Mouse Adenoviruses

Chapter 2 Mouse Adenoviruses Katherine R. Spindler, Martin L. Moore, and Angela N. Cauthen

49

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. History and Isolations of Mouse Adenoviruses, Antigenic Relationships, Virus Strains, and Virus Mutants . . . . . . . . . . . . . . . . . . . . . III. Physical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Molecular Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. MAV-1GenomeFeatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. MAV-1 E1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. MAV-1 E3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. MAV-1 E4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. MAV-1 Major Late Promoter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Growth of Mouse Adenoviruses In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . A. In Vitro Infection, wt MAV-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. In Vitro Infection, MAV-1 E1A Mutants . . . . . . . . . . . . . . . . . . . . . . . C. In Vitro Infection, MAV-1 E3 Mutants . . . . . . . . . . . . . . . . . . . . . . . . . VI. Clinical Disease and Pathogenesis of Mouse Adenoviruses . . . . . . . . . . . . A. Wild-Type MAV-1 Infection In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . B. MAV-1 E1A Mutant Infection In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . C. MAV-1 E3 Mutant Infection In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . D. Immune Response to MAV-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Innate Immune Response to MAV-1 . . . . . . . . . . . . . . . . . . . . . . . . 2. Cell-Mediated Immune Response to MAV-1 . . . . . . . . . . . . . . . . . 3. Humoral Immune Response to MAV-1 . . . . . . . . . . . . . . . . . . . . . 4. Model of MAV-1 Immunopathogenesis . . . . . . . . . . . . . . . . . . . . . E. MAV-2 Infection In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Host Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Host Range and Prevalence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Diagnosis, Control, and Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

INTRODUCTION

50 51 51 51 51 52 53 53 53 53 55 55 56 56 58 58 58 58 59 60 6o 6o 61 61 61 62 62

are useful for study of adenovirus

pathogenesis

in the n a t u r a l

host, in w h i c h t h e y c a u s e a c u t e a n d p e r s i s t e n t i n f e c t i o n s ( S m i t h T h e first m o u s e a d e n o v i r u s w a s i s o l a t e d b y H a r t l e y a n d R o w e

and

Spindler

w h i l e t r y i n g to e s t a b l i s h t h e F r i e n d l e u k e m i a v i r u s in t i s s u e c u l -

species-specific

ture from mice (Hartley and Rowe

of

1960). Mouse

THE MOUSEIN BIOMEDICALRESEARCH,2ND EDITION

adenoviruses

1999).

Such

human

immunocompetent

studies

adenoviruses and

are not possible

with

the

(hAds). The availability

immunodeficient

inbred

mouse

Copyright 9 2007, 1980,ElsevierInc. All rightsreserved.

49

50

KATHERINE

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strains, immunological reagents for mice, and tools for genetic mapping studies combine to make mouse adenovirus studies ideal for understanding virus-host interactions. Commercial mouse suppliers standardly monitor for mouse adenoviruses, and the viruses have been eliminated in commercial mouse colonies and are rare if not absent in institutional colonies (Richter 1986). Methods for propagating and titrating the virus have been described (Cauthen and Spindler 1999a). The hAds were isolated from patients with respiratory illness independently by Rowe et al. (1953) and Hilleman and Werner (1954). The molecular biology of the hAds has been extensively studied in the years since their discovery (Shenk 2001). In addition to their use as models for studying DNA replication and mRNA transcription and processing, hAds are being widely used to develop gene therapy and vaccine vectors (GomezRoman and Robert-Guroff 2003; Hart 2003; Imperiale and Kochanek 2004; Thomas et al. 2003). Far less is known about the pathogenesis of hAds, in part due to the strict species specificity of the adenoviruses. There are currently more than 51 distinct hAd serotypes, some of which cause respiratory disease; others are associated with diseases including conjunctivitis and gastroenteritis (reviewed in Horwitz 2001). Adenoviruses are associated with acute pneumonia in children in developing countries, where they are a major cause of illness and death (Kajon et al. 1996). Severe adenovirus infections occur in immunocompromised people (Kojaoghlanian et al. 2003), including AIDS patients or those undergoing bone marrow or solid organ transplantation (Blanke et al. 1995; Carrigan 1997; Flomenberg et al. 1994). Pediatric bone marrow transplant patients are particularly at risk of hAd infection and mortality (Gavin and Katz 2002; Hale et al. 1999; Walls et al. 2003). In productive infections, the mouth, nasopharynx, or ocular conjunctiva are the initial site of hAd entry, with replication in epithelial cell types (Horwitz 2001). Like the mouse adenoviruses, hAds also cause persistent infections (Lukashok and Horwitz 1999). A study using sensitive real-time PCR coupled with lymphocyte purification suggests that human mucosal T lymphocytes are the site of hAd persistence (Garnett et al. 2002).

II.

HISTORY AND ISOLATIONS OF MOUSE

ADENOVIRUSES, A N T I G E N I C RELATIONSHIPS, VIRUS STRAINS, AND VIRUS MUTANTS Although mouse adenoviruses have been isolated numerous times, there are only two serotypes, murine adenovirus 1 and murine adenovirus 2, as classified by the International Committee on Taxonomy of Viruses (2000). These are currently categorized as belonging to two different species, murine adenovirus A and murine adenovirus B, respectively. This is supported by data on serum neutralization (Lussier et al. 1987), growth characteristics (Smith et al. 1986), and genome restric-

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tion analysis (Hamelin et al. 1988; Hamelin and Lussier 1988; Jacques, Cousineau, et al. 1994). Because these viruses are not infectious for infant rats (Smith and Barthold 1987) and because of the host species specificity of adenoviruses, we favor nomenclature that uses "mouse" instead of "murine," and virus abbreviations as suggested by Ishibashi and Yasue (1984). Mouse adenovirus type 1 (MAV-1) was the first mouse adenovirus isolated (Hartley and Rowe 1960) and has also been designated in the literature as FL, MAdV-1, MAdV-FL, and MAd-1. Mouse adenovirus type 2 (MAV-2), also known as strain K87, was isolated from the feces of healthy mice by Hashimoto et al. (1966). In the work describing the isolation of MAV-1 and its classification as an adenovirus, Hartley and Rowe (1960) demonstrated a serologic relationship between MAV-1 and the hAds. Guinea pig serum from hAd-inoculated animals reacted against MAV-1 antigens. Similar results were obtained by Larsen and Nathans (1977) using serum against the hAd group antigen, hexon (a capsid protein). Hashimoto et al. (1966) used serum against hAd3 raised in guinea pigs to demonstrate a positive complement fixation reaction of MAV-2 antigen. For both MAV-1 and MAV-2, anti-MAV sera raised in mice are poorly reactive against hAd antigens (Hartley and Rowe 1960; Hashimoto et al. 1966). The antigenic relationships between MAV-1 and MAV-2 have been examined in several studies. In cross-neutralization tests between the two serotypes, there is a one-sided relationship between them (Wigand et al. 1977). MAV-2 antiserum neutralizes both MAV-1 and MAV-2, whereas MAV-1 antiserum neutralizes MAV-1 but only weakly neutralizes MAV-2. A similar partial serological relationship was identified by Lussier et al. (1987). Smith et al. (1986) found that MAV-1 antiserum neutralizing antibody titer is fourfold higher with MAV- 1 than with MAV-2. MAV-1 has been more extensively studied than MAV-2. MAV-1 is available from the American Type Culture Collection (cat. no. VR550), but it should be noted that genome sequence differences and slight pathogenesis differences were found between the "ATCC" strain and the strain that we and others obtained directly from Steven Larsen (referred to as "standard") (Ball et al. 1991). This was somewhat surprising, since the ATCC strain was deposited by Dr. Larsen. Evidently the ATCC and standard viruses have different passage histories. As discussed below (Section IV) the complete MAV-1 DNA sequence has been compiled, but there have been no sequences reported for MAV-2. A restriction map of MAV-2 has been determined (Jacques, D'Amours, et al. 1994). Mutant strains of MAV-1 have been constructed by sitedirected mutagenesis techniques using the standard strain as the starting virus. Because the standard MAV-1 genome has two E c o R I sites, these were mutated singly to obtain viruses that have a single E c o R I site in either early region 1 (El) (Smith et al. 1996) or early region 3 (E3) (Beard and Spindler 1996). The resulting viruses, pmE301 and p m E l O 1 , have been used to

