Age-Dependent Effects of Immunoproteasome Deficiency on Mouse Adenovirus Type 1 Pathogenesis.

Acute respiratory infection with mouse adenovirus type 1 (MAV-1) induces activity of the immunoproteasome, an inducible form of the proteasome that shapes CD8 T cell responses by enhancing peptide presentation by major histocompatibility complex (MHC) class I. We used mice deficient in all three immunoproteasome subunits (triple-knockout [TKO] mice) to determine whether immunoproteasome activity is essential for control of MAV-1 replication or inflammatory responses to acute infection. Complete immunoproteasome deficiency in adult TKO mice had no effect on MAV-1 replication, virus-induced lung inflammation, or adaptive immunity compared to C57BL/6 (B6) controls. In contrast, immunoproteasome deficiency in neonatal TKO mice was associated with decreased survival and decreased lung gamma interferon (IFN-γ) expression compared to B6 controls, although without substantial effects on viral replication, histological evidence of inflammation, or expression of the proinflammatory cytokines tumor necrosis factor alpha and interleukin-1ß in lungs or other organs. T cell recruitment and IFN-γ production was similar in lungs of infected B6 and TKO mice. In lungs of uninfected B6 mice, we detected low levels of immunoproteasome subunit mRNA and protein that increased with age. Immunoproteasome subunit expression was lower in lungs of adult IFN-γ-deficient mice compared to B6 controls. Together, these results demonstrate developmental regulation of the immunoproteasome that is associated with age-dependent differences in MAV-1 pathogenesis.IMPORTANCE MAV-1 infection is a useful model to study the pathogenesis of an adenovirus in its natural host. Host factors that control MAV-1 replication and contribute to inflammation and disease are not fully understood. The immunoproteasome is an inducible component of the ubiquitin proteasome system that shapes the repertoire of peptides presented by MHC class I to CD8 T cells, influences other aspects of T cell survival and activation, and promotes production of proinflammatory cytokines. We found that immunoproteasome activity is dispensable in adult mice. However, immunoproteasome deficiency in neonatal mice increased mortality and impaired IFN-γ responses in the lungs. Baseline immunoproteasome subunit expression in lungs of uninfected mice increased with age. Our findings suggest the existence of developmental regulation of the immunoproteasome, like other aspects of host immune function, and indicate that immunoproteasome activity is a critical protective factor early in life.

Enhanced Replication of Mouse Adenovirus Type 1 following Virus-Induced Degradation of Protein Kinase R (PKR).

Protein kinase R (PKR) plays a major role in activating host immunity during infection by sensing double-stranded RNA (dsRNA) produced by viruses. Once activated by dsRNA, PKR phosphorylates the translation factor eukaryotic initiation factor 2α (eIF2α), halting cellular translation. Many viruses have methods of inhibiting PKR activation or its downstream effects, circumventing protein synthesis shutdown. These include sequestering dsRNA or producing proteins that bind to and inhibit PKR activation. Here we describe our finding that in multiple cell types, PKR was depleted during mouse adenovirus type 1 (MAV-1) infection. MAV-1 did not appear to be targeting PKR at the transcriptional or translational level, because total PKR mRNA levels and levels of PKR mRNA bound to polysomes were unchanged or increased during MAV-1 infection. However, inhibiting the proteasome reduced the PKR depletion seen in MAV-1-infected cells, whereas inhibiting the lysosome had no effect. This suggests that proteasomal degradation alone is responsible for PKR degradation during MAV-1 infection. Time course experiments indicated that the degradation occurs early after infection. Infecting cells with UV-inactivated virus prevented PKR degradation, whereas inhibiting viral DNA replication did not. Together, these results suggest that an early viral gene is responsible. Degradation of PKR is a rare mechanism to oppose PKR activity, and it has been described in only six RNA viruses. To our knowledge, this is the first example of a DNA virus counteracting PKR by degrading it.IMPORTANCE The first line of defense in cells during viral infection is the innate immune system, which is activated by different viral products. PKR is a part of this innate immune system and is induced by interferon and activated by dsRNA produced by DNA and RNA viruses. PKR is such an important part of the antiviral response that many viral families have gene products to counteract its activation or the resulting effects of its activity. Although a few RNA viruses degrade PKR, this method of counteracting PKR has not been reported for any DNA viruses. MAV-1 does not encode virus-associated RNAs, a human adenoviral defense against PKR activation. Instead, MAV-1 degrades PKR, and it is the first DNA virus reported to do so. The innate immune evasion by PKR degradation is a previously unidentified way for a DNA virus to circumvent the host antiviral response.

