Oncogenesis of mammary glands, skin, and bones by polyomavirus correlates with viral persistence and prolonged genome replication potential.

A correlation between polyomavirus-induced oncogenesis and viral persistence on the one hand and/or prolonged genome replication potential on the other was established with respect to their respective organ distributions. Prolonged replication potential is defined as the capacity of a genome to replicate in a given organ from the time of infection up to the onset of oncogenesis. This conclusion was derived following intraperitoneal infection of BALB/c mice with wild-type strain A2. Viral genomes were used as parameters of persistence and replication and were detected by Southern blotting and PCR analysis. The major tumor target organs (mammary gland, skin, and bone), which have not been previously analyzed for persistence, were compared with other, non-tumor-prone organs (kidney, liver, lung, spleen, and salivary gland). A progressive loss of viral genomes was observed in all tissues as a function of time postinfection; however, genomes were shown to persist through 20 weeks postinfection in the mammary glands, skin, and bones to an extent similar to that in the previously described kidneys (D. J. McCance, J. Virol. 39:958-962, 1981; W. P. Rowe, J. W. Hartley, J. D. Estes, and R. J. Huebner, Natl. Cancer Inst. Monogr. 4:189-209, 1960). Thus, tumors arise among organs that sustain a persistent infection, but not all such organs develop tumors (e.g., the kidney). The capacity of organs to support de novo replication at various ages, including the age reached when the first tumors are detected, was also determined using a 3-day infection period for ages between 0 and 7 weeks. For all organs tested, a higher level of genomes was observed in organs of mice infected as neonates than in those infected after the age of 3 weeks. However, marked organ-specific differences were seen in the degree and timing of loss of replication. In particular, viral genome replication, although reduced, was maintained in the mammary glands, skin, and bones of adult animals, in contrast to the kidneys. We conclude that organ-specific oncogenesis correlates with two organ-specific parameters: persistence of viral genomes and prolonged viral genome replication potential. This may reflect a requirement for continued viral genome replication and/or gene expression for tumorigenesis. In turn, these parameters may be linked to the tissue-specific continued capacity for cellular division.     

Systemic polyomavirus genome increase and dissemination of capsid-defective genomes in mammary gland tumor-bearing mice

BALB/c mice that developed tumors 7 to 8 months following neonatal infection by polyomavirus (PYV) wild-type strain A2 were characterized with respect to the abundance and integrity of the viral genome in the tumors and in 12 nontumorous organs. These patterns were compared to those found in tumor-free mice infected in parallel. Six mice were analyzed in detail including four sibling females with mammary gland tumors. In four of five mammary gland tumors, the viral genome had undergone a unique deletion and/or rearrangement. Three tumor-resident genomes with an apparently intact large T coding region were present in abundant levels in an unintegrated state. Two of these had undergone deletions and rearrangements involving the capsid genes and therefore lacked the capacity to produce live virus. In the comparative organ survey, the tumors harboring replication-competent genomes contained by far the highest levels of genomes of any tissue. However, the levels of PYV genomes in other organs were elevated by up to 1 to 2 orders of magnitude compared to those detected in the same organs of tumor-free mice. The genomes found in the nontumorous organs had the same rearrangements as the genomes residing in the tumors. The original wild-type genome was detected at low levels in a few organs, particularly in the kidneys. The data indicate that a systemic increase in the level of viral genomes occurred in conjunction with the induction of tumors by PYV. The results suggest two novel hypotheses: (i) that genomes may spread from the tumors to the usual PYV target tissues and (ii) that this dissemination may take place in the absence of capsids, providing an important path for a virus to escape from the immune response. This situation may offer a useful model for the spread of HPV accompanying HPV-induced oncogenesis.     

Pattern of polyomavirus replication from infection until tumor formation in the organs of athymic nu/nu mice.