2. MOUSE ADENOVIRUSES

51

mapping of E 1, E3, early region 4 (E4), and identification of the major late promoter (MLP) (Ball et al. 1989, 1991, 1988; Beard et al. 1990; Cai et al. 1992; Cauthen and Spindler 1996; Kring et al. 1992; Kring and Spindler 1990; Raviprakash et al. 1989; Song et al. 1996, 1995; Weber et al. 1994). Davison et al. (2003) have analyzed the genetic content, phylogeny, and evolution of the family Adenoviridae, and they have submitted a third-party annotation for MAV-1 (AC_000012). It should be noted that their annotation includes some predicted genes for MAV-1 that are not in agreement with published experimental evidence. For example, their MAV-1 E1A annotation does not take into account E1A transcription mapping and cDNA sequencing data (Ball et al. 1989). Other differences are indicated for IVa2, the DNA polymerase, and pTP genes, which could be correct; these regions have not been transcription mapped. MAV-1 has inverted terminal repeats (ITRs) that are 93 nucleotides (nt) long (Ball et al. 1991, 1988; Temple et al. 1981). These ITRs have the first 18 nt that are highly conserved among the adenoviruses and that are essential for replication of the viral DNA (Challberg and Rawlins 1984; Lally et al. 1984; Tamanoi and Stillman 1983). Unlike hAds, MAV-1 does not encode virus-associated (VA) RNAs (Meissner et al. 1997). Although MAV-1 polypeptide III (penton base) shares high amino acid identity with the hAds, it does not have the arginineglycine-aspartic acid (RGD) motif found in many hAds and thought to be important for viral internalization via cellular integrins (Meissner et al. 1997). However, it has a leucine-aspartic acid-valine (LDV) motif recognized by other integrins, present as LDL and, like porcine adenoviruses, MAV-1 has an RGD sequence in the C-terminus of the fiber protein.

construct mutants in E1A and E3, respectively (Beard and Spindler 1996; Cauthen et aL 1999; Smith et aL 1996). These virus mutants were named based on the types of mutations that were introduced (pm, point mutation; dl, deletion) and the gene where the mutation lies, E1 or E3, followed by an isolation number. The in vitro and in vivo growth characteristics of these viruses are discussed below (Sections V, B and C and VI, B and C). A naturally occurring MAV-1 mutant was isolated by Winters et al. (1981). This variant exhibits a large-plaque phenotype in cell culture and causes clinical disease with a distinct pulmonary tropism in adult C3H/HeJ mice. The mutation(s) responsible for this phenotype have not been mapped.

III.

PHYSICAL P R O P E R T I E S

MAV-1 and MAV-2 share many physical properties, including ether resistance, thermal inactivation at 56~ and a size of --80-90 nm (Hartley and Rowe 1960; Hashimoto et al. 1966). Both mouse adenoviruses lack hemagglutinating activity, unlike the hAds. MAV-1 has a buoyant density in CsC1 like that of the hAds, 1.34 g/ml (Larsen and Nathans 1977; Wigand et al. 1977). MAV-1 is inactivated by 50% ethanol (Larsen and Nathans 1977).

IV. A.

MOLECULAR GENETICS MAV-1 Genome Features

The complete sequence (30,944 base pairs) of the doublestranded DNA genome of MAV-1 has been determined (Meissner et al. 1997) (accession number NC_000942). The ordering of genes in the MAV-1 genome, shown in Fig. 2-1, has been accomplished through sequence comparison with hAds, transcription

B.

MAV-1 E1

Genes expressed prior to early Ad viral replication are designated as "early," while those expressed at or after the time of DNA replication are "late." The E1 region in MAV-1

proteinase hexon lOOK, 3 3 K / p V I I I penton pVl I I ~fiber MLP I ! J I--'1 pllla \ \ I.... I I'~= '" i J I 52/55K\ E3 EIEll--pVII,V, pX

E1B ~ E1A

MAV-1

0

I 10

I 20 I

I V a 2 EEl

I

I 30 I

I 40

r----1pTP

D N A pol

I

I

I

5O

6O

70 I-'i

DBP

I 80

I

0

90

100

( E4

Fig. 2-1 Genomicorganizationof MAV-1.The genome is indicated by the horizontal lines in the center, and map units are indicated below the line. Circles on the end of the genome indicate the terminal protein. Genes whose transcription has been mapped are indicated by arrows; for E3 and E4, multiple transcripts are indicated by only a single arrow. Open reading frames with similarityto proteins of hAds are indicated by the boxes. Genes transcribedin the rightwarddirection are indicated above the genome, and genes transcribed in the leftward direction are indicated below. The major late promoter (MLP) is indicated by the arrowhead. DNApol, DNA polymerase;pTP, terminal protein precursor; DBP, DNA binding protein. (Adapted from Smith and Spindler, 1999, with permission.)

52

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corresponds in location to the E 1 region of hAds, at the left end of the genome (Ball et al. 1988), and the mRNAs are transcribed in a rightward direction (Ball et al. 1989). E1 encodes three mRNAs that overlap; one is designated E1A, and the other two correspond to hAd mRNAs that encode E1B 19K and E1B 55K proteins (Ball et al. 1989). The three mRNAs are 3' coterminal, and the three proteins share a common C-terminal sequence. The MAV-1 E1A protein sequence has little overall sequence similarity to the hAd E1A 289 aa protein (encoded by the 13S E1A mRNA). However, Ball et al. (1989) showed that MAV-1 E1A has approximately 40% sequence similarity in conserved regions 1 (CR1), CR2, and CR3 compared to CR1, CR2, and CR3, respectively, of the hAd 289 aa protein (Moran and Mathews 1987). MAV-1 CR2 is the most similar to that of hAds, with approximately 50% sequence similarity (Ball et al. 1988). The predicted amino acid sequence similarity of the MAV-1 E1B coding regions compared to that of the hAd 19K and 55K proteins is 37% and 42%, respectively (Ball et al. 1988). No further studies involving the E1B proteins have been reported. The E1A protein is detected in MAV-1 infections in cell culture at both early and late times by immunoprecipitation with polyclonal antiserum raised against amino acids 27-200 of the E1A protein (Smith et al. 1996; Ying et al. 1998). The E1A protein is predicted to have a molecular weight of 22 kDa, but migrates slightly larger than 30 kDa, likely due to its phosphorylation (Smith et al. 1996). hAd E1A proteins are also phosphorylated (Gaynor et al. 1982; Yee and Branton 1985a; Yee et al. 1983). However, the significance of the phosphorylation of E1A in MAV-1 infections is not known. MAV-1 viruses with mutations in E1A are described in Table 2-1. The E1A protein expression patterns were evaluated by Western blot analysis and immunoprecipitation (Smith et al. 1996; Ying et al. 1998). The d/E102 (CR2 deletion) and d/E106 (CR3 deletion) E1A proteins migrate faster than the wild-type (wt) E1A, as expected, pmE109 and p m E 1 1 2 , initiator methionine mutants, do not synthesize detectable levels of E1A protein.

TABLE 2-1

EFFECTS OF MAV-1 EIA MUTANTSIN CELL CULTUREAND OUTBRED SWISS MICE E1A mutant

Effect of the mutation a

d/E105 d/E102 d/E106 pmE 109 pmEll2

Deletion of CR1 region (amino acids 35-78) Deletion of CR2 region (amino acids 111-129) Deletion of CR3 region (amino acids 135-154) Mutation of initiator ATG to TTG Mutation of initiator ATG to CAC

Average LDs0 (log PFU) b 103.5 100.9 102.6 103.5 10 3.9

aSmith et aL 1996 bSmith et al. 1998; the average LDs0 for wt virus in these experiments was 10-1-5.