Characterization of a novel species of adenovirus from Japanese microbat and role of CXADR as its entry factor.

Recently, bat adenoviruses (BtAdVs) of genus Mastadenovirus have been isolated from various bat species, some of them displaying a wide host range in cell culture. In this study, we isolated two BtAdVs from Japanese wild microbats. While one isolate was classified as Bat mastadenovirus A, the other was phylogenetically independent of other BtAdVs. It was rather related to, but serologically different from, canine adenoviruses. We propose that the latter, isolated from Asian parti-colored bat, should be assigned to a novel species of Bat mastadenovirus. Both isolates replicated in various mammalian cell lines, implying their wide cell tropism. To gain insight into cell tropism of these BtAdVs, we investigated the coxsackievirus and adenovirus receptor (CXADR) for virus entry to the cells. We prepared CXADR-knockout canine kidney cells and found that replication of BtAdVs was significantly hampered in these cells. For confirmation, their replication in canine CXADR-addback cells was rescued to the levels with the original cells. We also found that viral replication was corrected in human or bat CXADR-transduced cells to similar levels as in canine CXADR-addback cells. These results suggest that BtAdVs were able to use several mammalian-derived CXADRs as entry factors.

Novel Divergent Polar Bear-Associated Mastadenovirus Recovered from a Deceased Juvenile Polar Bear.

Polar bears in captivity can be exposed to opportunistic pathogens not present in their natural environments. A 4-month-old polar bear (Ursus maritimus) living in an isolated enclosure with his mother in the Tierpark Berlin, Berlin, Germany, was suffering from severe abdominal pain, mild diarrhea, and loss of appetite and died in early 2017. Histopathology revealed severe hepatic degeneration and necrosis without evidence of inflammation or inclusion bodies, although a viral infection had been suspected on the basis of the clinical signs. We searched for nucleic acids of pathogens by shotgun high-throughput sequencing (HTS) from genomic DNA and cDNA extracted from tissue and blood. We identified a novel Mastadenovirus and assembled a nearly complete genome from the shotgun sequences. Quantitative PCR (qPCR) revealed that viral DNA was present in various concentrations in all tissues examined and that the highest concentrations were found in blood. Viral culture did not yield cytopathic effects, but qPCR suggested that virus replication was sustained for up to three passages. Positive immunofluorescence staining confirmed that the virus was able to replicate in the cells during early passage. Phylogenetic analysis demonstrated that the virus is highly divergent compared to other previously identified Mastadenovirus members and basal to most known viral clades. The virus was found only in the 4-month-old bear and not in other captive polar bears tested. We surmised, therefore, that the polar bear was infected from an unknown reservoir, illustrating that adenoviral diversity remains underestimated and that cross-species transmission of viruses can occur even under conditions of relative isolation.IMPORTANCE Cross-species transmission of viral pathogens is becoming an increasing problem for captive-animal facilities. This study highlights how animals in captivity are vulnerable to novel opportunistic pathogens, many of which do not result in straightforward diagnosis from symptoms and histopathology. In this study, a novel pathogen was suspected to have contributed to the death of a juvenile polar bear. HTS techniques were employed, and a novel Mastadenovirus was isolated. The virus was present in both the tissue and blood samples. Phylogenetic analysis of the virus at both the gene and genome levels revealed that it is highly divergent to other known mastadenoviruses. Overall, this study shows that animals in isolated conditions still come into contact with novel pathogens, and for many of these pathogens, the host reservoir and mode of transmission are yet to be determined.

Mouse adenovirus type 1 infection of adipose tissue.

Human adenovirus (HAdV) type 36 seropositivity has been linked to obesity in humans. That link is supported by a small number of studies using HAdV-36 infection of animals that are not natural hosts for HAdVs. In this study, we infected mice with mouse adenovirus type 1 (MAV-1), a mouse pathogen, to determine whether MAV-1 infected adipose tissue and was associated with adipose tissue inflammation and obesity. We detected MAV-1 in adipose tissue during acute MAV-1 infection, but we did not detect virus-induced increases in adipose tissue cytokine expression or histological evidence of adipose tissue inflammation during acute infection. MAV-1 did not persist in adipose tissue at later times, and we did not detect long-term adipose inflammation, increased adipose tissue mass, or body weight in infected mice. Our data indicate that MAV-1 is not associated with obesity in infected mice.