Inbred athymic nu/nu BALB/c mice were injected subcutaneously with the highly oncogenic polyomavirus A2 strain, and the sites of viral DNA replication were determined by whole mouse section hybridization (T. W. Dubensky, E. A. Murphy, and L. P. Villareal, J. Virol. 50:779-783, 1984) and Southern blot analysis. We show that infection is persistent in some epithelial tissues (skin, mammary, and salivary glands), in lymphoid organs (spleen and nodes), and in mesenchymal bone tissue. Only mammary glands and bones were targets for tumor formation. Although the same pattern of infection was observed in males and females, mammary adenocarcinomas were induced exclusively in females, while the frequency of osteosarcomas was similar in both sexes. No viral DNA or lytic lesion was detected in kidney, liver, or lung tissue. The restricted targeting of polyomavirus oncogenicity in nude mice, compared with newborn immunocompetent animals, inoculated via the same route with the same virus strain, therefore does not reflect selective tissue targeting of virus replication. These results further document the influence of the age, immunological status, and genetic background of the host on the pattern of viral infection and tumor formation.     

Enhancer dependence of polyomavirus persistence in mouse kidneys.

We previously showed that alterations in the enhancer sequence of polyomavirus DNA can alter both the level and the organ specificity of viral DNA replication during the acute phase of infection of newborn mice (R. Rochford, B. A. Campbell, and L. P. Villarreal, J. Virol. 64:476-485, 1990). In this study, we examined whether these enhancer sequence alterations can also affect polyomavirus replication during the persistent phase of infection in vivo. After infection of newborn mice with a mixture of three enhancer variants, the individual organs could select for enhancer-specific viral DNA replication during both the acute and the persistent phases of infection. Contrary to expectations, the ability of some variants to establish a high-level acute infection in some organs (e.g., the pancreas) did not necessarily lead to a persistent infection in those organs. Thus, enhancers can affect acute and persistent infections differently. In addition, some enhancer variants tended to establish a high-level persistent infection in the kidneys immediately following an acute infection; however, in all cases considerable histopathology was associated with these elevated long-term infections, and these mice were always runty. A persistent infection in the kidneys thus appears able to exist in two distinguishable states, a high-level pathological state and a low-level nonpathological state, which can be affected by the viral enhancer sequence.     

Genetic analysis of the enhancer requirements for polyomavirus DNA replication in mice.

In this report, we describe the first systematic analysis of the genetic requirements for polyomavirus (Py) enhancer-activated viral DNA replication during the acute phase of infection in mice. Four mutants were made which substituted XhoI sites for conserved enhancer consensus sequences (adenovirus type 5 E1A, c-fos, simian virus 40, and a glucocorticoidlike consensus sequence). Viral DNA replication in infected mouse organs was measured by DNA blot analysis. Only the loss of the glucocorticoidlike consensus sequence element significantly reduced Py DNA replication in the kidneys, the primary target organ for viral replication. The loss of the c-fos, adenovirus type 5 E1A, or simian virus 40 consensus sequences, however, expanded organ-specific viral DNA replication, relative to wild-type Py, by allowing high-level replication in the pancreas or heart or both. Analysis of Py variants selected for replication in undifferentiated embryonal carcinoma cell lines (PyF441, PyF111) showed that there was little change in levels of viral DNA replication in kidneys and other organs as compared with those in the wild-type virus. If the entire B enhancer is deleted, only low overall levels of viral replication are observed. Wild-type levels of replication in the kidneys can be reconstituted by addition of a single domain from within the A enhancer (nucleotides 5094 to 5132) to the B enhancer deletion virus, suggesting that a single domain from the A enhancer can functionally substitute for the entire B enhancer. This also indicates that the determinants for kidney-specific replication are not found in the B enhancer.     

The primary site of replication alters the eventual site of persistent infection by polyomavirus in mice.

Using DNA blot analysis, we monitored the course of polyomavirus infection in mice receiving an intranasal inoculation and compared this with the course of infection in mice receiving an intraperitoneal inoculation. Intranasal infection was characterized by an initial primary replication phase in the respiratory tract, followed by a systemic infection of the visceral organs. At 12 days postinfection, there was partial clearing of viral DNA in all organs; by 22 days postinfection, viral DNA persisted only in the lungs and kidneys, and the level of DNA slowly decreased during the next 3 months. Lungs have been a previously unrecognized site for polyomavirus persistent infection. In contrast to intranasal infection, intraperitoneal infection of mice was characterized by only three phases: an initial systemic phase in which viral DNA was found in the same respiratory and visceral organs as during intranasal infection, clearing of the virus from the organs, and ultimately, a persistent infection in the kidneys but not in the lungs. Thus, different organs became persistently infected when mice were inoculated via these different routes.     