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L. M O O R E ,

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N. C A U T H E N

Although d/E105 (CR1 deletion) has a 43 amino acid deletion, it produces protein that migrates slower than the wt protein; a similar phenomenon is seen in hAd 5 E1A CR1 deletion proteins (Egan et al. 1988). hAd E1A is a transcriptional regulator (reviewed in Gallimore and Turnel12001), and it is required for activation of early viral transcription (Berk et al. 1979; Jones and Shenk 1979). A plasmid encoding the left end of the MAV-1 genome (including the E1A coding region) transactivates the hAd5 E3 promoter in both HeLa cells and mouse L929 cells, albeit at a level lower than that of hAd E1A (Ball et al. 1988). Although MAV-1 E1A is able to transactivate the hAd5 E3 promoter, unlike h a d E1A, it does not appear to stimulate the expression of the other early mRNAs (E1A, E1B, E2, E3, and E4), at least in 3T6-infected cells at a multiplicity of infection (MOI) of 5 (Ying et al. 1998). Thus, the importance of the transactivation by MAV-1 E1A in reporter assays of transfected cells (Ball et al. 1988) is not understood. hAd E1A CR2 encodes a retinoblastoma protein (pRB) binding motif, (D)-L-X-C-X-E, that is conserved as (D)-L-R-C-Y-E in MAV-1 E1A CR2. MAV-1 E1A protein binds to mouse pRb and related proteins (Smith et al. 1996). This was shown both for in vitro transcribed and translated pRb protein (Smith et al. 1996) and for the endogenous pRb in virus-infected cells (L. Fang et al. 2004). This binding is primarily dependent on the presence of E1A CR2 (Smith et al. 1996). Similar experiments also showed that related pRb family proteins p107 and p130 bind to MAV-1 E1A (Smith et al. 1996; L. Fang et al. 2004). For the in vitro-produced p107, CR2 is necessary and sufficient for binding (Smith et al. 1996); p107 binds to hAd E1A in the CR2 region (Dyson, Guida, Mtinger, et al. 1992; Harlow et al. 1986; Whyte et al. 1989; Yee and Branton 1985b). In both the pRb and the p107 experiments in which infected cell lysates were mixed with in vitro translated pRb or p107, the E1A CR1 deletion mutant protein bound to pRb and p107 at greatly reduced levels compared to wt E1A (Smith et al. 1996). This suggests that CR1 may play a cooperative or stabilizing role in pRb and p107 binding to CR2 of E1A, as has been shown for hAd E1A (Dyson, Guida, McCall, et al. 1992; Ikeda and Nevins 1993). The functional relevance for the interaction between MAV-1 E1A and pRB was shown by experiments in SAOS-2 cells, which lack pRb (Smith et al. 1996). SAOS-2 cells transfected with mouse pRb alone exhibit an arrested growth phenotype that is reversed when MAV-1 E1A and pRb are introduced into the cells together.

C.

MAV-1 E3

Transcription mapping of the E3 region of MAV-1 indicated that this region produces three early mRNAs that are 5' and 3' coterminal (Beard et al. 1990). Each E3 mRNA has three exons; the first and second are identical among the three mRNAs, and the last intron of each is different due to differential splicing.

2.

MOUSE

53

ADENOVIRUSES

Thus, the predicted E3 proteins share a 72 amino acid N-terminus with unique C-terminal domains. The three mRNAs transcribed at early times are referred to as class 1 mRNA, which gives rise to the E3 gpl 1K protein, class 2, and class 3 mRNAs (Beard et al. 1990). Only the E3 gpl 1K protein has been detected in wt virus infection of cultured cells (Beard and Spindler 1995). A fourth type of mRNA transcribed at late times (after viral DNA replication) is also detected that encodes E3 g p l l K (Beard et al. 1990; Cauthen and Spindler 1999b). A polyclonal antiserum that specifically recognizes the unique portion of the E3 gpl 1K protein was used to demonstrate that E3 gpl 1K is detected in infected 3T6 cells beginning at 16 hours postinfection (h PI) and is detected until at least 48 h PI (Beard and Spindler 1995). The E3 gp 11K protein can also be immunoprecipitated from radiolabeled infected cell lysates at both early and late times in infection. Additionally, the gp 11K protein is recognized by polyclonal antiserum that is specific for the common portion of the E3 proteins (Beard and Spindler 1996). This antiserum fails to detect the other E3 proteins in MAV-1 infection of 3T6 cells, presumably because the proteins produced by the class 2 or class 3 mRNA are absent from infections in cell culture or are produced at levels too low for detection (Beard and Spindler 1996; Cauthen et al. 1999; Cauthen and Spindler 1999b). The E3 gpl 1K protein appears to be approximately 14K in infected cell lysates, but this is larger than the size of the product produced during in vitro transcription and translation of a plasmid containing the E3 gp 11K gene (Beard and Spindler 1995). This size difference can be attributed to modifications made to the protein in infected cells: the cleavage of the signal sequence (predicted at amino acids 1 to 37) at the N-terminus of the E3 gpl 1K protein, and glycosylation at the predicted N-glycosylation consensus site (N-X-S/T) located at amino acid 56. The E3 gp 11K protein localizes to the ER of the cell and is a peripheral membrane protein, as determined by alkaline extraction and phase separation experiments (Beard and Spindler 1995). E3 gpl 1K is transcribed and translated as an early mRNA and protein. However, a large mRNA expressed at late times can also encode E3 g p l l K (Cauthen and Spindler 1999b). This expression of E3 gp 11K is due to alternative splicing of a late transcript encoding a capsid protein, pVIII, pVIII is predicted to be translated from an unspliced late mRNA. However, if the primary pVIII transcript is spliced at the "E3" sites, a fusion protein consisting of sequences of pVIII and E3 gp 11K is predicted to occur (Cauthen and Spindler 1999b), since they are translated in the same reading frame and their coding regions partially overlap (Raviprakash et aL 1989). It is thought that the signal sequence of the E3 gpl 1K coding region allows the pVIII-E3 gpl 1K fusion protein to enter the ER and get processed, producing a mature E3 gpl 1K (Cauthen and Spindler 1999b). The relevance of transcription and translation of E3 gp 11K at late times is not known. The E3 mRNAs and their putative proteins have been identified or described, but the functions of these proteins have not

yet been discovered, hAd E3 proteins are involved in immune evasion (Fessler et al. 2004). For example, the hAd2/5 E3 gp 19K prevents the display of class I major histocompatibility complex (MHC) antigens on the surface of infected cells, and this has been proposed as a mechanism enabling persistence of hAds (Levine 1984; Wold and Gooding 1991). However, unlike hAds, MAV-1 does not prevent the display of class I MHC antigens on the surface of infected mouse cells in culture (Kring and Spindler 1996).

D. MAV-1 E4 The transcription map of MAV-1 E4 was determined and indicates that E4 encodes seven classes of mRNAs that are 3' coterminal (Kring et al. 1992). The predicted coding regions of three of these mRNAs have some sequence similarity to hAd E4 proteins. The MAV-1 E4 off a/b has 48% sequence similarity to the hAd2 E4 34K (off 6) protein (Ball et al. 1991; Kring et al. 1992). MAV-1 off a/c has 69% sequence similarity to the hAd2 E4 l l K (off 3) protein. The N-terminus of MAV-1 off d has 55% sequence similarity to hAd2 E4 off 2 (Kring et al. 1992), and the entire MAV-1 off d has 60% sequence similarity to hAdE4-orf6/7 (L. Fang and K. Spindler, unpublished data). Further studies involving the E4 region of MAV-1 have not been reported.

E. MAV-1 Major Late Promoter The MLP in hAds directs the synthesis of the late messages, and the MLP in MAV-1 was mapped to a similar region in the genome using ribonuclease protection assays and primer extension analysis (Song et al. 1996). The MAV-1 MLP has a TATA box, an inverted CAAT box, an SP1 binding site, and a DE1 element (Song et al. 1996). Notably, there is an absence of a USF-binding site, an initiator element (INR), and a DE2 element found in hAds and other mammalian viruses (Song and Young 1997). The lack of an INR may explain the finding of more than one start site of the MAV-1 MLP. Song and Young (1997) used a mutational analysis to demonstrate that the TATA box, SP1 site, and CAAT box elements are important for the MAV-1 MLP to function at normal levels in cells. Additionally, gel mobility shift assays were employed to show that the SP1 protein binds to the MAV-1 MLP with high affinity.