Pathogenesis of a Chinese strain of bovine adenovirus type 3 infection in albino guinea pigs.

Bovine adenovirus type 3 (BAV-3) is considered one of the most important respiratory tract agents of cattle and is widespread among cattle around the world. A BAV-3 strain was isolated from a bovine nasal swab for the first time in China in 2009 and named HLJ0955. Subsequently, BAV-3 has frequently been isolated from calves with respiratory diseases in China. To date, only limited study on the pathogenesis of BAV-3 infection in cotton rats has been conducted, and the pathogenesis of BAV-3 infection in guinea pigs has not been reported. Therefore, sixteen albino guinea pigs were inoculated intranasally with HLJ0955. All of the infected guinea pigs had apparently elevated rectal temperatures (39.2 °C-39.9 °C) at 2-7 days post-inoculation (PI). Consolidation and petechial hemorrhage were also observed in guinea pigs experimentally infected with HLJ0955. Viral replication was detectable by virus isolation and titration and by immunohistochemistry in the lungs of guinea pigs as early as 24 h PI. Viral DNA was detectable in the lungs of infected guinea pigs during 11 days of observation by real-time PCR. Virus-neutralizing antibodies against BAV-3 were detectable from 11 days PI and reached a peak titer at 15 days PI. Histopathological changes mainly occurred in the lungs of infected guinea pigs and were characterized by thickening of alveolar septa, mononuclear cell infiltration, hemorrhage and alveolar epithelial necrosis. These results indicate that HLJ0955 can replicate in the lungs of guinea pigs and cause fever and gross and histological lesions. The guinea pig infection model of BAV-3 would serve as a useful system for monitoring the infection process and pathogenesis of the Chinese BAV-3 strain HLJ0955, as well as immune responses to BAV-3 vaccines.

Evaluation of virucidal activity of three commercial disinfectants and formic acid using bovine enterovirus type 1 (ECBO virus), mammalian orthoreovirus type 1 and bovine adenovirus type 1.

A modified version of the test method of the Comité Européen de Normalisation (CEN) was developed using formic acid and three commercial disinfectants to evaluate virucidal activity against three non-enveloped viruses, bovine enterovirus type 1 (ECBO virus), mammalian orthoreovirus type 1 and bovine adenovirus type 1 (BAV 1). Determination of the effects of temperature was carried out at 20 and 10 degrees C. All tests with protein load used bovine serum albumin (BSA) and yeast extract. The investigations were performed in suspension tests and in carrier tests using poplar wood virus carriers. The carrier tests showed that ECBO virus could be inactivated at 20 degrees C with 1% formic acid within a 60 min reaction time. For disinfection of ECBO virus at 10 degrees C within 60 min, a 2% concentration of formic acid was necessary. Formic acid was ineffective against reovirus and bovine adenovirus and cannot be recommended as a reference disinfectant. Inactivation of ECBO virus and adenovirus type 1 using a disinfectant containing aldehydes and alcohols could be achieved, but only at room temperature. The disinfection of reovirus type 1 at room temperature with this product was possible without a protein load. This disinfectant exhibited disinfection ability at 10 degrees C at a concentration of more than 2% or with a longer exposure time. A disinfectant containing aldehydes was effective at room temperature but its effect was reduced in the presence of organic matter. Inactivation at 10 degrees C was found only against adenovirus. The fourth disinfectant, which contained peroxiacetic acid, inactivated all test viruses at a concentration of 0.5% within 15 min independent of temperature and protein load.

Bovine adenovirus type 3 pneumonia in dexamethasone-treated calves.