Susceptibility to acute mouse adenovirus type 1 respiratory infection and establishment of protective immunity in neonatal mice

There is an incomplete understanding of the differences between neonatal immune responses that contribute to the increased susceptibility of neonates to some viral infections. We tested the hypothesis that neonates are more susceptible than adults to mouse adenovirus type 1 (MAV-1) respiratory infection and are impaired in the ability to generate a protective immune response against a second infection. Following intranasal infection, lung viral loads were greater in neonates than in adults during the acute phase but the virus was cleared from the lungs of neonates as efficiently as it was from adult lungs. Lung gamma interferon (IFN-γ) responses were blunted and delayed in neonates, and lung viral loads were higher in adult IFN-γ(-/-) mice than in IFN-γ(+/+) controls. However, administration of recombinant IFN-γ to neonates had no effect on lung viral loads. Recruitment of inflammatory cells to the airways was impaired in neonates. CD4 and CD8 T cell responses were similar in the lungs of neonates and adults, although a transient increase in regulatory T cells occurred only in the lungs of infected neonates. Infection of neonates led to protection against reinfection later in life that was associated with increased effector memory CD8 T cells in the lungs. We conclude that neonates are more susceptible than adults to acute MAV-1 respiratory infection but are capable of generating protective immune responses.     

Role of IL-10 in a neonatal mouse listeriosis model.

This study was undertaken to test the hypothesis that altered IL-10 production plays a role in the increased susceptibility of neonates to listeriosis. Plasma IL-10 levels were measured in neonatal and adult mice at various times after infection with Listeria monocytogenes. Relative to adults, neonatal mice had markedly increased IL-10 levels early in the course of infection with Listeria using a 90% lethal dose. Higher neonatal IL-10 responses were also observed after injecting adults and pups with equal doses of killed organisms. Splenic macrophages from neonates produced higher IL-10 levels than those of adults after in vitro stimulation with killed bacteria, confirming in vivo observations. Moreover, IL-10 blockade had differential effects in neonates and adults infected with live Listeria. In adult mice, anti-IL-10 Abs decreased bacterial burden early in the course of infection, but were no longer effective at 6 days or later after challenge. In the pups, however, the same treatment had beneficial effects both early and late during infection and resulted in increased survival. Collectively, our data suggest that an overproduction of IL-10 by macrophages may at least partially explain the increased susceptibility of neonates to listeriosis, and provide further evidence that cytokine production is different in adults and neonates.     

Gamma interferon controls mouse polyomavirus infection in vivo

Human polyomaviruses are associated with substantial morbidity in immunocompromised patients, including those with HIV/AIDS, recipients of bone marrow and kidney transplants, and individuals receiving immunomodulatory agents for autoimmune and inflammatory diseases. No effective antipolyomavirus agents are currently available, and no host determinants have been identified to predict susceptibility to polyomavirus-associated diseases. Using the mouse polyomavirus (MPyV) infection model, we recently demonstrated that perforin-granzyme exocytosis, tumor necrosis factor alpha (TNF-α), and Fas did not contribute to control of infection or virus-induced tumors. Gamma interferon (IFN-γ) was recently shown to inhibit replication by human BK polyomavirus in primary cultures of renal tubular epithelial cells. In this study, we provide evidence that IFN-γ is an important component of the host defense against MPyV infection and tumorigenesis. In immortalized and primary cells, IFN-γ reduces expression of MPyV proteins and impairs viral replication. Mice deficient for the IFN-γ receptor (IFN-γR(-/-)) maintain higher viral loads during MPyV infection and are susceptible to MPyV-induced tumors; this increased viral load is not associated with a defective MPyV-specific CD8(+) T cell response. Using an acute MPyV infection kidney transplant model, we further show that IFN-γR(-/-) donor kidneys harbor higher MPyV levels than donor kidneys from wild-type mice. Finally, administration of IFN-γ to persistently infected mice significantly reduces MPyV levels in multiple organs, including the kidney, a major reservoir for persistent mouse and human polyomavirus infections. These findings demonstrate that IFN-γ is an antiviral effector molecule for MPyV infection.     