V.

GROWTH OF MOUSE ADENOVIRUSES IN VITRO A.

In Vitro Infection, wt MAV-1

Temple et al. (1981) reported that MAV-1 viral DNA synthesis is first observed at 35 h PI, even at MOIs of up to 800 PFU/cell

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when using [3H]thymidine labeling. However, using 3 2 p o 4 labeling and analysis of viral DNA prepared by the Hirt method (1967), we observed MAV-1 DNA synthesis as early as 20 h PI in L929 cells infected at a MOI of 10 PFU/cell (Fig. 2-2). Onestep growth curves in 3T6 cells and mouse brain microvascular endothelial cells (MBMEC) show that MAV-1 exhibits a typical eclipse period, with an increase in virus titer relative to input virus at 36-48 h PI (Cauthen et al. 1999; Ying et al. 1998). Similar results were seen in a two-step growth curve in mouse L929 cells (Fig. 2-3). Cytopathic effects are first detected at 36-48 h and are similar to other adenoviruses: Cells infected with wild-type MAV-1 round up, become refractile, and eventually detach from the substrate (Fig. 2-4). Analysis of [35S]-labeled infected cell proteins by electrophoresis indicates that unlike hAds, MAV-1 does not efficiently shut off host cell protein synthesis (Antoine et al. 1982; Ying et al. 1998). At least two mechanisms are proposed for the selective translation of viral mRNAs in hAd-infected cells resulting in shutoff of

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107

O_---() 106 -

5"

~

105

103 0

w 24

i 48

w 72

'

i 96

w 120

i 144

168

hPI

Fig. 2-3 Two-step growth curve of MAV-1 on L929 cells. Monolayers were infected with MAV-1 at a MOI of 0.1, harvested at the indicated times, and titrated by plaque assay on L929 cells.

Fig. 2-2 Time course of MAV-1 DNA replication. L929 cells were mock infected or infected at a MOI of 10. The cultures were labeled for one hour with 32po 4 and harvested at the indicated times PI. Hirt supernatants containing viral DNA were isolated (Hirt 1967) and digested with HindlII and RNase A and electrophoresed on 0.7% agarose gels, dried and autoradiographed. The sizes of the restriction fragments are indicated on the right. In a parallel experiment performed with h a d 5 in HeLa cells, DNA replication was first detected at 16 h PI (data not shown).

host protein synthesis (reviewed in Shenk 2001). One of these is via the VA RNAs; the lack of VA RNAs in MAV-1 infected cells (Meissner et al. 1997) could be responsible for defective host protein synthesis shutoff in MAV-1 infection. The other proposed mechanism of host protein synthesis shutoff in hAd-infected cells is via an inactivation of elF-4F by dephosphorylation observed late after hAd infection; whether this occurs in MAV-1 infection has not been reported. The receptor for MAV-1 has not been described, hAds use a two-step mechanism for entry. The protruding hAd fiber protein interacts with the cellular receptor (Philipson et al. 1968), which has been shown for a majority of hAd serotypes to be an immunoglobulin superfamily member, the Coxsackie-adenovirus receptor (CAR) (Bergelson et al. 1997; Tomko et al. 1997). In the second step, the penton base interacts with host integrin molecules to promote entry (Wickham et aL 1993). The murine homolog of CAR (mCAR) has been identified, and it can serve as a receptor for hAds (Bergelson et al. 1998; Tomko et al. 1997). However, it is not known whether mCAR is a receptor for MAV-1 or whether MAV-1 uses integrins for entry. Productive MAV-1 infections occur in cell lines that do not express mCAR, such as mouse L929 cells and NIH3T3 fibroblasts (Tomko et al. 1997), suggesting that mCAR is not required for entry. It is believed that MAV-1 does not replicate productively in cultured human cells (Antoine et al. 1982; Larsen and Nathans 1977; K. Spindler, unpublished data), although there is one early conflicting report (Sharon and Pollard 1964). Infection of human cells does not yield infectious virus (Larsen and Nathans 1977); viral DNA synthesis occurs, but there appears to be a defect at

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ADENOVIRUSES

Fig. 2-4 Cytopathic effects of MAV- 1 infection. Mouse 3T6 fibroblasts were mock-infected or infected with wt or pmE312 mutant virus at a MOI of 1 and photographed at 2.5 days PI. Wild-type virus consistently caused cells to release from the culture dish, whereas pmE312 did not, even upon prolonged incubation (data not shown). (Adapted from Cauthen 1998, with permission.)

the level of formation of virus particles due to defects in virus structural proteins, particularly hexon (Antoine et al. 1982).

B. In Vitro Infection, MAV-1 E1A Mutants Mouse 3T6 cells and 37.1 cells (a 3T6-derived cell line that expresses MAV-1 E1A protein) infected at a MOI of 5 with each of the E1A mutant viruses (Section IV, B) give yields of virus approximately like those of wt virus (Ying et al. 1998). Similarly, serum-starved 3T6 cells and transgenic mouse embryo fibroblasts (MEFs) from pRb +/+, pRb +/-, and pRb -/embryos infected at a MOI of 5 give yields of E1A mutant viruses similar to that of wt virus, indicating that at high MOIs, E1A is not required to stimulate progression of the cell cycle and DNA replication (Ying et al. 1998). However, infection of 3T6 cells, mouse brain microvascular endothelial cells (MBMECs), and MEFs at MOIs of 0.05 resulted in a two log unit reduction in viral yield for E1A null mutant pmE109 (Fang and Spindler 2005; M. Moore and K. Spindler, unpublished data). Multiplicity-dependent growth of mutant viruses has been observed previously for hAds (Gaynor and Berk 1983; Imperiale et al. 1984; Nevins 1981), human cytomegalovirus (Bresnahan and Shenk 2000; Oliveira and Shenk 2001), and

herpes simplex virus 1 (Cai and Schaffer 1992; Chen and Silverstein 1992; Everett et al. 2004), but its significance is unknown. The hAd E1A protein protects cells from the interferon (IFN) response (IFN-c~/I] and -7) by inhibiting the ISGF3 transcription factor, thereby reducing expression of IFN-stimulated genes (ISGs) (Ackrill et al. 1991; Gutch and Reich 1991; Leonard and Sen 1996). Similarly, MAV-1 E1A protects mouse 3T6 cells from IFN-~/~ and IFN- 7 (Kajon and Spindler 2000). E1A can provide this protection from IFN-c~/[~ in trans to vesicular stomatitis virus (VSV). The presence of MAV-1 E 1A (either in 37.1 cells or virus-infected cells) correlates with a reduction in steady-state levels of ISGs, suggesting that the mechanism of protection from IFN effects is like that of the hAds. We have preliminary evidence that MAV-1 E1A is able to bind to STAT-1 (L. Fang and K. Spindler, unpublished data), as has been suggested for hAd5 E1A (Look et al. 1998).

C.