The effects of immunosuppression were examined in 1.5-month-old calves that were given dexamethasone (DM) before endobronchial inoculation with bovine adenovirus type 3 (BAV-3). Immunohistopathologically, severe necrotizing bronchiolitis with eosinophilic and basophilic intranuclear inclusion bodies was observed both in DM-treated 1.5-month-old infected calves and in non-DM-treated 7-day-old infected calves. These inclusion bodies were correlated with the detection of BAV-3 antigen and viral particles. The presence of inclusion bodies in the desquamated epithelial cells or of BAV-3 antigen, or both, correlated well with the isolated level of BAV-3 in bronchoalveolar lavage (BAL) fluid. Few immunoglobulin (IgG, IgM, and IgA)-containing B lymphocytes or CD8+ T lymphocytes infiltrated the pneumonic lesion in both the 7-day-old and the DM-treated 1.5-month-old infected calves. Thus, depletion of CD8+ T lymphocytes in calves might influence the clearance of BAV-3 from respiratory tissues.

Immunohistopathology of calf pneumonia induced by endobronchial inoculation with bovine adenovirus 3.

Three 1-week-old and three 3-month-old Holstein calves that had received colostrum were inoculated endobronchially with bovine adenovirus 3 (BAV-3). The gross and histologic lesions in these six infected calves were localized mainly in the right caudal lobe of the lung and were closely associated with the site of the deposition of the inoculum. The pneumonic lesions were severe necrotizing bronchitis, bronchiolitis, and alveolitis, accompanied by infiltration of inflammatory cells and proliferation of type 2 pneumocytes. Intranuclear inclusion bodies, BAV-3 antigen, and virus particles were detected in the degenerated epithelial cells in the 1-week-old but not the 3-month-old calves. After infection, the total cell count in the bronchoalveolar lavage (BAL) fluid cells was increased. The results of BAV-3 isolation from BAL fluid were correlated with the detection of intranuclear inclusion bodies in the desquamated epithelial cells in the BAL fluid cells from the right caudal lobe but not in cells from the left caudal lobe. CD8+ T lymphocytes in the pneumonic lesion were found only in the 3-month-old infected calves. The difference in the immunopathologic reactions between the 1-week-old and the 3-month-old infected calves may be attributed to differences in immune system development.

Bovine adenovirus type 3 E1B(small) protein is essential for growth in bovine fibroblast cells.

In order to study the function of bovine adenovirus type 3 (BAV-3) E1A and E1B(small) proteins, we constructed two mutants: (a) BAV102A carries an in-frame deletion in the coding region for the E1A protein (nt 831-1080); (b) BAV102B carries an insertion of triple stop codons in the E1B region (nt 1654, 178 bp downstream of the E1B(small) start codon), which stops the translation of the E1B(small) gene. BAV102A virus could grow to the wild-type BAV-3 titer in transformed cell line VIDO R2 (HAV-5 E1 transformed) cells, but no progeny virus could be found in fetal bovine retina cells (FBRC). RT-PCR and Western blot analysis showed that neither mRNA transcripts nor protein expression of early genes [E1B(small) and DNA binding protein (DBP)] could be detected in BAV102A infected FBRC. The BAV102B grew 1.5 log less than wild-type BAV-3 in FBRC; however, no BAV102B progeny virus could be observed in bovine fibroblast (BFB) cells. No appreciable difference was observed in DBP transcript synthesis between wild-type BAV-3- or BAV102B-infected FBRC. However, compared to wild-type BAV-3, BAV102B viral DNA synthesis and fiber gene expression were found to be slightly reduced in FBRC. In contrast, compared to wild-type BAV-3, DBP transcripts and viral DNA synthesis were drastically reduced in BAV102B-infected BFB cells. In addition, no fiber gene expression could be detected in BAV102B-infected BFB cells. These results suggest that BAV-3 E1A is essential for virus replication and is required for activating the transcription of other BAV-3 early genes. However, the requirement for E1B(small) protein for BAV-3 replication appears to be cell type-dependent.

Determination of bovine adenovirus-3 titer based on immunohistochemical detection of DNA binding protein in infected cells.

DNA sequence coding for a portion of DNA binding protein (amino acids 3-58) of bovine adenovirus type-3 (BAV-3) was cloned and expressed in Escherichia coli as a fusion protein with Schistosoma japonicum glutathione S-transferase. The fusion protein was affinity purified and used to immunize rabbits. Immunoprecipitation and Western blot analysis showed that the antiserum could specifically recognize a protein of 48 kDa in BAV-3-infected cells, which was produced both in early and late phases of BAV-3 life cycle. Based on the ability of antiserum to recognize DNA binding protein, a novel assay for BAV-3 quantitation was established. The assay is less time consuming and can be performed on a wide variety of bovine cells. In addition, virus titers determined by this assay are comparable to the standard plaque assay.

