was severed up to the neck. The skin,lands, and muscle around the trachea were cut awayithout cutting the arteries on either side of the trachea.he rib cage was opened. A 28-gauge insulin syringeontaining 500 ml of buffered 10% formalin (Medicalhemical Corp., Santa Monica, CA) was inserted into the

rachea. The portion of the trachea above the syringeas clamped while the formalin was perfused slowly into

he lungs. Following perfusion, a thin cotton string wasied around the trachea below the needle entry point torevent backflow. The lungs were then removed, placed

nto a biopsy cassette, and submerged in 10% bufferedormalin overnight. In some cases, the lungs were placedn 70% ethanol to prevent overfixation prior to beingaraffin embedded. Six-micrometer sections were madend adhered to Silane-Prep slides (Sigma Diagnostics,

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St. Louis, MO). Paraffin embedding, sectioning, and H&Estaining were provided by Professional HistopathologyServices (Irvine, CA) and Pacific Coast Reference Labo-ratories (Cypress, CA).

Alternatively, lungs were perfused with a 1:1 OCT/PBSsolution and placed in 19% sucrose in PBS to preservemorphology. Sections were then frozen in OCT in vinylspecimen molds (Miles, Inc.) on dry ice and stored at220°C. Ten-micrometer sections were made and ad-hered to Silane-Prep slides. Hematoxylin and eosinstaining was performed using hematoxylin stain Gill’sformulation 2 (Fisher) and alcoholic eosin yellowish so-lution (Fisher).

In situ hybridization

DIG-labeled probe was synthesized by using the Ge-nius System nonradioactive DNA labeling kit (BoehringerMannheim) and protocol with PyA2 whole genomecloned into PAN7 as template. Unincorporated nucleo-tides were eliminated by purifying the probes over aSephadex G-50 column.

Paraffin sections were deparaffinized by repeated xy-lene washes and treated with proteinase K for 5 min priorto in situ hybridization. Frozen sections were fixed andpermeabilized with 3:1 ethanol/acetic acid for 15 minprior to in situ hybridization. Endogenous alkaline phos-phatase activity was removed by incubation in 0.2 M HCl.In situ hybridization was performed as previously de-scribed (Piatti et al., 1998).

Immunohistochemistry

OCT-preserved lung sections were fixed and perme-abilized by submersion in ice-cold 100% methanol for 10min, followed by two washes in water to remove OCTand a final dehydration in methanol. The sections wereincubated for 10 min in 10% BSA in PBS to block non-specific binding. They were then incubated in a humiditychamber at 37°C for 2 h with a 1:100 dilution of a ratanti-T-Ag antibody (a gift from W. Eckhart, Salk Institute,La Jolla, CA). Sections were washed, reblocked, andincubated for 2 h with anti-rat-IgG FITC conjugate(Sigma) in the humidity chamber at 37°C. Similarly im-munofluorescence for PyA2 VP1 protein was performedusing a primary rabbit anti-VP1 (a gift from R. Garcea,Harvard University).

ACKNOWLEDGMENTS

We thank Kent Osborn, D.V.M., Ph.D., Associate Director of theDepartment of Animal Resources at The Scripps Research Institute, forhis contributions to the histopathological observations of the mouselung. We also thank Siddiqua Hurst for excellent technical assistance.This research was supported by NIH Grant GM 36605 (L.P.V.) and by theIrvine Research Unit on Animal Viruses.

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the distribution and kinetics of polyomavirus in lungs of ... · using the perfusion methods...

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