In Vitro Infection, MAV-1 E3 Mutants

A series of mutations were made in E3 of MAV-1 to determine the individual effects of the three putative E3 proteins (Section IV, C) on virus replication in cell culture and on

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N. C A U T H E N

TABLE 2-2

EFFECTS OF M A V - 1 E 3 MUTANTS IN CELL CULTURE AND MICE E3 mutant virus

Effect of the mutation

pmE310 pmE312 pmE314 d/E303 d/E307 d/E309

No early expression of E3 protein; late expression of E3 gpl 1K No early expression of E3 protein; late expression of E3 gpl 1K No expression of E3 proteins at early or late times in infection Produces E3 gpl 1K mRNA only; E3 gpl 1K detected in cell culture Produces E3 class 2 mRNA only; no protein detected in cell culture Produces E3 class 3 mRNA only; no protein detected in cell culture

a

Average LDs0 (log PFU) 101.5 102.9a 105.2 103.7 103.6 104.7a

Reference Beard and Spindler 1996 Cauthen and Spindler 1999b Cauthen et al. 1999 Beard and Spindler 1996 Beard and Spindler 1996 Beard and Spindler 1996

This value is from a single experiment.

growth and pathogenesis in mice. The E3 mutant viruses and the effects of their mutations on the synthesis of the putative E3 proteins are shown in Table 2-2. The E3 mutant viruses have growth patterns similar to those of wt virus in mouse 3T6 cells at a high multiplicity (MOI of 5-10). p m E 3 1 0 , p m E 3 1 4 , and d/E309 grow to titers similar to that of wt virus (Beard and Spindler 1996; Cauthen and Spindler 1999b). In addition, pmE314 virus grew as well as wt virus in MBMECs (Cauthen and Spindler 1999b), which were tested since endothelial cells are one of the target cells of MAV-1 in vivo (Charles et al. 1998; Kajon et al. 1998). d/E303 and d/E307 grow to titers approximately 10- to 50-fold lower than that of wt virus (Beard and Spindler 1996). p m E 3 1 2 grows to titers approximately 10-fold lower than that of wt virus (Cauthen and Spindler 1999a). d/E303, d/E307, and pmE312 all exhibited slightly smaller plaques than wt virus (N. Cauthen and K. Spindler, unpublished data), and pmE312 showed a unique cytopathic effect in 3T6 cells (Fig. 2-4). The multiplicity dependence seen for MAV-1 E1A (Section V, B) is not seen for E3. E3 mutants has yields like wt virus, even at a MOI of 0.05 (M. Moore and K. Spindler, unpublished data).

VI.

CLINICAL DISEASE AND PATHOGENESIS OF MOUSE ADENOVIRUSES A.

Wild-Type MAV-1 Infection In Vivo

MAV-1 permits the study of a replicating Ad in vivo and provides a good model of Ad pathogenesis, hAds do not replicate in rodents, but high dose (108 to 101~PFU per animal) intranasal (i.n.) infection of cotton rats and mice induces pulmonary disease (Ginsberg et al. 1991; Pacini et al. 1984). In contrast to hAds, MAV-1 doses as low as 1-100 PFU cause fatal disease in newborn and adult mice (Spindler et al. 2001; van der Veen and Mes 1973). The outcome of MAV-1 infection depends on the virus dose, the mouse strain and age, the inoculation route, and the strain of virus.

When mice survive an acute MAV-1 infection, they can become persistently infected (reviewed in Smith and Spindler 1999). Infectious MAV-1 was found at high titers in urine of infected mice at 11 months PI (Rowe and Hartley 1962) and 24 months PI (van der Veen and Mes 1973). Infectious virus was found in kidney 70 days PI (Ginder 1964) and in liver 52 days PI (Wigand 1980). Virus particles were detected in urine, and viral DNA was detected in brains, spleens, and kidneys of mice 55 weeks PI (Smith et al. 1998). E1A mutants of MAV-1 persist for up to 55 weeks PI (Smith et al. 1998) (Section VI, B). The mechanism by which MAV-1 persists is not understood (Smith and Spindler 1999). This long-term shedding of MAV-1 is paralleled in human adenoviruses, which have been found shed in feces up to 2 years after infection (Fox et al. 1969). Human adenoviruses have also been isolated from urine of patients with AIDS (de Jong et al. 1983). Mouse adenoviruses have not been reported to be oncogenic, and MAV-1 virions and viral DNA do not cause transformation of cloned rat embryo fibroblast (CREF) cells, unlike hAd virions and viral DNA (K. Spindler, unpublished data). The effects of mouse age on infection are as follows. In adult mice MAV-1 induces clinical signs of disease and dose-dependent acute encephalomyelitis in outbred (Kring et al. 1995), C57BL/6 (B6) (Guida et al. 1995), 129 Sv/Ev (M. Moore and K. Spindler, unpublished data), and SJL/J (Spindler et al. 2001) mice. Adult BALB/c mice are resistant to MAV-1-induced disease, but high dose infection results in ruffled fur, hyperpnea, and conjunctivitis (Charles et al. 1998; Guida et al. 1995; Moore et al. 2004). In suckling mice MAV-1 produces fatal, disseminated disease and myocarditis (Blailock et al. 1967; Hartley and Rowe 1960), and intranuclear inclusions typical of adenovirus infections are seen in endothelial cells of the brain (Heck et al. 1972). Similar widely disseminated disease is seen in suckling mice infected with both the "standard" and "ATCC" strains of MAV-1 (Ball et al. 1991) (Section II). In both adult and suckling mice, regardless of dose, infection results in viremia as early as 1 day PI and is detected in tissues at 3 days PI (Heck et al. 1972; Spindler et al. 2001); viral DNA is found in tissues as early as 2 days PI (Ball et al. 1991). The effects of mouse strain differences in the disease outcome are discussed below (Section VII).

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Inoculation of MAV-1 by different routes does not result in major differences in pathogenesis. Outbred mice infected i.n. with MAV-1 exhibit slightly fewer disease signs and have protracted infection kinetics compared to mice infected intraperitoneally (i.p.) (Kajon et al. 1998; Wigand 1980). High dose (10 6 PFU) i.n. infection of newborn mice results in a robust macrophage infiltrate in the lung at 3 days PI (Gottlieb and Villarreal 2000). Intravenous (i.v.) infection of inbred mice results in levels of virus in brain and spleen, early antibody responses, and survival in B6 and B cell-deficient mice similar to what is seen in i.p. infection (Moore et al. 2004). Intracerebral (i.c.) inoculation of outbred mice results in significant infection of the adrenal gland (Margolis et al. 1974), an organ which also has significant viral DNA levels when infected i.p. (Kring et al. 1995). Similar to mice infected i.p. with MAV-1 (Spindler et al. 2001), susceptible and resistant adult inbred mice infected i.c. showed replication of the virus in brain and spleen (Fig. 2-5A). Interestingly, in the resistant BALB/c mice, virus was detected at high levels in the spleen as early as 2 days PI and was even detected in one susceptible SJL spleen at 2 days PI. The high levels of virus found earlier in BALB/c spleens were surprising, since BALB/c mice are resistant to the virus (Guida et al. 1995; Spindler et al. 2001). One interpretation of the data is that earlier high levels of virus in spleen correlate with or stimulate a stronger innate immune response in BALB/c mice that is able to control subsequent replication in resistant mice. To determine whether

57

the i.c. injections would result in a difference in viral titers at 8 days PI, mice were infected with three low doses of MAV-1 (Fig. 2-5B). Higher levels of virus were found in the brains and spleens of SJL mice than BALB/c mice at every dose (except spleens at the lowest dose), similar to results of infection of SJL mice by the i.p. route (Spindler et al. 2001; Welton et al. 2005). Taken together, the data suggest a model where MAV-1 replicates equally well in susceptible SJL and resistant BALB/c mouse brains, but as the infection proceeds, viral replication is controlled in resistant mice but not in susceptible mice, resulting in higher viral loads at later times after infection. Pregnant mice infected with MAV-1 show histological signs of infection, but the virus does not cross the placenta (Lipps and Mayor 1980; Margolis et al. 1974). Maternal antibodies to MAV-1 are protective for suckling mice (Hartley and Rowe 1960; Lipps and Mayor 1982). In most strains of MAV-1-infected adult mice, MAV-1 infects cells of the monocyte/macrophage lineage and endothelial cells of the vasculature throughout the mouse; highest levels of virus are found in the spleen and central nervous system (CNS) (Charles et al. 1998; Guida et al. 1995; Kajon et al. 1998; Kring and Spindler 1990). MAV-1 nucleic acid is also detected by in situ hybridization (ISH) in the renal tubular epithelium of adult outbred mice infected i.p. or i.n. (Kajon et al. 1998; Smith et al. 1998). Viral DNA is detected in organ homogenates of bowel, pancreas, spleen, adrenal gland, kidney, liver, lung, heart, brain,

Fig. 2-5 IntracerebralMAV-1 infections of mice. A. Anesthetized susceptible SJL/J and resistant BALB/c mice were injected i.c. with 104 PFU MAV-1 in a volume of 30 gl. Mice were euthanized at the indicated days PI (dpi), and brain and spleen titers were determined by plaque assay. Each symbol represents an individual mouse; the short horizontal lines indicate the mean values. The arrow indicates the input dose in PFU/g, assuming brains have a mass of 0.3 g. The asterisk and dotted line indicate the level of detection. B. Mice were infected i.c. with the indicated dose of virus, and brain and spleen titers were determined at 8 days PI. P < 0.03 for the 3 PFU brain and spleen titers between SJL and BALB/c mice; P = 0.002 for the 30-PFU brain and spleen titers.