The role of the L4 33K gene in adenovirus infection.

The late phase of adenovirus infection is characterized not only by the synthesis of late proteins and the assembly of new virions, but also by the inhibition of early gene expression and host cell translation. Previous work has demonstrated that both of these inhibitory effects depend upon expression from the major late transcription unit (MLTU), controlled by the major late promoter (MLP). Furthermore, the repression of early gene expression has been shown to be mediated in trans, suggesting a role for one or more MLTU-encoded soluble factor(s). A possible candidate for such a factor is the L4-encoded 33K gene product, a protein conserved throughout the Mastadenoviridae, but of no known function. To test the role of this protein in viral infection, a stop codon was placed at the 20th position of the 33K ORF. Viable virus with genomes containing the mutation were recovered in an overlap recombination assay. Phenotypic analysis revealed that the mutant virus had a significant deficiency in both kinetics of replication and final yield, as compared to the wild-type virus. Detailed analysis of infected cells showed that there was no detectable change in the regulation of expression of several early genes and the pIX gene. This suggests either that 33K is not involved in this late phase phenomenon or that this function is replaceable by another late protein(s). Late protein synthesis and accumulation were similar to those in wild-type-infected cells. However, the reduced yield of infectious mutant virus could be accounted for by a marked deficiency in the accumulation of intermediate particles and completed capsids, suggesting a role for 33K in the process of assembly. In addition there was a small but reproducible deficiency in the shutoff of host cell translation. These results show that the 33K protein plays an important, although apparently not essential, function in the late phase of virus infection.

Development of porcine adenovirus-3 as an expression vector.

Porcine adenovirus-3 (PAV-3) was developed as an expression vector using homologous recombination in Escherichia coli BJ 5183. As a prerequisite, the complete genome of PAV-3 was first introduced as a PacI restriction fragment into a bacterial plasmid. The plasmid, when PacI restricted and transfected into swine testicular cells, produces an infectious virus. The potential of this procedure was demonstrated by the construction of several PAV-3 recombinants. Part of the E3 region, which is nonessential for virus replication under cell culture conditions, was identified and deleted from the virus genome. The gene for glycoprotein D (gD) of pseudorabies virus (PRV), which elicits PRV-neutralizing antibodies in pigs, was cloned and expressed from the E3 region of PAV-3. A 50 kDa polypeptide was identified in recombinant PAV-3-infected cell lysates by immunoprecipitation assays using gD-specific monoclonal antibodies. In another experiment, a region between the right inverted terminal repeat and the promoter of the E4 region was used to clone and express the chloramphenicol acetyltransferase (CAT) gene under the control of SV40 immediate early promoter. CAT gene expression was observed irrespective of the orientation of the CAT gene. These results indicate that the helper-independent recombinant PAV-3 could be used as an expression vector and has potential as a recombinant vaccine vector in pigs.

Mouse adenovirus type 1 early region 1A is dispensable for growth in cultured fibroblasts.

Mouse adenovirus type 1 (MAV-1) mutants with deletions of conserved regions of early region 1A (E1A) or with point mutations that eliminate translation of E1A were used to determine the role of E1A in MAV-1 replication. MAV-1 E1A mutants expressing no E1A protein grew to titers comparable to wild-type MAV-1 titers on mouse fibroblasts (3T6 fibroblasts and fibroblasts derived from Rb+/+, Rb+/-, and Rb-/- transgenic embryos). To test the hypothesis that E1A could induce a quiescent cell to reenter the cell cycle, fibroblasts were serum starved to stop DNA replication and cellular replication and then infected with the E1A mutant and wild-type viruses. All grew to equivalent titers. Steady-state levels of MAV-1 early mRNAs (E1A, E1B, E2, E3, and E4) from 3T6 cells infected with wild-type or E1A mutant virus were examined by Northern analysis. Steady-state levels of mRNAs from the mutant-infected cells were comparable to or greater than the levels found in wild-type virus infections for most of the early regions and for two late genes. The E2 mRNA levels were slightly reduced in all mutant infections relative to wild-type infections. E1A mRNA was not detected from infections with the MAV-1 E1A null mutant, pmE109, or from infections with similar MAV-1 E1A null mutants, pmE112 and pmE113. The implications for the lack of a requirement of E1A in cell culture are discussed.