In vivo replication of the hamster polyomavirus genome and generation of specific deletions in the process of lymphomagenesis.

Hamster polyomavirus (HaPV) causes lymphomas when injected into newborn hamsters. These tumors are virus-free but accumulate large amounts of deleted extrachromosomal viral genomes. In order to identify the major sites of virus replication in animals, we have monitored the HaPV DNA present in different organs at various times after injection. The data demonstrate that viral replication preferentially occurs in lymphoid organs. Lymphoma-associated viral genomes display specific deletions. PCR analysis shows that such viral genomes are the only variants detectable in infected animals, suggesting that they are generated by a specific cellular mechanism. We have tested the possible role of the lymphoid cell-specific V(D)J recombination activity in the generation of these specific variants. Our results indicate that this mechanism is not solely responsible for the viral genome rearrangement, if involved at all.     

Bioluminescence imaging reveals systemic dissemination of herpes simplex virus type 1 in the absence of interferon receptors

Herpes simplex virus type 1 (HSV-1) can produce disseminated, systemic infection in neonates and patients with AIDS or other immunocompromising diseases, resulting in significant morbidity and mortality in spite of antiviral therapy. Components of host immunity that normally limit HSV-1 to localized epithelial and neuronal infection remain incompletely defined. We used in vivo bioluminescence imaging to determine effects of type I and II interferons (IFNs) on replication and tropism of HSV-1 infection in mice with genetic deficiency of type I, type II, or both type I and II IFN receptors. Following footpad or ocular infection of mice lacking type I IFN receptors, HSV-1 spread to parenchymal organs, including lung, liver, spleen, and regional lymph nodes, but mice survived. Deletion of type I and II IFN receptors produced quantitatively greatest and most widespread dissemination of virus to visceral organs and the nervous system, and these mice invariably died after ocular or footpad infection. Type II receptor knockout and wild-type mice had comparable viral replication and localization, with no systemic spread of HSV-1 or lethality. Therefore, while isolated deficiency of type II IFN receptors did not affect pathogenesis, loss of these receptors in combination with genetic deletion of type I receptors had a profound effect on susceptibility to HSV-1. These data demonstrate different effects of type I and II IFNs in limiting systemic dissemination of HSV-1 and further validate the use of bioluminescence imaging for studies of viral pathogenesis.     

A mouse model for Chikungunya: young age and inefficient type-I interferon signaling are risk factors for severe disease

Chikungunya virus (CHIKV) is a re-emerging arbovirus responsible for a massive outbreak currently afflicting the Indian Ocean region and India. Infection from CHIKV typically induces a mild disease in humans, characterized by fever, myalgia, arthralgia, and rash. Cases of severe CHIKV infection involving the central nervous system (CNS) have recently been described in neonates as well as in adults with underlying conditions. The pathophysiology of CHIKV infection and the basis for disease severity are unknown. To address these critical issues, we have developed an animal model of CHIKV infection. We show here that whereas wild type (WT) adult mice are resistant to CHIKV infection, WT mouse neonates are susceptible and neonatal disease severity is age-dependent. Adult mice with a partially (IFN-alpha/betaR(+/-)) or totally (IFN-alpha/betaR(-/-)) abrogated type-I IFN pathway develop a mild or severe infection, respectively. In mice with a mild infection, after a burst of viral replication in the liver, CHIKV primarily targets muscle, joint, and skin fibroblasts, a cell and tissue tropism similar to that observed in biopsy samples of CHIKV-infected humans. In case of severe infections, CHIKV also disseminates to other tissues including the CNS, where it specifically targets the choroid plexuses and the leptomeninges. Together, these data indicate that CHIKV-associated symptoms match viral tissue and cell tropisms, and demonstrate that the fibroblast is a predominant target cell of CHIKV. These data also identify the neonatal phase and inefficient type-I IFN signaling as risk factors for severe CHIKV-associated disease. The development of a permissive small animal model will expedite the testing of future vaccines and therapeutic candidates.