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and spinal cord of i.p. infected outbred mice (Kring et al. 1995); most likely this is due to infection of endothelium throughout the mice. MAV-1 has not been observed in the brain parenchyma; it is restricted to the vascular endothelium (Charles et al. 1998; Guida et al. 1995; Kajon et al. 1998). Mice with MAV-1-induced encephalomyelitis exhibit histological evidence of perivascular edema in the CNS, and moribund infected mice also exhibit endothelial cell reactivity, vasculitis, vascular wall degeneration, and viral inclusion bodies in microvascular endothelial cells (Charles et al. 1998; Guida et al. 1995; Kajon et al. 1998; Kring et al. 1995; Spindler et al. 2001).

B.

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CAUTHEN

Sublethal doses of ),-irradiation result in increased levels of MAV-1 in infected mice 42-55 weeks PI (Smith et al. 1998). The percentage of mice with viral DNA-positive organs (brain, kidney, and spleen) increases in post-irradiation mice previously infected with wt or E1A mutant viruses, peaks in the first week following irradiation, and returns to pre-irradiation percentages by 3 weeks PI. Thus MAV-1 can persist in the brains, spleens, and kidneys of infected mice for up to 55 weeks PI, the virus can be shed in the urine, and E1A is not required for the persistence of the virus. It is not known whether the virus produced is infectious virus, nor is it known whether persistence is due to a chronic or latent infection.

MAV-1 E1A Mutant Infection In Vivo C.

The 50% lethal dose (LDs0) of each E1A mutant virus (Section IV, B) was determined and is shown in Table 2-1. The LDs0 values for the E1A mutants are 1.5-5 log units higher than that of wt virus, indicating that the E1A protein is important in MAV-1 pathogenesis, pmE112, the virus that does not synthesize the E1A protein, is the least virulent (Smith et al. 1998). The disease signs observed in the mutant virus-infected mice are identical to those of wt-infected mice, and the onset of disease is dose-dependent. Studies of the E1A null mutants, pmE109 and pmE112, were carried out in mice to evaluate histopathology and the presence and quantity of viral DNA in mice (Smith et al. 1998). Neither wt nor E1A null mutant viruses produce detectable levels of virus, as measured by dot blot analysis of DNA, in the spleens at 5 d PI in outbred mice infected with 104 PFU. Brains of wt and pmE109-infected mice have similar levels of viral DNA, but the other E1A null mutant, pmE112, produces significantly less viral DNA in the mouse. At lower dose infections, near the LDs0 for the wt virus, no viral DNA is detected in brains or spleens of the p m E 109- or p m E 112-infected mice at 5 or 14 d PI. Mutations in the E1A null mutants did not revert in vivo, because PCR amplification and sequencing of viral DNAs recovered from infected mice showed they were identical to the starting mutant viruses (K. Smith and K. Spindler, unpublished data). The E1A mutant viruses exhibit histopathology similar to that of wt virus during the acute phase of disease (Smith et al. 1998). When doses of 104 PFU are used, the tropism of pmE109 and p m E 1 1 2 is similar to that of wt virus, with the exception that p m E l l 2 is also found in thymus of infected mice. Persistence of wt and E1A mutant MAV-1 was evaluated using an immunocapture assay of urine from virus-infected mice (Smith et al. 1998). During the 12-22 week period PI, mice infected with wt and E1A mutant viruses all shed MAV-1. From 42-55 weeks PI, only wt-infected mice shed detectable levels of virus. At 42 weeks PI viral DNA is detected in brains, spleens, and kidneys of mice infected with wt and E1A mutant viruses by PCR amplification and ISH, indicating that a persistent MAV-1 infection can be established in the absence of E1A.

MAV-1 E3 Mutant Infection In Vivo

Table 2-2 shows the LDs0 values for infection of outbred mice by E3 mutant viruses (Section V, C), which are all less virulent than wt virus; the E3 null mutant p m E 3 1 4 has the most severe defect in virulence. The elevated LDs0 levels for each of the E3 mutant viruses suggest that each of the three E3 gene products plays a role in the pathogenesis of MAV-1 in vivo. Infections with E3 mutant viruses result in the same clinical signs of disease in mice as wt virus, and the onset of disease is dependent on the dose of virus (Cauthen et al. 1999; and C. Beard, N. Cauthen, and K. Spindler, unpublished data). Similar to wt virus, pmE314 is found primarily in endothelial cells of the brain and spinal cord and in endothelial cells and stationary macrophages in the spleen (Cauthen et al. 1999). Outbred mice given 105 or 106 PFU ofpmE314 die 3 or 4 d PI with large numbers of viral inclusion bodies and ISH evidence of virus; it is thought that at this dose the mice die of an overwhelming infection of endothelial cells. Lower doses (103 or 104 PFU) of p m E 3 1 4 given to outbred mice result in fewer and less severe histopathological changes than wt virus, particularly with respect to inflammation and endothelial damage. Viral nucleic acid is distributed in a similar pattern in the brains and spinal cords of both p m E 3 1 4 - and wt-infected mice, albeit at slightly lower levels in pmE314-infected brains and spinal cords. The functions of the E3 gene products have not yet been elucidated, thus the mechanism by which the lack of E3 results in reduced inflammation in p m E 3 1 4 infection is unknown.

D. 1.

Immune Response to MAV-1

I n n a t e I m m u n e Response to MAV-1

The innate immune response to MAV-1 includes early increases in the steady-state levels of the mRNAs of cytokines and chemokines (Charles et al. 1999; Charles et al. 1998). Charles and coworkers quantitated cytokine and chemokine mRNAs in mock-infected and MAV-l-infected B6 and BALB/c brains because BALB/c mice are more resistant to MAV-l-induced encephalomyelitis than B6 mice (Guida et al. 1995). MAV-1

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increases the mRNA steady-state levels of the cytokines IFN-y, tumor necrosis factor alpha (TNF-o0, interleukin-1 (IL-1), IL-6, and lymphotoxin (LT) in B6 and BALB/c brains 4 days PI (Charles et al. 1998) and transiently increases expression of IL-12 mRNA by macrophages in CBA/Ht mice (Coutelier et al. 1995). MAV-1 also increases the mRNA steady-state levels of the chemokine receptors CCR1-CCR5 in B6 and BALB/c brains 4 days PI (Charles et al. 1999). Steady-state mRNA levels of the chemokines IFN-y-induced protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), and T cell activation gene 3 (TCA-3) are increased by MAV-1 infection in B6 but not BALB/c brains 4 days PI, suggesting that innate immune responses contribute to the MAV-1-induced encephalomyelitis in B6 mice. IFN-a/[3 signaling plays a role in MAV-1 pathogenesis and is a determinant of MAV-1 organ tropism. In single-cycle infectious yield reduction assays in vitro, MAV-1 is more resistant than VSV to pretreatment with mouse IFN-t~/[3 (Kajon and Spindler 2000). However, MAV-1 replication is not completely resistant to IFN-tx/[3, since virus yields are reduced about 10-fold in high concentrations of IFN. E1A mutants of MAV-1 are more sensitive to IFN-tx/[3 in vitro than wt MAV-1. These data indicate that MAV-1 E1A counteracts the IFN-o~/~i antiviral response in vitro. The role of IFN-t~/[3 in MAV-l-induced encephalomyelitis was examined by comparing the pathogenesis of MAV-1 in 129 Sv/Ev and IFN-ct/[3R-/- mice (M. Moore and K. Spindler, unpublished data), which are defective for type I IFN signaling (Miiller et al. 1994). IFN-a/~R -/- mice had higher levels of infectious MAV-1 in spleens at 4 and 7 days PI and exhibited a more disseminated MAV-1 infection at 7 days PI than control mice (M. Moore and K. Spindler, unpublished data). However, this disseminated MAV-1 infection in IFN-a/[3R-/mice did not show clinical or histopathological differences from infection of control mice, and survival was not different between IFN-a/~R-/-mice and controls given a 700 PFU dose of virus. Virus levels in brain were high in both 129 Sv/Ev (control) and I F N - ~ R -/- mice. These results suggest that IFN-~[3 signaling is correlated with reduced MAV-1 replication in the spleen and prevention of widespread infection of vascular endothelial cells. However, WN-a/~R signaling does not prevent MAV-l-induced encephalomyelitis or limit MAV-1 replication in the brain. ISGs whose steady-state RNA levels were increased by MAV-1 infection in vitro and in vivo were identified first by cDNA arrays and confirmed by Northern analysis (M. Moore and K. Spindler, unpublished data). These ISGs included the transcription factors interferon regulatory factor 7 (IRF-7), interferon regulatory factor 1 (IRF-1), and signal transducer and activator of transcription 1 (STAT-1).

2. Cell-Mediated Immune Response to MAV-1

Outbred mice infected i.p. with a sublethal dose develop MAV-l-specific cytotoxic T cells (CTL) that are detectable 4 days PI, peak at 10 days PI, then rapidly decline (Inada and Uetake 1978a, 1978c). These kinetics are paralleled by

59

cell-mediated immunity measured by induction of macrophage migration inhibitory factor (Inada and Uetake 1978b) and are typical of acute viral infections in mice. Several studies implicate a protective role for adaptive immunity in MAV-1-induced disease. MAV-1-infected athymic nu/nu (T cell-deficient) mice on a mixed NIH Swiss and C3H/HeN background succumb to a wasting disease with characteristic duodenal hemorrhage and intranuclear adenovirus particles in endothelial cells (Winters and Brown 1980). Mice that are homozygous for the severe combined immunodeficiency (SCID) mutation (T cell- and B cell-deficient) on a CB.17 or BALB/c background succumb to MAV-1 infection with diffuse hepatic injury that resembles Reye syndrome pathology (Charles et al. 1998; Pirofski et al. 1991). RAG-1 -/- (T cell- and B cell-deficient) mice are more susceptible to MAV-1 infection than B6 controls (Moore et al. 2004). These studies suggest that adaptive immunity protects adult mice from MAV-l-induced disease. Furthermore, sublethal irradiation of inbred mice that are resistant to MAV-1 infection renders them susceptible, suggesting that resistance to MAV-1 infection has an immunological basis (Spindler et al. 2001). Infection of mice deficient for T cells, T cell subsets, and T cell-related functions revealed that T cells cause acute immunopathology and are required for long-term survival in MAV-l-induced encephalomyelitis (Moore et al. 2003). Brains harvested from MAV-l-infected mice lacking tx/[3 T cells or perforin have less histological evidence of MAV-1 encephalomyelitis and less cellular inflammation than brains harvested from control mice. Mice lacking o~/~ T cells, MHC class I (~2m-/-), or perforin have fewer disease signs at 8 days PI than control B6 mice, whereas mice lacking MHC class II have acute disease signs like B6 controls, such as hunched posture, ataxia, and ruffled fur. Thus, CD8 + CTL contribute to disease severity in the acute phase of MAV-1 infection. Similar to virus-induced disease in other virus infections, MAV-1-induced disease in B6 mice depends on virus dose and cell-mediated immunity (Moore et al. 2003), supporting the view that antigen quantity controls T cell-mediated immunity (Zinkernagel and Hengartner 2001). Mice lacking cz/[3 T cells succumb to MAV-1 infection 9 to 16 weeks PI. These mice have detectable viral loads in spleens and brains at 3 weeks PI and high viral loads in spleen and brains when moribund (Moore et al. 2003). In contrast, control B6 mice clear MAV-1 to a level below the plaque assay detection limit by approximately 12 days PI, and no infectious virus is recovered 12 weeks PI from B6 mice (Moore et al. 2003; M. Moore and K. Spindler, unpublished data). Somewhat surprisingly, neither MHC class-I deficient, MHC class II-deficient, CD8 -/-, CD4 -/-, perforin-deficient, nor IFN-y-deficient mice have any detectable infectious virus in spleens or brains at 12 weeks PI. Since almost all a/~l T cells are either CD8 + or CD4 + (Mombaerts et al. 1992), these results suggest that having either CD8 + or CD4 + effector a/[~ T cells is sufficient for a/J3 T cell-mediated clearance of MAV- 1.

60 3.

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Humoral Immune Response to MAV-1

Early studies showed that MAV-1, like hAds, induces a strong humoral immune response. Outbred mice infected i.p. with a sublethal dose of MAV-1 develop high neutralizing antibody (nAb) titers 2 weeks PI, and nAb titers increase for one year then decline (van der Veen and Mes 1973). MAV-1 infection of CBMHt mice with 107 50% infectious doses (IDs0) results in significant splenic B cell proliferation at 10 days PI (Coutelier et al. 1990) and, like other viral infections of mice, stimulates predominantly antiviral IgG of the IgG2a subtype (Coutelier et al. 1990, 1988, 1987). MAV-1 acts as a T cell-independent (TI) Ag of the TI-2 type (Moore et al. 2004); that is, like polyomavirus, it induces TI antibodies in immunocompetent but not Xid mice (SzomolanyiTsuda and Welsh 1998). Early TI antiviral IgM plays a crucial role in protection against disseminated MAV-1 infection (Moore et al. 2004). In contrast to mice lacking T cells, mice lacking B cells are more susceptible to acute MAV-1-induced disease than B6 controls. B cell-deficient mice die early (7 to 10 days PI), and T cells are not required for survival of acute infection (Moore et al. 2003). These findings are consistent with TI B cell responses being critical for protection against MAV-l-induced encephalomyelitis. Mice lacking Bruton's tyrosine kinase (Btk) have reductions in serum immunoglobulin, conventional B cells, and peritoneal B-1 cells (Khan et al. 1995). Btk is required for survival of acute MAV-1 infection, since Btk-deficient mice succumb to MAV-1 infection, (Moore et al. 2004). This was the first demonstration that Btk plays a role in protection from virus-induced disease in mice. Btk-deficient and gMT mice, deficient for B cells and on B6.129 and B6 strain backgrounds, respectively, succumb to acute MAV-1 infection with systemically high viral loads and histological evidence of hepatitis in addition to the histological evidence of MAV-l-induced encephalomyelitis (Moore et al. 2004). B cell--deficient mice on a BALB/c background (Jh mice) are more susceptible to acute MAV-l-induced disease than BALB/c controls, and succumb with systemically high viral loads and evidence of significant hepatitis. However, moribund Jh mice do not exhibit encephalomyelitis; MAV-l-infected Jh mice likely die of hemorrhagic enteritis. SCID (T cell- and B cell-deficient) mice on a BALB/c background succumb to acute MAV-1 infection with evidence of liver infection but no histological evidence of hepatitis (Charles et al. 1998). One explanation for this strain-specific pathology is as follows. In the absence of B cells, MAV-1 replicates to high titers throughout the mouse (Moore et al. 2004). In the presence of T cells, acute MAV-1 infection elicits dose-dependent immunopathology (Moore et al. 2003). T cell-mediated immunopathology may be directed to various organs in different mouse strains by strain-specific innate immune responses, for example, differential chemokine expression in the brains of MAV-l-infected B6 and BALB/c brains (Charles et al. 1999). Data showing that MAV-1 replicates to high levels in the brains of Jh mice without inducing encephalomyelitis (Moore et al. 2004) support this

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model rather than a model in which receptor differences account for differential pathology in MAV-l-infected B6 and BALB/c mice (Charles et al. 1998).

4.

Model of MAV-1 Immunopathogenesis

T cells, B cells, and type I IFN each have a distinct protective role in MAV-1 pathogenesis, and the data suggest their roles are interrelated. B cell-deficient mice succumb to disseminated infection with high virus loads throughout the mouse (Moore et al. 2004). T cell-mediated immunopathology is implicated in exacerbating disease in B cell-deficient mice (Moore et al. 2003, 2004). Similar to B cell-deficient mice, type I IFN-deficient mice also had a more disseminated infection with higher virus loads than control mice (M. Moore and K. Spindler, unpublished data). However, unlike B cell--deficient mice, type I IFN-deficient mice did not exhibit more clinical disease or more histopathological evidence of disease than control mice, even in organs with high virus loads, such as the liver (M. Moore and K. Spindler, unpublished data). This suggests that type I IFN controls MAV-1 replication but also contributes to T cell-mediated immunopathology.

E. MAV-2 Infection In Vivo Hashimoto and colleagues studied the pathogenesis of MAV-2 in mice infected perorally (Hashimoto et al. 1970). The virus grows in the intestinal tract and is shed in feces for 3 weeks after infection, but there are no clinical signs of disease. Inbred DK1 mice given 2 x 105 TCIDs0 of virus show virus replication from 3-14 days PI, with a peak of virus yield from 7-14 days PI. At the peak times of infection, virus is seen by immunofluorescence and electron microscopy in epithelial (columnar, goblet, and Paneth) cells of the ileum (Takeuchi and Hashimoto 1976). Mesenchymal cells are not infected; the virus has a specific tropism for the villus epithelium. The infection results in little cytopathic effect, but infected epithelial cells are shed at a high rate into the gut lumen. Interestingly, MAV-2 peroral infection of BALB/c nude (nu/nu) mice, which lack T cells, results in prolonged viral proliferation in the gut, but viral replication is suppressed 6 weeks PI (Umehara et al. 1984). MAV-2 is not found in organs other than the gut. Antiviral resistance in BALB/c nu/nu mice 6 weeks PI does not correlate with interferon levels or NK cell activity, and the resistance is not affected by administration of anti-asialo GM1 antibody or carrageenan (Umehara et al. 1987). However, antiviral resistance is completely abolished by cyclophosphamide treatment. Cyclophosphamide is a carcinogen and mutagen that is toxic to actively cycling cells, reduces peripheral lymphocytes, and reduces serum IgG levels in nu/nu mice. Mice rechallenged with MAV-2 28 days after initial infection are resistant to virus growth (Hashimoto et al. 1970). The data suggest that antibody

2.

MOUSE

responses are responsible for the antiviral resistance of nu/nu mice to MAV-2.

VII.

HOST G E N E T I C S

Different strains of outbred and inbred mice have been shown to differ in their susceptibility to MAV-1 (Guida et al. 1995; Kring et al. 1995; Spindler et al. 2001). Adult B6 mice infected with MAV-1 succumb to a fatal hemorrhagic encephalitis whereas BALB/c mice do not (Guida et al. 1995). B6 mice show clinical signs of acute CNS disease accompanied by histological evidence of hemorrhage and inflammation, and high levels of virus in brain and spinal cord. In contrast, infected BALB/c mice do not have detectable levels of viral mRNA in brain or spleen, and they lack histological and clinical signs of disease. Enteritis has been seen in BALB/SCID (T and B lymphocyte-deficient) mice (Charles et al. 1998), and B lymphocyte-deficient mice on a BALB background (Jh) (Moore et al. 2004) likely succumb due to a hemorrhagic enteritis. These results suggest that there are mouse strain differences in tropism and cause of death. SJL/J mice are more than four log units more susceptible to MAV-1 than most inbred strains of mice (Spindler et al. 2001). A sublethal dose of gamma irradiation renders resistant mice susceptible, and there are no differences in viral yield in ex vivo MAV-1 infection of primary cells from susceptible and resistant mice. Susceptible mice have higher virus loads in brain and spleen than resistant mice but only modest differences in histopathology. The results suggest that immune response differences may account for differences in susceptibility. A genetic mapping approach (positional cloning) is being used to identify the host gene(s) for susceptibility to MAV-1 (Welton et al. 2005).

VIII.

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ADENOVIRUSES

HOST R A N G E AND PREVALENCE

The mouse strain used when MAV-1 was isolated was not specified by Hartley and Rowe (1960), but MAV-1 infects inbred and outbred strains of mice in the M u s genus. There is a report of adenovirus isolation from Peromyscus (Reeves et al. 1967) and a report of a seropositive wild rodent (Kaplan et al. 1980), but these do not provide strong support for infections of n o n - M u s mice. The host range of MAV-1 in cells in culture is limited to mouse cells; infection of a variety of human and monkey cells does not result in infectious virus (Antoine et al. 1982; Larsen and Nathans 1977). A survey reported in 1966 testing mice in U.S. laboratory colonies indicated that evidence of MAV-1 infection was only found in 4 of 34 colonies, much less frequently than other viruses tested (Parker et al. 1966). A survey from 1984-1988 of

laboratory colonies in 10 European countries showed no mouse adenovirus infections (Kraft and Meyer 1990). MAV-1 is virtually absent from commercial colonies, which are now monitored routinely for MAV-1 (Otten and Tennant 1982). Commercial and laboratory colonies are not usually tested for MAV-2 (A. Smith, personal communication). It has been postulated that MAV-1 occurred enzootically in infected laboratory colonies as a silent infection that was transmitted orally to cage mates (Richter 1986). Although infection can be transmitted to cage mates, there is no seroconversion for animals held in the same animal room but in separate cages (Hartley and Rowe 1960). Mice kept in close contact with MAV-l-infected mice seroconvert by day 21 PI (Lussier et al. 1987). Animals placed on bedding obtained from cages of MAV-1-infected animals do not show signs of disease, seroconversion, or shedding of virus in urine (Smith et al. 1998). The prevalence of mouse adenoviruses in the wild has not been systematically studied. However, a serological survey of wild M. domesticus in southeastern Australia indicated that of mice isolated from 14 sites, 37% were seropositive for MAV-2 and 0% were seropositive for MAV-1 (Smith et al. 1993). There was significant variation in MAV-2 seroprevalence at two study sites in southeastern Australia investigated over 13 months, and again, no evidence of MAV-1 was seen (Singleton et al. 1993). M. domesticus was introduced to an island off the northwest coast of western Australia and first identified in 1986 (Moro et al. 1999). A study of mice from this island in 1994-1996 indicated no serological evidence of MAV-1 or MAV-2 in M. domesticus or in an indigenous mammal, the short-tailed mouse Leggadina lakedownensis.

IX.

DIAGNOSIS, CONTROL, AND P R E V E N T I O N

MAV-1 infection can be diagnosed by serological testing by ELISA; test kits are available commercially (Charles River Laboratories). Complement-fixing antibodies are detected in mice inoculated i.p. with 104 TCIDs0 as early as 14 days PI (Lussier et al. 1987). Neutralizing Ab is detected in mice infected with 1 PFU at 12 days PI (Moore et al. 2004). Because commercial colonies are free of mouse adenoviruses (Section VIII), control is unlikely to be needed. If necessary, control in an infected colony can be achieved by embryo rederivation (Richter 1986; Trentin et al. 1966). Mice to be infected with MAV-1 should be maintained in a room where their cages and bedding can be autoclaved after use. Because the virus appears to require close mouse-mouse contact for transmission (Hartley and Rowe 1960; Lussier et al. 1987), it is straightforward to work with these mice using standard microisolator techniques. In 20 years of performing MAV-1 infections in animal facilities in four different buildings at two institutions, we have never had mock-infected experimental mice (in separate cages) or sentinel mice (even in open cages) in the same

62

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R.

SPINDLER,

room as infected mice show disease signs or become seropositive for MAV-1 (K. Spindler, unpublished data).

ACKNOWLEDGMENTS We thank Lei Fang for critical reading of the manuscript. This work was supported by NIH R01 AI023762.

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